Journal: Cardiovasc Res

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Abstract

Testosterone therapy and cardiovascular diseases.

Cittadini A, Isidori AM, Salzano A
Since it was first synthesised in 1935, testosterone (T) has been viewed as the mythical Fountain of Youth, promising rejuvenation, restoring sexual appetites, growing stronger muscles, and quicker thinking. T is endowed with direct effects on myocardial and vascular structure and function, as well as on risk factors for cardiovascular (CV) disease. Indeed, low serum T levels are a risk factor for diabetes, metabolic syndrome, inflammation, and dyslipidaemia. Moreover, many studies have shown that T deficiency per se is an independent risk factor of CV and all-cause mortality. On this background and due to direct-to-patient marketing by drug companies, we have witnessed to the widespread use of T replacement therapy (TT) without clear indications particularly in late-life onset hypogonadism. The current review will dwell upon current evidence and controversies surrounding the role of T in the pathophysiology of CV diseases, the link between circulating T levels and CV risk, and the use of replacing T as a possible adjuvant treatment in specific CV disorders. Specifically, recent findings suggest that heart failure and type 2 diabetes mellitus represent two potential targets of T therapy once that a state of hypogonadism is diagnosed. However, only if ongoing studies solve the CV safety issue the T orchid may eventually \'bloom\'.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 21 Jul 2021; epub ahead of print
Cittadini A, Isidori AM, Salzano A
Cardiovasc Res: 21 Jul 2021; epub ahead of print | PMID: 34293112
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Abstract

Nicotine promotes vascular calcification via intracellular Ca2+-mediated, Nox5-induced oxidative stress and extracellular vesicle release in vascular smooth muscle cells.

Petsophonsakul P, Burgmaier M, Willems B, Heeneman S, ... Furmanik M, Schurgers L
Aims
Smokers are at increased risk of cardiovascular events. However, the exact mechanisms through which smoking influences cardiovascular disease resulting in accelerated atherosclerosis and vascular calcification are unknown. The aim of this study was to investigate effects of nicotine on initiation of vascular smooth muscle cell (VSMC) calcification and to elucidate underlying mechanisms.
Methods and results
We assessed vascular calcification of 62 carotid lesions of both smoking and non-smoking patients using ex vivo micro-computed-tomography (µCT)-scanning. Calcification was present more often in carotid plaques of smokers (n = 22 of 30, 73.3%) compared to non-smokers (n = 11 of 32, 34.3%; p < 0.001), confirming higher atherosclerotic burden. The difference was particularly profound for microcalcifications, which was 17-fold higher in smokers compared to non-smokers. In vitro, nicotine induced human primary VSMC calcification, increased osteogenic gene expression (Runx2, Osx, BSP and OPN), and extracellular vesicle (EV) secretion. The pro-calcifying effects of nicotine were mediated by Ca2+-dependent Nox5. SiRNA knock-down of Nox5 inhibited nicotine-induced EV release and calcification. Moreover, pre-treatment of hVSMCs with vitamin K2 ameliorated nicotine-induced intracellular oxidative stress, EV secretion, and calcification. Using nicotinic acetylcholine receptor (nAChR) blockers α-bungarotoxin and hexamethonium bromide we found that the effects of nicotine on intracellular Ca2+ and oxidative stress were mediated by α7 and α3 nAChR. Finally, we showed that Nox5 expression was higher in carotid arteries of smokers and correlated with calcification levels in these vessels.
Conclusion
In this study we provide evidence that nicotine induces Nox5-mediated pro-calcific processes as novel mechanism of increased atherosclerotic calcification. We identified that activation of α7 and α3 nAChR by nicotine increases intracellular Ca2+ and initiates calcification of hVSMCs through increased Nox5 activity, leading to oxidative stress-mediated EV release. Identifying the role of Nox5-induced oxidative stress opens novel avenues for diagnosis and treatment of smoking-induced cardiovascular disease.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 16 Jul 2021; epub ahead of print
Petsophonsakul P, Burgmaier M, Willems B, Heeneman S, ... Furmanik M, Schurgers L
Cardiovasc Res: 16 Jul 2021; epub ahead of print | PMID: 34273166
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Abstract

Natriuretic peptide receptor B maintains heart rate and sinoatrial node function via cyclic GMP-mediated signaling.

Dorey TW, Mackasey M, Jansen HJ, McRae MD, ... Atkinson L, Rose RA
Aims
Heart rate (HR) is a critical indicator of cardiac performance that is determined by sinoatrial node (SAN) function and regulation. Natriuretic peptides, including C-type NP (CNP) have been shown to modulate ion channel function in the SAN when applied exogenously. CNP is the only NP that acts as a ligand for natriuretic peptide receptor-B (NPR-B). Despite these properties, the ability of CNP and NPR-B to regulate HR and intrinsic SAN automaticity in vivo, and the mechanisms by which it does so, are incompletely understood. Thus, the objective of this study was to determine the role of NPR-B signaling in regulating HR and SAN function.
Methods and results
We have used NPR-B deficient mice (NPR-B+/-) to study HR regulation and SAN function using telemetry in conscious mice, intracardiac electrophysiology in anesthetized mice, high resolution optical mapping in isolated SAN preparations, patch-clamping in isolated SAN myocytes, and molecular biology in isolated SAN tissue. These studies demonstrate that NPR-B+/- mice exhibit slow HR, increased corrected SAN recovery time, and slowed SAN conduction. Spontaneous AP firing frequency in isolated SAN myocytes was impaired in NPR-B+/- mice due to reductions in the hyperpolarization activated current (If) and L-type Ca2+ current (ICa,L). If and ICa,L were reduced due to lower cGMP levels and increased hydrolysis of cAMP by phosphodiesterase 3 (PDE3) in the SAN. Inhibiting PDE3 or restoring cGMP signaling via application of 8-Br-cGMP abolished the reductions in cAMP, AP firing, If, and ICa,L, and normalized SAN conduction, in the SAN in NPR-B+/- mice. NPR-B+/- mice did not exhibit changes in SAN fibrosis and showed no evidence of cardiac hypertrophy or changes in ventricular function.
Conclusions
NPR-B plays an essential physiological role in maintaining normal HR and SAN function by modulating ion channel function in SAN myocytes via a cGMP/PDE3/cAMP signaling mechanism.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 16 Jul 2021; epub ahead of print
Dorey TW, Mackasey M, Jansen HJ, McRae MD, ... Atkinson L, Rose RA
Cardiovasc Res: 16 Jul 2021; epub ahead of print | PMID: 34273155
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Abstract

Cyclic GMP modulating drugs in cardiovascular diseases: Mechanism-based network pharmacology.

Petraina A, Nogales C, Krahn T, Mucke H, ... Hobbs AJ, Schmidt HHHW
Mechanism-based therapy centred on the molecular understanding of disease-causing pathways in a given patient is still the exception rather than the rule in medicine, even in cardiology. However, recent successful drug developments centred around the second messenger cyclic guanosine-3\'-5\'-monophosphate (cGMP), which is regulating a number of cardiovascular disease modulating pathways, are about to provide novel targets for such a personalised cardiovascular therapy. Whether cGMP breakdown is inhibited or cGMP synthesis is stimulated via guanylyl cyclases or their upstream regulators in different cardiovascular disease phenotypes, the outcomes seem to be so far uniformly protective. Thus, a network of cGMP modulating drugs has evolved that act in a mechanism-based, possibly causal manner in a number of cardiac conditions. What remains a challenge is the detection of cGMPopathy endotypes amongst cardiovascular disease phenotypes. Here we review the growing clinical relevance of cGMP and provide a glimpse into the future on how drugs interfering with this pathway may change how we treat and diagnose cardiovascular diseases altogether.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 15 Jul 2021; epub ahead of print
Petraina A, Nogales C, Krahn T, Mucke H, ... Hobbs AJ, Schmidt HHHW
Cardiovasc Res: 15 Jul 2021; epub ahead of print | PMID: 34270705
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Abstract

Bcar1/p130Cas is essential for ventricular development and neural crest cell remodelling of the cardiac outflow tract.

Mahmoud M, Evans I, Wisniewski L, Tam Y, ... Scambler P, Zachary I
Aim
The adapter protein p130Cas, encoded by the Bcar1 gene, is a key regulator of cell movement, adhesion, and cell cycle control in diverse cell types. Bcar1 constitutive knockout mice are embryonic lethal by embryonic days (E) 11.5-12.5, but the role of Bcar1 in embryonic development remains unclear. Here, we investigated the role of Bcar1 specifically in cardiovascular development and defined the cellular and molecular mechanisms disrupted following targeted Bcar1 deletions.
Methods and results
We crossed Bcar1 floxed mice with Cre transgenic lines allowing for cell-specific knockout either in smooth muscle and early cardiac tissues (SM22-Cre), mature smooth muscle cells (smMHC-Cre), endothelial cells (Tie2-Cre), second heart field cells (Mef2c-Cre), or neural crest cells (NCC) (Pax3-Cre) and characterised these conditional knock outs using a combination of histological and molecular biology techniques.Conditional knockout of Bcar1 in SM22-expressing smooth muscle cells and cardiac tissues (Bcar1SM22KO) was embryonically lethal from E14.5-15.5 due to severe cardiovascular defects, including abnormal ventricular development and failure of outflow tract (OFT) septation leading to a single outflow vessel reminiscent of persistent truncus arteriosus. SM22-restricted loss of Bcar1 was associated with failure of OFT cushion cells to undergo differentiation to septal mesenchymal cells positive for SMC-specific α-actin, and disrupted expression of proteins and transcription factors involved in epithelial-to-mesenchymal transformation (EMT). Furthermore, knockout of Bcar1 specifically in NCC (Bcar1PAX3KO) recapitulated part of the OFT septation and aortic sac defects seen in the Bcar1SM22KO mutants, indicating a cell-specific requirement for Bcar1 in NCC essential for OFT septation. In contrast, conditional knockouts of Bcar1 in differentiated smooth muscle, endothelial cells, and second heart field cells survived to term and were phenotypically normal at birth and post-natally.
Conclusions
Our work reveals a cell-specific requirement for Bcar1 in NCC, early myogenic and cardiac cells, essential for OFT septation, myocardialisation and EMT/cell cycle regulation and differentiation to myogenic lineages.
Translational perspective
The molecular pathways coordinating cardiogenesis and the remodelling of the OFT are complex, and dysregulation of these pathways causes human heart defects. Our findings highlight a specific requirement for Bcar1 essential for cardiogenesis. Furthermore, the failure of OFT septation in Bcar1SM22KO mice resembles persistent truncus arteriosus (PTA), a feature of several human congenital heart diseases, including DiGeorge Syndrome. Our findings have implications for the mechanisms underlying the pathogenesis of congenital heart disease, and suggest that mice with conditional Bcar1 deletions may be useful models for dissecting mechanisms involved in the pathogenesis of human heart defects.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 15 Jul 2021; epub ahead of print
Mahmoud M, Evans I, Wisniewski L, Tam Y, ... Scambler P, Zachary I
Cardiovasc Res: 15 Jul 2021; epub ahead of print | PMID: 34270692
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Abstract

HDAC1: an environmental sensor regulating endothelial function.

Dunaway LS, Pollock JS
The histone deacetylases (HDACs) are a family of enzymes that catalyze lysine deacetylation of both histone and non-histone proteins. Here we review, summarize, and provide perspectives on the literature regarding one such HDAC, HDAC1, in endothelial biology. In the endothelium, HDAC1 mediates the effects of external and environmental stimuli by regulating major endothelial functions such as angiogenesis, inflammatory signaling, redox homeostasis, and nitric oxide signaling. Angiogenesis is most often, but not exclusively, repressed by endothelial HDAC1. The regulation of inflammatory signaling is more complex as HDAC1 promotes or suppresses inflammatory signaling depending upon the environmental stimuli. HDAC1 is protective in models of atherosclerosis where loss of HDAC1 results in increased cytokine and cell adhesion molecule abundance. In other models, HDAC1 promotes inflammation by increasing cell adhesion molecules and repressing claudin-5 expression. Consistently, from many investigations, HDAC1 decreases antioxidant enzyme expression and nitric oxide production in the endothelium. HDAC1 decreases antioxidant enzyme expression through the deacetylation of histones and transcription factors, and also regulates nitric oxide production through regulating both the expression and activity of nitric oxide synthase 3. The HDAC1-dependent regulation of endothelial function through the deacetylation of both histone and non-histone proteins ultimately impacts whole animal physiology and health.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 14 Jul 2021; epub ahead of print
Dunaway LS, Pollock JS
Cardiovasc Res: 14 Jul 2021; epub ahead of print | PMID: 34264338
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Abstract

Multimarker profiling identifies protective and harmful immune processes in heart failure: findings from BIOSTAT-CHF.

Markousis-Mavrogenis G, Tromp J, Ouwerkerk W, Fereirra JP, ... Voors AA, van der Meer P
Aims
The exploration of novel immunomodulatory interventions to improve outcome in heart failure (HF) is hampered by the complexity/redundancies of inflammatory pathways, which remain poorly understood. We thus aimed to investigate the associations between the activation of diverse immune processes and outcomes in patients with HF.
Methods and results
We measured 355 biomarkers in 2,022 patients with worsening HF and an independent validation cohort (n = 1,691) (BIOSTAT-CHF index and validation cohorts), and classified them according to their functions into biological processes based on the Gene Ontology classification. Principal component analyses were used to extract weighted scores per process. We investigated the association of these processes with all-cause mortality at 2-year follow-up. The contribution of each biomarker to the weighted score(s) of the processes was used to identify potential therapeutic targets. Mean age was 69 (±12.0) years and 537 (27%) patients were women. We identified 64 unique overrepresented immune-related processes representing 188 of 355 biomarkers. Of these processes, 19 were associated with all-cause mortality (10 positively and 9 negatively). Increased activation of \"T-cell costimulation\" and \"response to interferon gamma/positive regulation of interferon gamma production\" showed the most consistent positive and negative associations with all-cause mortality respectively, after external validation. Within T-cell costimulation, inducible co-stimulator-ligand (ICOSLG), CD28, CD70, and tumor necrosis factor superfamily member-14 (TNFSF14) were identified as potential therapeutic targets.
Conclusions
We demonstrate the divergent protective and harmful effects of different immune processes in HF and suggest novel therapeutic targets. These findings constitute a rich knowledge base for informing future studies of inflammation in HF.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 14 Jul 2021; epub ahead of print
Markousis-Mavrogenis G, Tromp J, Ouwerkerk W, Fereirra JP, ... Voors AA, van der Meer P
Cardiovasc Res: 14 Jul 2021; epub ahead of print | PMID: 34264317
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Abstract

Determining a minimum set of variables for machine learning cardiovascular event prediction: results from REFINE SPECT registry.

Rios R, Miller RJH, Hu LH, Otaki Y, ... Berman DS, Slomka P
Aims
Optimal risk stratification with machine learning (ML) from myocardial perfusion imaging (MPI) includes both clinical and imaging data. While most imaging variables can be derived automatically, clinical variables require manual collection, which is time consuming and prone to error. We determined the fewest manually input and imaging variables required to maintain the prognostic accuracy for major adverse cardiac events (MACE) in patients undergoing single-photon emission computed tomography (SPECT) MPI.
Methods and results
This study included 20,414 patients from the multicenter REFINE SPECT registry and 2,984 from the University of Calgary for training and external testing of the ML models, respectively. ML models were trained using all variables (ML-All) and all image-derived variables (including age and sex, ML-Image). Next, ML models were sequentially trained by incrementally adding manually input and imaging variables to baseline ML models based on their importance ranking. The fewest variables were determined as the ML models (ML-Reduced, ML-Minimum, and ML-Image-Reduced) that achieved comparable prognostic performance to ML-All and ML-Image. Prognostic accuracy of the ML models was compared with visual diagnosis, stress total perfusion deficit (TPD), and traditional multivariable models using area under the receiver-operating characteristic curve (AUC).ML-Minimum (AUC 0.798) obtained comparable prognostic accuracy to ML-All (AUC 0.798, p = 0.18) by including 12 of 40 manually input variables and 11 of 58 imaging variables. ML-Reduced achieved comparable accuracy (AUC 0.795) with a reduced set of manually input variables and all imaging variables. In external validation, the ML models also obtained comparable or higher prognostic accuracy than traditional multivariable models.
Conclusion
Reduced ML models, including a minimum set of manually collected or imaging variables, achieved slightly lower accuracy compared to a full ML model, but outperformed standard interpretation methods and risk models. ML models with fewer collected variables may be more practical for clinical implementation.
Translational perspective
A reduced machine learning model, with 12 out of 40 manually collected variables and 11 of 58 imaging variables, achieved >99% of the prognostic accuracy of the full model. Models with fewer manually collected features require less infrastructure to implement, are easier for physicians to utilize, and are potentially critical to ensuring broader clinical implementation. Additionally, these models can integrate mechanisms to explain patient-specific risk estimates to improve physician confidence in the machine learning prediction.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 13 Jul 2021; epub ahead of print
Rios R, Miller RJH, Hu LH, Otaki Y, ... Berman DS, Slomka P
Cardiovasc Res: 13 Jul 2021; epub ahead of print | PMID: 34259870
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Abstract

Interleukin-5-induced eosinophil population improves cardiac function after myocardial infarction.

Xu JY, Xiong YY, Tang RJ, Jiang WY, ... Li XD, Yang YJ
Aims
Interleukin (IL)-5 mediates the development of eosinophils (EOS) that are essential for tissue post-injury repair. It remains unknown whether IL-5 plays a role in heart repair after myocardial infarction (MI). This study aims to test whether IL-5-induced EOS population promotes the healing and repair process post-MI and to reveal the underlying mechanisms.
Method and results
MI was induced by permanent ligation of the left anterior descending coronary artery in wild-type C57BL/6 mice. Western blot and real-time polymerase chain reaction revealed elevated expression of IL-5 in the heart at 5 days post-MI. Immunohistostaining indicated that IL-5 was secreted mainly from macrophages and type 2 innate lymphoid cells in the setting of experimental MI. External supply of recombinant mouse IL-5 (20 min, 1 day, and 2 days after MI surgery) reduced the infarct size and increased ejection fraction and angiogenesis in the border zone. A significant expansion of EOS was detected in both the peripheral blood and infarcted myocardium after IL-5 administration. Pharmacological depletion of EOS by TRFK5 pretreatment muted the beneficial effects of IL-5 in MI mice. Mechanistic studies demonstrated that IL-5 increased the accumulation of CD206+ macrophages in infarcted myocardium at 7 days post-MI. In vitro co-culture experiments showed that EOS shifted bone marrow-derived macrophage polarization towards the CD206+ phenotypes. This activity of EOS was abolished by IL-4 neutralizing antibody, but not IL-10 or IL-13 neutralization. Western blot analyses demonstrated that EOS promoted the macrophage downstream signal transducer and activator of transcription 6 (STAT6) phosphorylation.
Conclusion
IL-5 facilitates the recovery of cardiac dysfunction post-MI by promoting EOS accumulation and subsequent CD206+ macrophage polarization via the IL-4/STAT6 axis.
Translational perspective
Accumulating evidence suggests that modulation of innate and adaptive immune responses is a promising therapeutic strategy for myocardial infarction. In this study, we demonstrate that IL-5 exerts cardioprotective effects on infarcted myocardium by promoting eosinophil accumulation and subsequent CD206+ macrophage polarization via the IL-4/STAT6 axis. Hence, regulation of cardiac IL-5 level or eosinophil count may become a therapeutic approach for post-myocardial infarction cardiac repair in humans.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 13 Jul 2021; epub ahead of print
Xu JY, Xiong YY, Tang RJ, Jiang WY, ... Li XD, Yang YJ
Cardiovasc Res: 13 Jul 2021; epub ahead of print | PMID: 34259869
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Abstract

Focus on the road to modelling cardiomyopathy in muscular dystrophy.

Canonico F, Chirivi M, Maiullari F, Milan M, ... Bearzi C, D\'Amario D
Alterations in the DMD gene, which codes for the protein dystrophin, cause forms of dystrophinopathies such as Duchenne muscular dystrophy, an X-linked disease. Cardiomyopathy linked to DMD mutations is becoming the leading cause of death in patients with dystrophinopathy. Since phenotypic pathophysiological mechanisms are not fully understood, the improvement and development of new disease models, considering their relative advantages and disadvantages, is essential. The application of genetic engineering approaches on induced pluripotent stem cells, such as gene editing technology, enables the development of physiologically relevant human cell models for in vitro dystrophinopathy studies. The combination of induced pluripotent stem cells-derived cardiovascular cell types and 3 D bioprinting technologies hold great promise for the study of dystrophin-linked cardiomyopathy. This combined approach enables the assessment of responses to physical or chemical stimuli, and the influence of pharmaceutical approaches. The critical objective of in vitro microphysiological systems is to more accurately reproduce the microenvironment observed in vivo. Ground-breaking methodology involving the connection of multiple microphysiological systems comprised of different tissues would represent a move toward precision body-on-chip disease modelling could lead to a critical expansion in what is known about inter-organ responses to disease and novel therapies that have the potential to replace animal models. In this review, we will focus on the generation, development, and application of current cellular, animal and potential for bio-printed models, in the study of the pathophysiological mechanisms underlying dystrophin-linked cardiomyopathy in the direction of personalized medicine.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 11 Jul 2021; epub ahead of print
Canonico F, Chirivi M, Maiullari F, Milan M, ... Bearzi C, D'Amario D
Cardiovasc Res: 11 Jul 2021; epub ahead of print | PMID: 34254111
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Abstract

Management of cardiac fibrosis is the largest unmet medical need in heart failure Cardiac fibrosis in heart failure.

Díez J, de Boer RA
Cardiac fibrosis is a major driver associated with the growing burden of heart failure, especially in older people. However, integrating cardiac fibrosis in heart failure management is still an unmet medical need, which may be explained by its high tissue heterogeneity and clinical diversity, and, as a consequence, the very real limitations of its diagnosis and treatment. In this viewpoint article we summarize the challenges and requirements in the clinical management of cardiac fibrosis in heart failure patients.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 08 Jul 2021; epub ahead of print
Díez J, de Boer RA
Cardiovasc Res: 08 Jul 2021; epub ahead of print | PMID: 34244741
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Abstract

Interleukin-7 and interleukin-15 drive CD4+CD28null T lymphocyte expansion and function in patients with acute coronary syndrome.

Bullenkamp J, Mengoni V, Kaur S, Chhetri I, ... Kaski JC, Dumitriu IE
Aims
Inflammation has important roles in atherosclerosis. CD4+CD28null (CD28null) T cells are a specialized T lymphocyte subset that produce inflammatory cytokines and cytotoxic molecules. CD28null T cells expand preferentially in patients with acute coronary syndrome (ACS) rather than stable angina and are barely detectable in healthy subjects. Importantly, ACS patients with CD28null T-cell expansion have increased risk for recurrent acute coronary events and poor prognosis, compared to ACS patients in whom this cell subset does not expand. The mechanisms regulating CD28null T-cell expansion in ACS remain elusive. We therefore investigated the role of cytokines in CD28null T-cell expansion in ACS.
Methods and results
High-purity sorted CD4+ T cells from ACS patients were treated with a panel of cytokines (TNF-α, IL-1β, IL-6, IL-7, and IL-15), and effects on the number, phenotype, and function of CD28null T cells were analysed and compared to the control counterpart CD28+ T-cell subset. IL-7- and IL-15-induced expansion of CD28null T cells from ACS patients, while inflammatory cytokines TNF-α, IL-1β, and IL-6 did not. The mechanisms underlying CD28null T-cell expansion by IL-7/IL-15 were preferential activation and proliferation of CD28null T cells compared to control CD28+ T cells. Additionally, IL-7/IL-15 markedly augmented CD28null T-cell cytotoxic function and interferon-γ production. Further mechanistic analyses revealed differences in baseline expression of component chains of IL-7/IL-15 receptors (CD127 and CD122) and increased baseline STAT5 phosphorylation in CD28null T cells from ACS patients compared to the control CD28+ T-cell subset. Notably, we demonstrate that CD28null T-cell expansion was significantly inhibited by Tofacitinib, a selective JAK1/JAK3 inhibitor that blocks IL-7/IL-15 signalling.
Conclusion
Our novel data show that IL-7 and IL-15 drive the expansion and function of CD28null T cells from ACS patients suggesting that IL-7/IL-15 blockade may prevent expansion of these cells and improve patient outcomes.

© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 06 Jul 2021; 117:1935-1948
Bullenkamp J, Mengoni V, Kaur S, Chhetri I, ... Kaski JC, Dumitriu IE
Cardiovasc Res: 06 Jul 2021; 117:1935-1948 | PMID: 32647892
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Abstract

Adult zebrafish ventricular electrical gradients as tissue mechanisms of ECG patterns under baseline vs. oxidative stress.

Zhao Y, James NA, Beshay AR, Chang EE, ... Nguyen B, Nguyen TP
Aims
In mammalian ventricles, electrical gradients establish electrical heterogeneities as essential tissue mechanisms to optimize mechanical efficiency and safeguard electrical stability. Electrical gradients shape mammalian electrocardiographic patterns; disturbance of electrical gradients is proarrhythmic. The zebrafish heart is a popular surrogate model for human cardiac electrophysiology thanks to its remarkable recapitulation of human electrocardiogram and ventricular action potential features. Yet, zebrafish ventricular electrical gradients are largely unexplored. The goal of this study is to define the zebrafish ventricular electrical gradients that shape the QRS complex and T wave patterns at baseline and under oxidative stress.
Methods and results
We performed in vivo electrocardiography and ex vivo voltage-sensitive fluorescent epicardial and transmural optical mapping of adult zebrafish hearts at baseline and during acute H2O2 exposure. At baseline, apicobasal activation and basoapical repolarization gradients accounted for the polarity concordance between the QRS complex and T wave. During H2O2 exposure, differential regional impairment of activation and repolarization at the apex and base disrupted prior to baseline electrical gradients, resulting in either reversal or loss of polarity concordance between the QRS complex and T wave. KN-93, a specific calcium/calmodulin-dependent protein kinase II inhibitor (CaMKII), protected zebrafish hearts from H2O2 disruption of electrical gradients. The protection was complete if administered prior to oxidative stress exposure.
Conclusions
Despite remarkable apparent similarities, zebrafish and human ventricular electrocardiographic patterns are mirror images supported by opposite electrical gradients. Like mammalian ventricles, zebrafish ventricles are also susceptible to H2O2 proarrhythmic perturbation via CaMKII activation. Our findings suggest that the adult zebrafish heart may constitute a clinically relevant model to investigate ventricular arrhythmias induced by oxidative stress. However, the fundamental ventricular activation and repolarization differences between the two species that we demonstrated in this study highlight the potential limitations when extrapolating results from zebrafish experiments to human cardiac electrophysiology, arrhythmias, and drug toxicities.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 06 Jul 2021; 117:1891-1907
Zhao Y, James NA, Beshay AR, Chang EE, ... Nguyen B, Nguyen TP
Cardiovasc Res: 06 Jul 2021; 117:1891-1907 | PMID: 32735330
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Abstract

Impaired retinal microvascular function predicts long-term adverse events in patients with cardiovascular disease.

Theuerle JD, Al-Fiadh AH, Amirul Islam FM, Patel SK, ... Wong TY, Farouque O
Aims
Endothelial dysfunction is a precursor to the development of symptomatic atherosclerosis. Retinal microvascular reactivity to flicker light stimulation is a marker of endothelial function and can be quantified in vivo. We sought to determine whether retinal microvascular endothelial dysfunction predicts long-term major adverse cardiovascular events (MACE).
Methods and results
In a single-centre prospective observational study, patients with coronary artery disease (CAD) or cardiovascular risk factors underwent dynamic retinal vessel assessment in response to flicker light stimulation and were followed up for MACE. Retinal microvascular endothelial dysfunction was quantified by measuring maximum flicker light-induced retinal arteriolar dilatation (FI-RAD) and flicker light-induced retinal venular dilatation (FI-RVD). In total, 252 patients underwent dynamic retinal vessel assessment and 242 (96%) had long-term follow-up. Of the 242 patients, 88 (36%) developed MACE over a median period of 8.6 years (interquartile range 6.0-9.1). After adjustment for traditional risk factors, patients within the lowest quintile of FI-RAD had the highest risk of MACE [odds ratio (OR) 5.21; 95% confidence interval (CI) 1.78-15.28]. Patients with lower FI-RAD were also more likely to die (OR 2.09; 95% CI 1.00-4.40, per standard deviation decrease in FI-RAD). In Kaplan-Meier analysis, patients with FI-RAD responses below the cohort median of 1.4% exhibited reduced MACE-free survival (55.5 vs. 71.5%; log-rank P = 0.004). FI-RVD was not predictive of MACE.
Conclusion
Retinal arteriolar endothelial dysfunction is an independent predictor of MACE in patients with CAD or cardiovascular risk factors. Dynamic retinal vessel analysis may provide added benefit to traditional risk factors in stratifying patients at risk for cardiovascular events.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 06 Jul 2021; 117:1949-1957
Theuerle JD, Al-Fiadh AH, Amirul Islam FM, Patel SK, ... Wong TY, Farouque O
Cardiovasc Res: 06 Jul 2021; 117:1949-1957 | PMID: 32750111
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Abstract

Preclinical evidence for the therapeutic value of TBX5 normalization in arrhythmia control.

Rathjens FS, Blenkle A, Iyer LM, Renger A, ... Zelarayan LC, Zafeiriou MP
Aims 
Arrhythmias and sudden cardiac death (SCD) occur commonly in patients with heart failure. We found T-box 5 (TBX5) dysregulated in ventricular myocardium from heart failure patients and thus we hypothesized that TBX5 reduction contributes to arrhythmia development in these patients. To understand the underlying mechanisms, we aimed to reveal the ventricular TBX5-dependent transcriptional network and further test the therapeutic potential of TBX5 level normalization in mice with documented arrhythmias.
Methods and results 
We used a mouse model of TBX5 conditional deletion in ventricular cardiomyocytes. Ventricular (v) TBX5 loss in mice resulted in mild cardiac dysfunction and arrhythmias and was associated with a high mortality rate (60%) due to SCD. Upon angiotensin stimulation, vTbx5KO mice showed exacerbated cardiac remodelling and dysfunction suggesting a cardioprotective role of TBX5. RNA-sequencing of a ventricular-specific TBX5KO mouse and TBX5 chromatin immunoprecipitation was used to dissect TBX5 transcriptional network in cardiac ventricular tissue. Overall, we identified 47 transcripts expressed under the control of TBX5, which may have contributed to the fatal arrhythmias in vTbx5KO mice. These included transcripts encoding for proteins implicated in cardiac conduction and contraction (Gja1, Kcnj5, Kcng2, Cacna1g, Chrm2), in cytoskeleton organization (Fstl4, Pdlim4, Emilin2, Cmya5), and cardiac protection upon stress (Fhl2, Gpr22, Fgf16). Interestingly, after TBX5 loss and arrhythmia development in vTbx5KO mice, TBX5 protein-level normalization by systemic adeno-associated-virus (AAV) 9 application, re-established TBX5-dependent transcriptome. Consequently, cardiac dysfunction was ameliorated and the propensity of arrhythmia occurrence was reduced.
Conclusions 
This study uncovers a novel cardioprotective role of TBX5 in the adult heart and provides preclinical evidence for the therapeutic value of TBX5 protein normalization in the control of arrhythmia.

© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 06 Jul 2021; 117:1908-1922
Rathjens FS, Blenkle A, Iyer LM, Renger A, ... Zelarayan LC, Zafeiriou MP
Cardiovasc Res: 06 Jul 2021; 117:1908-1922 | PMID: 32777030
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Impact:
Abstract

Impaired cytoplasmic domain interactions cause co-assembly defect and loss of function in the p.Glu293Lys KNCJ2 variant isolated from an Andersen-Tawil syndrome patient.

Déri S, Borbás J, Hartai T, Hategan L, ... Sepp R, Ördög B
Aims
Subunit interactions at the cytoplasmic domain interface (CD-I) have recently been shown to control gating in inward rectifier potassium channels. Here we report the novel KCNJ2 variant p.Glu293Lys that has been found in a patient with Andersen-Tawil syndrome type 1 (ATS1), causing amino acid substitution at the CD-I of the inward rectifier potassium channel subunit Kir2.1. Neither has the role of Glu293 in gating control been investigated nor has a pathogenic variant been described at this position. This study aimed to assess the involvement of Glu293 in CD-I subunit interactions and to establish the pathogenic role of the p.Glu293Lys variant in ATS1.
Methods and results
The p.Glu293Lys variant produced no current in homomeric form and showed dominant-negative effect over wild-type (WT) subunits. Immunocytochemical labelling showed the p.Glu293Lys subunits to distribute in the subsarcolemmal space. Salt bridge prediction indicated the presence of an intersubunit salt bridge network at the CD-I of Kir2.1, with the involvement of Glu293. Subunit interactions were studied by the NanoLuc® Binary Technology (NanoBiT) split reporter assay. Reporter constructs carrying NanoBiT tags on the intracellular termini produced no bioluminescent signal above background with the p.Glu293Lys variant in homomeric configuration and significantly reduced signals in cells co-expressing WT and p.Glu293Lys subunits simultaneously. Extracellularly presented reporter tags, however, generated comparable bioluminescent signals with heteromeric WT and p.Glu293Lys subunits and with homomeric WT channels.
Conclusions
Loss of function and dominant-negative effect confirm the causative role of p.Glu293Lys in ATS1. Co-assembly of Kir2.1 subunits is impaired in homomeric channels consisting of p.Glu293Lys subunits and is partially rescued in heteromeric complexes of WT and p.Glu293Lys Kir2.1 variants. These data point to an important role of Glu293 in mediating subunit assembly, as well as in gating of Kir2.1 channels.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 06 Jul 2021; 117:1923-1934
Déri S, Borbás J, Hartai T, Hategan L, ... Sepp R, Ördög B
Cardiovasc Res: 06 Jul 2021; 117:1923-1934 | PMID: 32810216
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Abstract

High spatial endothelial shear stress gradient independently predicts site of acute coronary plaque rupture and erosion.

Thondapu V, Mamon C, Poon EKW, Kurihara O, ... Barlis P, Jang IK
Aims
To investigate local haemodynamics in the setting of acute coronary plaque rupture and erosion.
Methods and results
Intracoronary optical coherence tomography performed in 37 patients with acute coronary syndromes caused by plaque rupture (n = 19) or plaque erosion (n = 18) was used for three-dimensional reconstruction and computational fluid dynamics simulation. Endothelial shear stress (ESS), spatial ESS gradient (ESSG), and oscillatory shear index (OSI) were compared between plaque rupture and erosion through mixed-effects logistic regression. Lipid, calcium, macrophages, layered plaque, and cholesterol crystals were also analysed. By multivariable analysis, only high ESSG [odds ratio (OR) 5.29, 95% confidence interval (CI) 2.57-10.89, P < 0.001], lipid (OR 12.98, 95% CI 6.57-25.67, P < 0.001), and layered plaque (OR 3.17, 95% CI 1.82-5.50, P < 0.001) were independently associated with plaque rupture. High ESSG (OR 13.28, 95% CI 6.88-25.64, P < 0.001), ESS (OR 2.70, 95% CI 1.34-5.42, P = 0.005), and OSI (OR 2.18, 95% CI 1.33-3.54, P = 0.002) independently associated with plaque erosion. ESSG was higher at rupture sites than erosion sites [median (interquartile range): 5.78 (2.47-21.15) vs. 2.62 (1.44-6.18) Pa/mm, P = 0.009], OSI was higher at erosion sites than rupture sites [1.04 × 10-2 (2.3 × 10-3-4.74 × 10-2) vs. 1.29 × 10-3 (9.39 × 10-5-3.0 × 10-2), P < 0.001], but ESS was similar (P = 0.29).
Conclusions
High ESSG is independently associated with plaque rupture while high ESSG, ESS, and OSI associate with plaque erosion. While ESSG is higher at rupture sites than erosion sites, OSI is higher at erosion sites and ESS was similar. These results suggest that ESSG and OSI may play critical roles in acute plaque rupture and erosion, respectively.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 06 Jul 2021; 117:1974-1985
Thondapu V, Mamon C, Poon EKW, Kurihara O, ... Barlis P, Jang IK
Cardiovasc Res: 06 Jul 2021; 117:1974-1985 | PMID: 32832991
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Abstract

A combined microRNA and target protein-based panel for predicting the probability and severity of uraemic vascular calcification: a translational study.

Chao CT, Yeh HY, Tsai YT, Chiang CK, Chen HW
Aims 
Vascular calcification (VC) increases the future risk of cardiovascular events in uraemic patients, but effective therapies are still unavailable. Accurate identification of those at risk of developing VC using pathogenesis-based biomarkers is of particular interest and may facilitate individualized risk stratification. We aimed to uncover microRNA (miRNA)-target protein-based biomarker panels for evaluating uraemic VC probability and severity.
Methods and results 
We created a three-tiered in vitro VC model and an in vivo uraemic rat model receiving high phosphate diet to mimic uraemic VC. RNAs from the three-tiered in vitro and in vivo uraemic VC models underwent miRNA and mRNA microarray, with results screened for differentially expressed miRNAs and their target genes as biomarkers. Findings were validated in original models and additionally in an ex vivo VC model and human cells, followed by functional assays of identified miRNAs and target proteins, and tests of sera from end-stage renal disease (ESRD) and non-dialysis-dependent chronic kidney disease (CKD) patients without and with VC. Totally 122 down-regulated and 119 up-regulated miRNAs during calcification progression were identified initially; further list narrowing based on miRNA-mRNA pairing, anti-correlation, and functional enrichment left 16 and 14 differentially expressed miRNAs and mRNAs. Levels of four miRNAs (miR-10b-5p, miR-195, miR-125b-2-3p, and miR-378a-3p) were shown to decrease throughout all models tested, while one mRNA (SULF1, a potential target of miR-378a-3p) exhibited the opposite trend concurrently. Among 96 ESRD (70.8% with VC) and 59 CKD patients (61% with VC), serum miR-125b2-3p and miR-378a-3p decreased with greater VC severity, while serum SULF1 levels increased. Adding serum miR-125b-2-3p, miR-378a-3p, and SULF1 into regression models for VC substantially improved performance compared to using clinical variables alone.
Conclusion 
Using a translational approach, we discovered a novel panel of biomarkers for gauging the probability/severity of uraemic VC based on miRNAs/target proteins, which improved the diagnostic accuracy.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 06 Jul 2021; 117:1958-1973
Chao CT, Yeh HY, Tsai YT, Chiang CK, Chen HW
Cardiovasc Res: 06 Jul 2021; 117:1958-1973 | PMID: 32866261
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Abstract

The physiological and pathological functions of VEGFR3 in cardiac and lymphatic development and related diseases.

Monaghan RM, Page DJ, Ostergaard P, Keavney BD
Vascular endothelial growth factor receptors (VEGFRs) are part of the evolutionarily conserved VEGF signalling pathways that regulate the development and maintenance of the body\'s cardiovascular and lymphovascular systems. VEGFR3, encoded by the FLT4 gene, has an indispensable and well-characterized function in development and establishment of the lymphatic system. Autosomal dominant VEGFR3 mutations, that prevent the receptor functioning as a homodimer, cause one of the major forms of hereditary primary lymphoedema; Milroy disease. Recently, we and others have shown that FLT4 variants, distinct to those observed in Milroy disease cases, predispose individuals to Tetralogy of Fallot, the most common cyanotic congenital heart disease, demonstrating a novel function for VEGFR3 in early cardiac development. Here, we examine the familiar and emerging roles of VEGFR3 in the development of both lymphovascular and cardiovascular systems, respectively, compare how distinct genetic variants in FLT4 lead to two disparate human conditions, and highlight the research still required to fully understand this multifaceted receptor.

© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 06 Jul 2021; 117:1877-1890
Monaghan RM, Page DJ, Ostergaard P, Keavney BD
Cardiovasc Res: 06 Jul 2021; 117:1877-1890 | PMID: 33067626
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Impact:
Abstract

Cell senescence: basic mechanisms and the need for computational networks in vascular ageing.

Regnault V, Challande P, Pinet F, Li Z, Lacolley P
This review seeks to provide an update of the mechanisms of vascular cell senescence, from newly identified molecules to arterial ageing phenotypes, and finally to present a computational approach to connect these selected proteins in biological networks. We will discuss current key signalling and gene expression pathways by which these focus proteins and networks drive normal and accelerated vascular ageing. We also review the possibility that senolytic drugs, designed to restore normal cell differentiation and function, could effectively treat multiple age-related vascular diseases. Finally, we discuss how cell senescence is both a cause and a consequence of vascular ageing because of the possible feedback controls between identified networks.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 06 Jul 2021; 117:1841-1858
Regnault V, Challande P, Pinet F, Li Z, Lacolley P
Cardiovasc Res: 06 Jul 2021; 117:1841-1858 | PMID: 33206947
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Impact:
Abstract

Obesity, kidney dysfunction, and inflammation: interactions in hypertension.

Hall JE, Mouton AJ, da Silva AA, Omoto ACM, ... Li X, do Carmo JM
Obesity contributes 65-75% of the risk for human primary (essential) hypertension (HT) which is a major driver of cardiovascular and kidney diseases. Kidney dysfunction, associated with increased renal sodium reabsorption and compensatory glomerular hyperfiltration, plays a key role in initiating obesity-HT and target organ injury. Mediators of kidney dysfunction and increased blood pressure include (i) elevated renal sympathetic nerve activity (RSNA); (ii) increased antinatriuretic hormones such as angiotensin II and aldosterone; (iii) relative deficiency of natriuretic hormones; (iv) renal compression by fat in and around the kidneys; and (v) activation of innate and adaptive immune cells that invade tissues throughout the body, producing inflammatory cytokines/chemokines that contribute to vascular and target organ injury, and exacerbate HT. These neurohormonal, renal, and inflammatory mechanisms of obesity-HT are interdependent. For example, excess adiposity increases the adipocyte-derived cytokine leptin which increases RSNA by stimulating the central nervous system proopiomelanocortin-melanocortin 4 receptor pathway. Excess visceral, perirenal and renal sinus fat compress the kidneys which, along with increased RSNA, contribute to renin-angiotensin-aldosterone system activation, although obesity may also activate mineralocorticoid receptors independent of aldosterone. Prolonged obesity, HT, metabolic abnormalities, and inflammation cause progressive renal injury, making HT more resistant to therapy and often requiring multiple antihypertensive drugs and concurrent treatment of dyslipidaemia, insulin resistance, diabetes, and inflammation. More effective anti-obesity drugs are needed to prevent the cascade of cardiorenal, metabolic, and immune disorders that threaten to overwhelm health care systems as obesity prevalence continues to increase.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 06 Jul 2021; 117:1859-1876
Hall JE, Mouton AJ, da Silva AA, Omoto ACM, ... Li X, do Carmo JM
Cardiovasc Res: 06 Jul 2021; 117:1859-1876 | PMID: 33258945
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Abstract

CENTRAL AND PERIPHERAL SYMPATHETIC ACTIVATION IN HEART FAILURE.

Grassi G, Mancia G, Esler M
The sympathetic nervous system overdrive occurring in heart failure has been reported since more than half a century. Refinements in the methodological approaches to assess human sympathetic neural function have allowed during recent years to better define various aspects related to the neuroadrenergic alteration. These include 1) the different participation of the individual regional sympathetic cardiovascular districts at the process, 2) the role of the central nervous system in determining the neuroadrenergic overdrive, 3) the involvement of baroreflex, cardiopulmonary reflex and chemoreflex mechanisms in the phoenomenon, which is also closely linked to inflammation and the immune reaction, 4) the relationships with the severity of the disease, its ischaemic or idiopathic nature and the preserved or reduced left ventricular ejection fraction and 5) the adverse functional and structural impact of the sympathetic activation on cardiovascular organs, such as the brain, the heart and the kidneys. Information have been also gained on the active role exerted by the sympathetic activation on the disease outcome and its potential relevance as target of the therapeutic interventions based on non-pharmacological, pharmacological and invasive approaches, including the renal denervation, the splanchnic sympathetic nerve ablation and the carotid baroreflex stimulation. The still undefined aspects of the neurogenic alterations and the unmet goals of the therapeutic approach having the sympathetic activation as a target of the intervention will be finally mentioned.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 06 Jul 2021; epub ahead of print
Grassi G, Mancia G, Esler M
Cardiovasc Res: 06 Jul 2021; epub ahead of print | PMID: 34240147
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Abstract

Dietary recommendations for prevention of atherosclerosis.

Riccardi G, Giosuè A, Calabrese I, Vaccaro O
This review aims at summarizing updated evidence on cardiovascular disease (CVD) risk associated with consumption of specific food items to substantiate dietary strategies for atherosclerosis prevention. A systematic search on PubMed was performed to identify meta-analyses of cohort studies and RCTs with CVD outcomes. The evidence is highly concordant in showing that, for the healthy adult population, low consumption of salt and foods of animal origin, and increased intake of plant-based foods-whole grains, fruits, vegetables, legumes, and nuts-are linked with reduced atherosclerosis risk. The same applies for the replacement of butter and other animal/tropical fats with olive oil and other unsaturated-fat-rich oil. Although the literature reviewed overall endorses scientific society dietary recommendations, some relevant novelties emerge. With regard to meat, new evidence differentiates processed and red meat-both associated with increased CVD risk-from poultry, showing a neutral relationship with CVD for moderate intakes. Moreover, the preferential use of low-fat dairies in the healthy population is not supported by recent data, since both full-fat and low-fat dairies, in moderate amounts and in the context of a balanced diet, are not associated with increased CVD risk; furthermore, small quantities of cheese and regular yogurt consumption are even linked with a protective effect. Among other animal protein sources, moderate fish consumption is also supported by the latest evidence, although there might be sustainability concerns. New data endorse the replacement of most high glycemic index (GI) foods with both whole grain and low GI cereal foods. As for beverages, low consumption not only of alcohol, but also of coffee and tea is associated with a reduced atherosclerosis risk while soft drinks show a direct relationship with CVD risk. This review provides evidence-based support for promoting appropriate food choices for atherosclerosis prevention in the general population.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 05 Jul 2021; epub ahead of print
Riccardi G, Giosuè A, Calabrese I, Vaccaro O
Cardiovasc Res: 05 Jul 2021; epub ahead of print | PMID: 34229346
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Impact:
Abstract

Human coronary microvascular contractile dysfunction associates with viable synthetic smooth muscle cells.

Dora KA, Borysova L, Ye X, Powell C, ... Smart N, Ascione R
Aims
Coronary microvascular smooth muscle cells (SMCs) respond to luminal pressure by developing myogenic tone (MT), a process integral to the regulation of microvascular perfusion. The cellular mechanisms underlying poor myogenic reactivity in patients with heart valve disease are unknown and form the focus of this study.
Methods and results
Intramyocardial coronary micro-arteries (IMCAs) isolated from human and pig right atrial appendage (RA) and left ventricular (LV) biopsies were studied using pressure myography combined with confocal microscopy. All RA- and LV-IMCAs from organ donors and pigs developed circa 25% MT. In contrast, 44% of human RA-IMCAs from 88 patients with heart valve disease had poor (<10%) MT yet retained cell viability and an ability to raise cytoplasmic Ca2+ in response to vasoconstrictor agents. Comparing across human heart chambers and species we found that based on patient medical history and six tests, the strongest predictor of poor MT in IMCAs was increased expression of the synthetic marker caldesmon relative to the contractile marker SM-myosin heavy chain. In addition, high resolution imaging revealed a distinct layer of longitudinally-aligned SMCs between ECs and radial SMCs, and we show poor MT was associated with disruptions in these cellular alignments.
Conclusions
These data demonstrate the first use of atrial and ventricular biopsies from patients and pigs to reveal that impaired coronary MT reflects a switch of viable SMCs towards a synthetic phenotype, rather than a loss of SMC viability. These arteries represent a model for further studies of coronary microvascular contractile dysfunction.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 25 Jun 2021; epub ahead of print
Dora KA, Borysova L, Ye X, Powell C, ... Smart N, Ascione R
Cardiovasc Res: 25 Jun 2021; epub ahead of print | PMID: 34173824
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Impact:
Abstract

Culprit site extracellular DNA and microvascular obstruction in ST-elevation myocardial infarction.

Mangold A, Ondracek AS, Hofbauer TM, Scherz T, ... Testori C, Lang IM
Aims
Extracellular chromatin and deoxyribonuclease (DNase) have been identified as important players of thrombosis, inflammation and homeostasis in a murine model. We previously demonstrated that activated neutrophils release neutrophil extracellular traps (NETs) at the culprit site in ST elevation myocardial infarction (STEMI), which significantly contribute to extracellular chromatin burden, and are associated with larger infarcts. To understand the correlation between neutrophil activation, extracellular chromatin and infarct size (IS), we investigated these parameters in a porcine myocardial infarction model, and at different time points and sites in a prospective STEMI trial with cardiac magnetic resonance (CMR) endpoints.
Methods and results
In a prospective STEMI trial (NCT01777750), 101 STEMI patients were included and blood samples were obtained from first medical contact until 6 months after primary percutaneous coronary intervention (pPCI) including direct sampling from the culprit site. CMR was performed 4 ± 2 days and 6 months after pPCI. Neutrophil counts, markers of extracellular chromatin and inflammation were measured. Double-stranded DNA (dsDNA), citrullinated histone 3, nucleosomes, myeloperoxidase, neutrophil elastase and interleukin (IL)-6 were significantly increased, while DNase activity was significantly decreased at the culprit site in STEMI patients. High neutrophil counts and dsDNA levels at the culprit site correlated with high microvascular obstruction (MVO) and low ejection fraction (EF). High DNase activity at the culprit site correlated with low MVO and high EF.In correspondence, dsDNA correlated with IS in the porcine myocardial infarction model. In porcine infarcts, neutrophils and extracellular chromatin were detected in congested small arteries corresponding with MVO. Markers of neutrophil activation, extracellular chromatin, DNase activity and CMR measurements correlated with markers of systemic inflammation C-reactive protein and IL-6 in patients.
Conclusions
NETs and extracellular chromatin are important determinants of MVO in STEMI. Rapid degradation of extracellular chromatin by DNases appears to be crucial for microvascular patency and outcome.
Translational perspective
We show that NETs and extracellular DNA obstruct microvessels in the porcine myocardial infarction model and is connected to increased infarct size. We are able to prove this observation in human STEMI patients. DNase is capable to counteract these effects. Extracellular DNA could be a new treatment target in STEMI.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 25 Jun 2021; epub ahead of print
Mangold A, Ondracek AS, Hofbauer TM, Scherz T, ... Testori C, Lang IM
Cardiovasc Res: 25 Jun 2021; epub ahead of print | PMID: 34173822
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Impact:
Abstract

Murine models of clonal hematopoiesis to assess mechanisms of cardiovascular disease.

Wang Y, Sano S, Ogawa H, Horitani K, ... Doviak H, Walsh K
Clonal hematopoiesis (CH) is a phenomenon whereby somatic mutations confer a fitness advantage to hematopoietic stem and progenitor cells (HSPC) and thus facilitate their aberrant clonal expansion. These mutations are carried into progeny leukocytes leading to a situation whereby a substantial fraction of an individual\'s blood cells originate from the HSPC mutant clone. Although this condition rarely progresses to a hematological malignancy, circulating blood cells bearing the mutation have the potential to affect other organ systems as they infiltrate into tissues under both homeostatic and disease conditions. Epidemiological and clinical studies have revealed that CH is highly prevalent in the elderly and is associated with an increased risk of cardiovascular disease and mortality. Recent experimental studies in murine models have assessed the most commonly mutated \"driver\" genes associated with CH, and have provided evidence for mechanistic connections between CH and cardiovascular disease. A deeper understanding of the mechanisms by which specific CH mutations promote disease pathogenesis is of importance, as it could pave the way for individualized therapeutic strategies targeting the pathogenic CH gene mutations in the future. Here, we review the epidemiology of CH and the mechanistic work from studies using murine disease models, with a particular focus on the strengths and limitations of these experimental systems. We intend for this review to help investigators select the most appropriate models to study CH in the setting of cardiovascular disease.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 22 Jun 2021; epub ahead of print
Wang Y, Sano S, Ogawa H, Horitani K, ... Doviak H, Walsh K
Cardiovasc Res: 22 Jun 2021; epub ahead of print | PMID: 34164655
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Impact:
Abstract

Hooked on heart regeneration: the zebrafish guide to recovery.

Ross Stewart KM, Walker SL, Baker AH, Riley PR, Brittan M
While humans lack sufficient capacity to undergo cardiac regeneration following injury, zebrafish can fully recover from a range of cardiac insults. Over the past two decades our understanding of the complexities of both the independent and co-ordinated injury responses by multiple cardiac tissues during zebrafish heart regeneration has increased exponentially. Although cardiomyocyte regeneration forms the cornerstone of the reparative process in the injured zebrafish heart, recent studies have shown that this is dependent on prior neovascularisation and lymphangiogenesis, which in turn require epicardial, endocardial and inflammatory cell signalling within an extracellular milieu that is optimised for regeneration. Indeed, it is the amalgamation of multiple regenerative systems and gene regulatory patterns that drives the much-heralded success of the adult zebrafish response to cardiac injury. Increasing evidence supports the emerging paradigm that developmental transcriptional programmes are reactivated during adult tissue regeneration, including in the heart, and the zebrafish represents an optimal model organism to explore this concept. In this review we summarise recent advances from the zebrafish cardiovascular research community with novel insight into the mechanisms associated with endogenous cardiovascular repair and regeneration, which may be of benefit to inform future strategies for patients with cardiovascular disease.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 22 Jun 2021; epub ahead of print
Ross Stewart KM, Walker SL, Baker AH, Riley PR, Brittan M
Cardiovasc Res: 22 Jun 2021; epub ahead of print | PMID: 34164652
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Impact:
Abstract

Circulating microRNAs predispose to takotsubo syndrome following high-dose adrenaline exposure.

Couch LS, Fiedler J, Chick G, Clayton R, ... Thum T, Harding SE
Aims
Takotsubo syndrome (TTS) is an acute heart failure, typically triggered by high adrenaline during physical or emotional stress. It is distinguished from myocardial infarction (MI) by a characteristic pattern of ventricular basal hypercontractility with hypokinesis of apical segments, and absence of coronary occlusion. We aimed to understand whether recently discovered circulating biomarkers miR-16 and miR-26a, which differentiate TTS from MI at presentation, were mechanistically involved in the pathophysiology of TTS.
Methods and results
miR-16 and miR-26a were co-overexpressed in rats with AAV and TTS induced with an adrenaline bolus. Untreated isolated rat cardiomyocytes were transfected with pre-/anti-miRs and functionally assessed. Ventricular basal hypercontraction and apical depression were accentuated in miR-transfected animals after induction of TTS. In vitro miR-16 and/or miR-26a overexpression in isolated apical (but not basal) cardiomyocytes produced strong depression of contraction, with loss of adrenaline sensitivity. They also enhanced the initial positive inotropic effect of adrenaline in basal cells. Decreased contractility after TTS-miRs was reproduced in non-failing human apical cardiomyocytes. Bioinformatic profiling of miR targets, followed by expression assays and functional experiments, identified reductions of CACNB1 (L-type calcium channel Cavβ subunit), RGS4 (regulator of G-protein signalling 4) and G-protein subunit Gβ (GNB1) as underlying these effects.
Conclusion
miR-16 and miR-26a sensitise the heart to TTS-like changes produced by adrenaline. Since these miRs have been associated with anxiety and depression, they could provide a mechanism whereby priming of the heart by previous stress causes an increased likelihood of TTS in the future.
Translational perspective
TTS-associated miRs have the potential to be active players predisposing to TTS. Feasibly, their measurement in recovered TTS patients during subsequent periods of stress could be used to predict likelihood of recurrence, a significant risk in this population, and allow preventative action. Since they have been reported as raised in anxiety and depression, they could be part of a priming mechanism where chronic stress predisposes to an acute episode. Understanding the mechanistic basis for the sensitisation may also allow design of other prophylactic pharmacological therapies, including the pre/anti-miR constructs which are now starting to reach the clinic.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 21 Jun 2021; epub ahead of print
Couch LS, Fiedler J, Chick G, Clayton R, ... Thum T, Harding SE
Cardiovasc Res: 21 Jun 2021; epub ahead of print | PMID: 34155498
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Impact:
Abstract

Human BIN1 isoforms grow, maintain and regenerate excitation-contraction couplons in adult rat and human stem cell-derived cardiomyocytes.

Guo J, Tian Q, Barth M, Xian W, ... Laugwitz KL, Lipp P
Aims
In ventricular myocytes, Transverse-tubules (T-tubules) are instrumental for excitation-contraction (EC) coupling and their disarray is a hallmark of cardiac diseases. BIN1 is a key contributor to their biogenesis. Our study set out to investigate the role of human BIN1 splice variants in the maintenance and regeneration of EC-coupling in rat adult ventricular myocytes and human induced pluripotent stem cell-derived cardiac myocytes (hiPS-CMs).
Methods and results
In heart samples from healthy human donors expression patterns of 5 BIN1 splice variants were identified. Following viral transduction of human BIN1 splice variants in cellular models of T-tubular disarray we employed high-speed confocal calcium imaging and Ca-CLEAN analysis to identify functional EC-coupling sites and T-tubular architecture. Adult rat ventricular myocytes were used to investigate the regeneration after loss and maintenance of EC-coupling while we studied the enhancement of EC-coupling in hiPS-CMs. All five human BIN1 splice variants induced de novo generation of T-tubules in both cell types. Isoforms with the phosphoinositide binding motif (PI) were most potent in maintenance and regeneration of T-tubules and functional EC-coupling in adult rat myocytes. In hiPSC-CMs, BIN1 variants with PI motiv induced de-novo generation of T-tubules, functional EC-coupling sites and enhanced calcium handling.
Conclusion(s)
BIN1 is essential for the maintenance, regeneration, and de-novo generation of functional T-tubules, especially isoforms with PI motifs. These T-tubules trigger the development of functional EC couplons resulting in enhanced calcium handling.
Translational perspective
Cardiomyopathy and heart failure are among the most frequent causes of death in modern societies. Gene therapies and hiPSC technology are becoming increasingly promising, both for treatment and therapy development. On the cellular level, one of the common denominators of cardiac diseases is the concurrent loss of T-tubules essential for efficient EC-coupling. While initial approaches in animal models employing gene therapy with BIN1 have depicted encouraging improvements the expression pattern of BIN1 isoforms in the human heart is still elusive. The present study identifies a unique set of five distinct BIN1 isoforms in healthy human hearts and demonstrates their potency in both, T-tubule maintenance and re-generation after loss resulting in efficient EC-coupling. Noteworthy, PI-motif containing isoforms were potent trigger of de-novo generation of T-tubules and establishment of efficient EC-coupling in hiPSC-CMs. Therefore, the expression of BIN1 might be novel and promising for pharmaceutical treatment and gene therapy.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 20 Jun 2021; epub ahead of print
Guo J, Tian Q, Barth M, Xian W, ... Laugwitz KL, Lipp P
Cardiovasc Res: 20 Jun 2021; epub ahead of print | PMID: 34152414
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Impact:
Abstract

Epicardial origin of cardiac arrhythmias: clinical evidences and pathophysiology.

Chaumont C, Suffee N, Gandjbakhch E, Balse E, Anselme F, Hatem SN
Recent developments in imaging, mapping and ablation techniques have shown that the epicardial region of the heart is a key player in the occurrence of ventricular arrhythmic events in several cardiac diseases such as Brugada syndrome, arrhythmogenic cardiomyopathy or dilated cardiomyopathy. At the atrial level as well, the epicardial region has emerged as an important determinant of the substrate of atrial fibrillation, pointing to common underlying pathophysiological mechanisms. Alteration in the gradient of repolarization between myocardial layers favoring the occurrence of re-entry circuits has largely been described. The fibro-fatty infiltration of the subepicardium is another shared substrate between ventricular and atrial arrhythmias. Recent data have emphasized the role of the epicardial reactivation in the formation of this arrhythmogenic substrate. There are new evidences supporting this structural remodeling process to be regulated by the recruitment of epicardial progenitor cells that can differentiate into adipocytes or fibroblasts under various stimuli. In addition, immune-inflammatory processes can also contribute to fibrosis of the subepicardial layer. A better understanding of such \"electrical fragility\" of the epicardial area will open perspectives for novel biomarkers and therapeutic strategies. In this review article, a pathophysiological scheme of epicardial-driven arrhythmias will be proposed.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 20 Jun 2021; epub ahead of print
Chaumont C, Suffee N, Gandjbakhch E, Balse E, Anselme F, Hatem SN
Cardiovasc Res: 20 Jun 2021; epub ahead of print | PMID: 34152392
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Impact:
Abstract

Non-invasive transcutaneous vagal nerve stimulation improves myocardial performance in doxorubicin-induced cardiotoxicity.

Lai Y, Zhou X, Guo F, Jin X, ... Yu L, Jiang H
Aims
The clinical use of antitumor agent doxorubicin (DOX) is hampered by its dose-dependent cardiotoxicity. Development of highly efficient and safe adjuvant intervention for preventing DOX-induced adverse cardiac events is urgently needed. We aimed to investigate whether transcutaneous vagal nerve stimulation (tVNS) plays a cardio-protective role in DOX-induced cardiotoxicity.
Methods and results
Healthy male adult Sprague Dawley rats were used in the experiment and were randomly divided into four groups including control, DOX, tVNS and DOX+tVNS groups. A cumulative dose of 15 mg/kg DOX was intraperitoneally injected into rats to generate cardiotoxicity. Non-invasive tVNS was conducted for 6 weeks (30 min/day). After six-week intervention, the indices from the echocardiography revealed that tVNS significantly improved left ventricular function compared to the DOX group. The increased malondialdehyde (MDA) and Interleukin-1β (IL-1β), and decreased superoxide dismutase (SOD) were observed in the DOX group, while tVNS significantly prevented these changes. From cardiac histopathological analysis, the DOX+tVNS group showed a mild myocardial damage, and decreases in cardiac fibrosis and myocardial apoptosis compared to the DOX group. Heart rate variability (HRV) analysis showed that tVNS significantly inhibited DOX-induced sympathetic hyperactivity compared to the DOX group. Additionally, the results of RNA-sequencing analysis showed that there were 245 differentially expressed genes in the DOX group compared to the control group, among which 39 genes were downregulated by tVNS and most of these genes were involved in immune system. Moreover, tVNS significantly downregulated the relative mRNA expressions of chemokine-related genes and macrophages recruitment compared to the DOX group.
Conclusion
These results suggest that tVNS prevented DOX-induced cardiotoxicity by rebalancing autonomic tone, ameliorating cardiac dysfunction and remodeling. Notably, crosstalk between autonomic neuromodulation and innate immune cells macrophages mediated by chemokines might be involved in the underlying mechanisms.
A translational perspective
Non-invasive tVNS has been identified an effective neuromodulation strategy exerting beneficial effects on rebalancing autonomic tone and cardiac pathological conditions. The present study provided direct evidence for a beneficial role of tVNS in preventing DOX-induced autonomic dysfunction and cardiotoxicity in vivo. Additionally, recent studies revealed the importance of sympathetic nerve fibers involving in tumorigenesis and the benefits of higher vagal tone for tumor prognosis either in animal or human trials. Together, tVNS may not only become a novel, nonpharmacological adjuvant therapy for preventing doxorubicin-induced cardiotoxicity, but also may be beneficial for prognosis of cancer patients during chemotherapy. In our future study, we would investigate the effect of tVNS on both combined chemotherapy-induced cardiotoxicity and the antitumor efficacy of DOX in tumor models.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 17 Jun 2021; epub ahead of print
Lai Y, Zhou X, Guo F, Jin X, ... Yu L, Jiang H
Cardiovasc Res: 17 Jun 2021; epub ahead of print | PMID: 34145895
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Impact:
Abstract

Efficacy and limitations of senolysis in atherosclerosis.

Garrido AM, Kaistha A, Uryga AK, Oc S, ... Jørgensen H, Bennett M
Aims
Traditional markers of cell senescence including p16, Lamin B1, and senescence-associated beta galactosidase (SAβG) suggest very high frequencies of senescent cells in atherosclerosis, while their removal via \'senolysis\' has been reported to reduce atherogenesis. However, selective killing of a variety of different cell types can exacerbate atherosclerosis. We therefore examined the specificity of senescence markers in vascular smooth muscle cells (VSMCs) and the effects of genetic or pharmacological senolysis in atherosclerosis.
Methods and results
We examined traditional senescence markers in human and mouse VSMCs in vitro, and in mouse atherosclerosis. p16 and SAβG increased and Lamin B1 decreased in replicative senescence (RS) and stress-induced premature senescence (SIPS) of cultured human VSMCs. In contrast, mouse VSMCs undergoing SIPS showed only modest p16 upregulation, and proliferating mouse monocyte/macrophages also expressed p16 and SAβG. Single cell RNA-sequencing (scRNA-seq) of lineage-traced mice showed increased p16 expression in VSMC-derived cells in plaques vs. normal arteries, but p16 localized to Stem cell antigen-1 (Sca1)+ or macrophage-like populations. Activation of a p16-driven suicide gene to remove p16+ vessel wall- and/or bone marrow-derived cells increased apoptotic cells, but also induced inflammation and did not change plaque size or composition. In contrast, the senolytic ABT-263 selectively reduced senescent VSMCs in culture, and markedly reduced atherogenesis. However, ABT-263 did not reduce senescence markers in vivo, and significantly reduced monocyte and platelet counts and IL6 as a marker of systemic inflammation.
Conclusions
We show that genetic and pharmacological senolysis have variable effects on atherosclerosis, and may promote inflammation and non-specific effects respectively. In addition, traditional markers of cell senescence such as p16 have significant limitations to identify and remove senescent cells in atherosclerosis, suggesting that senescence studies in atherosclerosis and new senolytic drugs require more specific and lineage-restricted markers before ascribing their effects entirely to senolysis.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 16 Jun 2021; epub ahead of print
Garrido AM, Kaistha A, Uryga AK, Oc S, ... Jørgensen H, Bennett M
Cardiovasc Res: 16 Jun 2021; epub ahead of print | PMID: 34142149
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Impact:
Abstract

Pro-arrhythmic effects of elevated branched chain amino acid levels.

Portero V, Nicol T, Podliesna S, Marchal GA, ... Potter PK, Remme CA
Aim
Cardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke and sudden cardiac death (SCD). Development of efficient preventive and therapeutic strategies is hampered by incomplete knowledge of disease mechanisms and pathways. Our aim is to identify novel mechanisms underlying cardiac arrhythmia and SCD using an unbiased approach.
Methods and results
We employed a phenotype-driven N-ethyl-N-nitrosourea (ENU) mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6-9 weeks) in the absence of prior symptoms. Affected mice were found to be homozygous for the nonsense mutation Bcat2p.Q300*/p.Q300* in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4-5 weeks, Bcat2p.Q300*/p.Q300* mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs - leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. In line with these findings, plasma BCAA levels were positively correlated to ECG indices of conduction and repolarization in the German community-based KORA F4 Study. Isolated cardiomyocytes from Bcat2p.Q300*/p.Q300* mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs) and dysregulated calcium homeostasis. Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation.
Conclusions
Our findings identify for the first time a causative link between elevated BCAAs and arrhythmia, which has implications for arrhythmogenesis in conditions associated with BCAA metabolism dysregulation such as diabetes, metabolic syndrome and heart failure.
Translational perspectives
Development of efficient anti-arrhythmic strategies is hampered by incomplete knowledge of disease mechanisms. Using an unbiased approach, we here identified for the first time a pro-arrhythmic effect of increased levels of branched chain amino acids (BCAAs). This is of particular relevance for conditions associated with BCAA dysregulation and increased arrhythmia risk, including heart failure, obesity and diabetes, as well as for athletes supplementing their diet with BCAAs. Such metabolic dysregulation is potentially modifiable through dietary interventions, paving the way for novel preventive strategies. Our findings furthermore identify mTOR inhibition as a potential anti-arrhythmic strategy in patients with metabolic syndrome.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 16 Jun 2021; epub ahead of print
Portero V, Nicol T, Podliesna S, Marchal GA, ... Potter PK, Remme CA
Cardiovasc Res: 16 Jun 2021; epub ahead of print | PMID: 34142125
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Impact:
Abstract

Altered atrial cytosolic calcium handling contributes to the development of postoperative atrial fibrillation.

Fakuade FE, Steckmeister V, Seibertz F, Gronwald J, ... Mason FE, Voigt N
Aims 
Atrial fibrillation (AF) is a commonly occurring arrhythmia after cardiac surgery (postoperative AF, poAF) and is associated with poorer outcomes. Considering that reduced atrial contractile function is a predictor of poAF and that Ca2+ plays an important role in both excitation-contraction coupling and atrial arrhythmogenesis, this study aims to test whether alterations of intracellular Ca2+ handling contribute to impaired atrial contractility and to the arrhythmogenic substrate predisposing patients to poAF.
Methods and results 
Right atrial appendages were obtained from patients in sinus rhythm undergoing open-heart surgery. Cardiomyocytes were investigated by simultaneous measurement of [Ca2+]i and action potentials (APs, patch-clamp). Patients were followed-up for 6 days to identify those with and without poAF. Speckle-tracking analysis of preoperative echocardiography revealed reduced left atrial contraction strain in poAF patients. At the time of surgery, cellular Ca2+ transients (CaTs) and the sarcoplasmic reticulum (SR) Ca2+ content were smaller in the poAF group. CaT decay was slower in poAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting preserved sodium-calcium exchanger function. In agreement, western blots revealed reduced SERCA2a expression in poAF patients but unaltered phospholamban expression/phosphorylation. Computational modelling indicated that reduced SERCA activity promotes occurrence of CaT and AP alternans. Indeed, alternans of CaT and AP occurred more often and at lower stimulation frequencies in atrial myocytes from poAF patients. Resting membrane potential and AP duration were comparable between both groups at various pacing frequencies (0.25-8 Hz).
Conclusions 
Biochemical, functional, and modelling data implicate reduced SERCA-mediated Ca2+ reuptake into the SR as a major contributor to impaired preoperative atrial contractile function and to the pre-existing arrhythmogenic substrate in patients developing poAF.

© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 15 Jun 2021; 117:1790-1801
Fakuade FE, Steckmeister V, Seibertz F, Gronwald J, ... Mason FE, Voigt N
Cardiovasc Res: 15 Jun 2021; 117:1790-1801 | PMID: 32520995
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Impact:
Abstract

The inflammation-resolution promoting molecule resolvin-D1 prevents atrial proarrhythmic remodelling in experimental right heart disease.

Hiram R, Xiong F, Naud P, Xiao J, ... Tardif JC, Nattel S
Aims
Inflammation plays a role in atrial fibrillation (AF), but classical anti-inflammatory molecules are ineffective. Recent evidence suggests that failure of inflammation-resolution causes persistent inflammatory signalling and that a novel drug-family called resolvins promotes inflammation-resolution. Right heart disease (RHD) is associated with AF; experimental RHD shows signs of atrial inflammatory-pathway activation. Here, we evaluated resolvin-therapy effects on atrial arrhythmogenic remodelling in experimental RHD.
Methods and results
Pulmonary hypertension and RHD were induced in rats with an intraperitoneal injection of 60 mg/kg monocrotaline (MCT). An intervention group received daily resolvin-D1 (RvD1), starting 1 day before MCT administration. Right atrial (RA) conduction and gene-expression were analysed respectively by optical mapping and qPCR/gene-microarray. RvD1 had no or minimal effects on MCT-induced pulmonary artery or right ventricular remodelling. Nevertheless, in vivo transoesophageal pacing induced atrial tachyarrhythmias in no CTRL rats vs. 100% MCT-only rats, and only 33% RvD1-treated MCT rats (P < 0.001 vs. MCT-only). Conduction velocity was significantly decreased by MCT, an effect prevented by RvD1. RHD caused RA dilation and fibrosis. RvD1 strongly attenuated RA fibrosis but had no effect on RA dilation. MCT increased RA expression of inflammation- and fibrosis-related gene-expression pathways on gene-microarray transcriptomic analysis, effects significantly attenuated by RvD1 (334 pathways enriched in MCT-rats vs. control; only 177 dysregulated by MCT with RvD1 treatment). MCT significantly increased RA content of type 1 (proinflammatory) CD68-positive M1 macrophages without affecting type 2 (anti-inflammatory) M2 macrophages. RvD1-treated MCT-rat RA showed significant reductions in proinflammatory M1 macrophages and increases in anti-inflammatory M2 macrophages vs. MCT-only. MCT caused statistically significant increases in protein-expression (western blot) of COL3A1, ASC, CASP1, CASP8, IL1β, TGFβ3, CXCL1, and CXCL2, and decreases in MMP2, vs. control. RvD1-treatment suppressed all these MCT-induced protein-expression changes.
Conclusion
The inflammation-resolution enhancing molecule RvD1 prevents AF-promoting RA remodelling, while suppressing inflammatory changes and fibrotic/electrical remodelling, in RHD. Resolvins show potential promise in combating atrial arrhythmogenic remodelling by suppressing ongoing inflammatory signalling.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 15 Jun 2021; 117:1776-1789
Hiram R, Xiong F, Naud P, Xiao J, ... Tardif JC, Nattel S
Cardiovasc Res: 15 Jun 2021; 117:1776-1789 | PMID: 32866246
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Impact:
Abstract

Transcriptome and proteome mapping in the sheep atria reveal molecular featurets of atrial fibrillation progression.

Alvarez-Franco A, Rouco R, Ramirez RJ, Guerrero-Serna G, ... Jalife J, Manzanares M
Aims
Atrial fibrillation (AF) is a progressive cardiac arrhythmia that increases the risk of hospitalization and adverse cardiovascular events. There is a clear demand for more inclusive and large-scale approaches to understand the molecular drivers responsible for AF, as well as the fundamental mechanisms governing the transition from paroxysmal to persistent and permanent forms. In this study, we aimed to create a molecular map of AF and find the distinct molecular programmes underlying cell type-specific atrial remodelling and AF progression.
Methods and results
We used a sheep model of long-standing, tachypacing-induced AF, sampled right and left atrial tissue, and isolated cardiomyocytes (CMs) from control, intermediate (transition), and late time points during AF progression, and performed transcriptomic and proteome profiling. We have merged all these layers of information into a meaningful three-component space in which we explored the genes and proteins detected and their common patterns of expression. Our data-driven analysis points at extracellular matrix remodelling, inflammation, ion channel, myofibril structure, mitochondrial complexes, chromatin remodelling, and genes related to neural function, as well as critical regulators of cell proliferation as hallmarks of AF progression. Most important, we prove that these changes occur at early transitional stages of the disease, but not at later stages, and that the left atrium undergoes significantly more profound changes than the right atrium in its expression programme. The pattern of dynamic changes in gene and protein expression replicate the electrical and structural remodelling demonstrated previously in the sheep and in humans, and uncover novel mechanisms potentially relevant for disease treatment.
Conclusions
Transcriptomic and proteomic analysis of AF progression in a large animal model shows that significant changes occur at early stages, and that among others involve previously undescribed increase in mitochondria, changes to the chromatin of atrial CMs, and genes related to neural function and cell proliferation.

© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 15 Jun 2021; 117:1760-1775
Alvarez-Franco A, Rouco R, Ramirez RJ, Guerrero-Serna G, ... Jalife J, Manzanares M
Cardiovasc Res: 15 Jun 2021; 117:1760-1775 | PMID: 33119050
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Impact:
Abstract

NLRP3 inflammasome is a key driver of obesity-induced atrial arrhythmias.

Scott L, Fender AC, Saljic A, Li L, ... Dobrev D, Li N
Aims
Obesity, an established risk factor of atrial fibrillation (AF), is frequently associated with enhanced inflammatory response. However, whether inflammatory signaling is causally linked to AF pathogenesis in obesity remains elusive. We recently demonstrated that the constitutive activation of the \'NACHT, LRR, and PYD Domains-containing Protein 3\' (NLRP3) inflammasome promotes AF susceptibility. In this study, we hypothesized that the NLRP3 inflammasome is a key driver of obesity-induced AF.
Methods and results
Western blotting was performed to determine the level of NLRP3 inflammasome activation in atrial tissues of obese patients, sheep, and diet-induced obese (DIO) mice. The increased body weight in patients, sheep, and mice was associated with enhanced NLRP3-inflammasome activation. To determine whether NLRP3 contributes to the obesity-induced atrial arrhythmogenesis, wild-type (WT) and NLRP3 homozygous knockout (NLRP3-/-) mice were subjected to high-fat-diet (HFD) or normal chow (NC) for 10 weeks. Relative to NC-fed WT mice, HFD-fed WT mice were more susceptible to pacing-induced AF with longer AF duration. In contrast, HFD-fed NLRP3-/- mice were resistant to pacing-induced AF. Optical mapping in DIO mice revealed an arrhythmogenic substrate characterized by abbreviated refractoriness and action potential duration (APD), two key determinants of reentry-promoting electrical remodeling. Upregulation of ultra-rapid delayed-rectifier K+-channel (Kv1.5) contributed to the shortening of atrial refractoriness. Increased profibrotic signaling and fibrosis along with abnormal Ca2+ release from sarcoplasmic reticulum (SR) accompanied atrial arrhythmogenesis in DIO mice. Conversely, genetic ablation of Nlrp3 (NLRP3-/-) in HFD-fed mice prevented the increases in Kv1.5 and the evolution of electrical remodeling, the upregulation of profibrotic genes, and abnormal SR Ca2+ release in DIO mice.
Conclusion
These results demonstrate that the atrial NLRP3 inflammasome is a key driver of obesity-induced atrial arrhythmogenesis and establishes a mechanistic link between obesity-induced AF and NLRP3-inflammasome activation.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 15 Jun 2021; 117:1746-1759
Scott L, Fender AC, Saljic A, Li L, ... Dobrev D, Li N
Cardiovasc Res: 15 Jun 2021; 117:1746-1759 | PMID: 33523143
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Impact:
Abstract

Novel approaches to mechanism-based atrial fibrillation ablation.

Quintanilla JG, Shpun S, Jalife J, Filgueiras-Rama D
Modern cardiac electrophysiology has reported significant advances in the understanding of mechanisms underlying complex wave propagation patterns during atrial fibrillation (AF), although disagreements remain. One school of thought adheres to the long-held postulate that AF is the result of randomly propagating wavelets that wonder throughout the atria. Another school supports the notion that AF is deterministic in that it depends on a small number of high-frequency rotors generating three-dimensional scroll waves that propagate throughout the atria. The spiralling waves are thought to interact with anatomic and functional obstacles, leading to fragmentation and new wavelet formation associated with the irregular activation patterns documented on AF tracings. The deterministic hypothesis is consistent with demonstrable hierarchical gradients of activation frequency and AF termination on ablation at specific (non-random) atrial regions. During the last decade, data from realistic animal models and pilot clinical series have triggered a new era of novel methodologies to identify and ablate AF drivers outside the pulmonary veins. New generation electroanatomical mapping systems and multielectrode mapping catheters, complimented by powerful mathematical analyses, have generated the necessary platforms and tools for moving these approaches into clinical procedures. Recent clinical data using such platforms have provided encouraging evidence supporting the feasibility of targeting and effectively ablating driver regions in addition to pulmonary vein isolation in persistent AF. Here, we review state-of-the-art technologies and provide a comprehensive historical perspective, characterization, classification, and expected outcomes of current mechanism-based methods for AF ablation. We discuss also the challenges and expected future directions that scientists and clinicians will face in their efforts to understand AF dynamics and successfully implement any novel method into regular clinical practice.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 15 Jun 2021; 117:1662-1681
Quintanilla JG, Shpun S, Jalife J, Filgueiras-Rama D
Cardiovasc Res: 15 Jun 2021; 117:1662-1681 | PMID: 33744913
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Impact:
Abstract

New biomarkers from multiomics approaches: improving risk prediction of atrial fibrillation.

Kornej J, Hanger VA, Trinquart L, Ko D, ... Benjamin EJ, Lin H
Atrial fibrillation (AF) is a common cardiac arrhythmia leading to many adverse outcomes and increased mortality. Yet the molecular mechanisms underlying AF remain largely unknown. Recent advances in high-throughput technologies make large-scale molecular profiling possible. In the past decade, multiomics studies of AF have identified a number of potential biomarkers of AF. In this review, we focus on the studies of multiomics profiles with AF risk. We summarize recent advances in the discovery of novel biomarkers for AF through multiomics studies. We also discuss limitations and future directions in risk assessment and discovery of therapeutic targets for AF.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 15 Jun 2021; 117:1632-1644
Kornej J, Hanger VA, Trinquart L, Ko D, ... Benjamin EJ, Lin H
Cardiovasc Res: 15 Jun 2021; 117:1632-1644 | PMID: 33751041
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Impact:
Abstract

New aspects of endocrine control of atrial fibrillation and possibilities for clinical translation.

Aguilar M, Rose RA, Takawale A, Nattel S, Reilly S
Hormones are potent endo-, para-, and autocrine endogenous regulators of the function of multiple organs, including the heart. Endocrine dysfunction promotes a number of cardiovascular diseases, including atrial fibrillation (AF). While the heart is a target for endocrine regulation, it is also an active endocrine organ itself, secreting a number of important bioactive hormones that convey significant endocrine effects, but also through para-/autocrine actions, actively participate in cardiac self-regulation. The hormones regulating heart-function work in concert to support myocardial performance. AF is a serious clinical problem associated with increased morbidity and mortality, mainly due to stroke and heart failure. Current therapies for AF remain inadequate. AF is characterized by altered atrial function and structure, including electrical and profibrotic remodelling in the atria and ventricles, which facilitates AF progression and hampers its treatment. Although features of this remodelling are well-established and its mechanisms are partly understood, important pathways pertinent to AF arrhythmogenesis are still unidentified. The discovery of these missing pathways has the potential to lead to therapeutic breakthroughs. Endocrine dysfunction is well-recognized to lead to AF. In this review, we discuss endocrine and cardiocrine signalling systems that directly, or as a consequence of an underlying cardiac pathology, contribute to AF pathogenesis. More specifically, we consider the roles of products from the hypothalamic-pituitary axis, the adrenal glands, adipose tissue, the renin-angiotensin system, atrial cardiomyocytes, and the thyroid gland in controlling atrial electrical and structural properties. The influence of endocrine/paracrine dysfunction on AF risk and mechanisms is evaluated and discussed. We focus on the most recent findings and reflect on the potential of translating them into clinical application.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 15 Jun 2021; 117:1645-1661
Aguilar M, Rose RA, Takawale A, Nattel S, Reilly S
Cardiovasc Res: 15 Jun 2021; 117:1645-1661 | PMID: 33723575
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Impact:
Abstract

Why translation from basic discoveries to clinical applications is so difficult for atrial fibrillation and possible approaches to improving it.

Nattel S, Sager PT, Hüser J, Heijman J, Dobrev D
Atrial fibrillation (AF) is the most common sustained clinical arrhythmia, with a lifetime incidence of up to 37%, and is a major contributor to population morbidity and mortality. Important components of AF management include control of cardiac rhythm, rate, and thromboembolic risk. In this narrative review article, we focus on rhythm-control therapy. The available therapies for cardiac rhythm control include antiarrhythmic drugs and catheter-based ablation procedures; both of these are presently neither optimally effective nor safe. In order to develop improved treatment options, it is necessary to use preclinical models, both to identify novel mechanism-based therapeutic targets and to test the effects of putative therapies before initiating clinical trials. Extensive research over the past 30 years has provided many insights into AF mechanisms that can be used to design new rhythm-maintenance approaches. However, it has proven very difficult to translate these mechanistic discoveries into clinically applicable safe and effective new therapies. The aim of this article is to explore the challenges that underlie this phenomenon. We begin by considering the basic problem of AF, including its clinical importance, the current therapeutic landscape, the drug development pipeline, and the notion of upstream therapy. We then discuss the currently available preclinical models of AF and their limitations, and move on to regulatory hurdles and considerations and then review industry concerns and strategies. Finally, we evaluate potential paths forward, attempting to derive insights from the developmental history of currently used approaches and suggesting possible paths for the future. While the introduction of successful conceptually innovative new treatments for AF control is proving extremely difficult, one significant breakthrough is likely to revolutionize both AF management and the therapeutic development landscape.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 15 Jun 2021; 117:1616-1631
Nattel S, Sager PT, Hüser J, Heijman J, Dobrev D
Cardiovasc Res: 15 Jun 2021; 117:1616-1631 | PMID: 33769493
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Impact:
Abstract

Computational models of atrial fibrillation: achievements, challenges, and perspectives for improving clinical care.

Heijman J, Sutanto H, Crijns HJGM, Nattel S, Trayanova NA
Despite significant advances in its detection, understanding and management, atrial fibrillation (AF) remains a highly prevalent cardiac arrhythmia with a major impact on morbidity and mortality of millions of patients. AF results from complex, dynamic interactions between risk factors and comorbidities that induce diverse atrial remodelling processes. Atrial remodelling increases AF vulnerability and persistence, while promoting disease progression. The variability in presentation and wide range of mechanisms involved in initiation, maintenance and progression of AF, as well as its associated adverse outcomes, make the early identification of causal factors modifiable with therapeutic interventions challenging, likely contributing to suboptimal efficacy of current AF management. Computational modelling facilitates the multilevel integration of multiple datasets and offers new opportunities for mechanistic understanding, risk prediction and personalized therapy. Mathematical simulations of cardiac electrophysiology have been around for 60 years and are being increasingly used to improve our understanding of AF mechanisms and guide AF therapy. This narrative review focuses on the emerging and future applications of computational modelling in AF management. We summarize clinical challenges that may benefit from computational modelling, provide an overview of the different in silico approaches that are available together with their notable achievements, and discuss the major limitations that hinder the routine clinical application of these approaches. Finally, future perspectives are addressed. With the rapid progress in electronic technologies including computing, clinical applications of computational modelling are advancing rapidly. We expect that their application will progressively increase in prominence, especially if their added value can be demonstrated in clinical trials.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 15 Jun 2021; 117:1682-1699
Heijman J, Sutanto H, Crijns HJGM, Nattel S, Trayanova NA
Cardiovasc Res: 15 Jun 2021; 117:1682-1699 | PMID: 33890620
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Impact:
Abstract

Genetics of atrial fibrillation-practical applications for clinical management: if not now, when and how?

Kany S, Reissmann B, Metzner A, Kirchhof P, Darbar D, Schnabel RB
The prevalence and economic burden of atrial fibrillation (AF) are predicted to more than double over the next few decades. In addition to anticoagulation and treatment of concomitant cardiovascular conditions, early and standardized rhythm control therapy reduces cardiovascular outcomes as compared with a rate control approach, favouring the restoration, and maintenance of sinus rhythm safely. Current therapies for rhythm control of AF include antiarrhythmic drugs (AADs) and catheter ablation (CA). However, response in an individual patient is highly variable with some remaining free of AF for long periods on antiarrhythmic therapy, while others require repeat AF ablation within weeks. The limited success of rhythm control therapy for AF is in part related to incomplete understanding of the pathophysiological mechanisms and our inability to predict responses in individual patients. Thus, a major knowledge gap is predicting which patients with AF are likely to respond to rhythm control approach. Over the last decade, tremendous progress has been made in defining the genetic architecture of AF with the identification of rare mutations in cardiac ion channels, signalling molecules, and myocardial structural proteins associated with familial (early-onset) AF. Conversely, genome-wide association studies have identified common variants at over 100 genetic loci and the development of polygenic risk scores has identified high-risk individuals. Although retrospective studies suggest that response to AADs and CA is modulated in part by common genetic variation, the development of a comprehensive clinical and genetic risk score may enable the translation of genetic data to the bedside care of AF patients. Given the economic impact of the AF epidemic, even small changes in therapeutic efficacy may lead to substantial improvements for patients and health care systems.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 15 Jun 2021; 117:1718-1731
Kany S, Reissmann B, Metzner A, Kirchhof P, Darbar D, Schnabel RB
Cardiovasc Res: 15 Jun 2021; 117:1718-1731 | PMID: 33982075
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Impact:
Abstract

How machine learning is impacting research in atrial fibrillation: implications for risk prediction and future management.

Olier I, Ortega-Martorell S, Pieroni M, Lip GYH
There has been an exponential growth of artificial intelligence (AI) and machine learning (ML) publications aimed at advancing our understanding of atrial fibrillation (AF), which has been mainly driven by the confluence of two factors: the advances in deep neural networks (DeepNNs) and the availability of large, open access databases. It is observed that most of the attention has centred on applying ML for dvsetecting AF, particularly using electrocardiograms (ECGs) as the main data modality. Nearly a third of them used DeepNNs to minimize or eliminate the need for transforming the ECGs to extract features prior to ML modelling; however, we did not observe a significant advantage in following this approach. We also found a fraction of studies using other data modalities, and others centred in aims, such as risk prediction, AF management, and others. From the clinical perspective, AI/ML can help expand the utility of AF detection and risk prediction, especially for patients with additional comorbidities. The use of AI/ML for detection and risk prediction into applications and smart mobile health (mHealth) technology would enable \'real time\' dynamic assessments. AI/ML could also adapt to treatment changes over time, as well as incident risk factors. Incorporation of a dynamic AI/ML model into mHealth technology would facilitate \'real time\' assessment of stroke risk, facilitating mitigation of modifiable risk factors (e.g. blood pressure control). Overall, this would lead to an improvement in clinical care for patients with AF.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 15 Jun 2021; 117:1700-1717
Olier I, Ortega-Martorell S, Pieroni M, Lip GYH
Cardiovasc Res: 15 Jun 2021; 117:1700-1717 | PMID: 33982064
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Abstract

Neuroscientific therapies for atrial fibrillation.

Hanna P, Buch E, Stavrakis S, Meyer C, ... Ardell JL, Shivkumar K
The cardiac autonomic nervous system (ANS) plays an integral role in normal cardiac physiology as well as in disease states that cause cardiac arrhythmias. The cardiac ANS, comprised of a complex neural hierarchy in a nested series of interacting feedback loops, regulates atrial electrophysiology and is itself susceptible to remodelling by atrial rhythm. In light of the challenges of treating atrial fibrillation (AF) with conventional pharmacologic and myoablative techniques, increasingly interest has begun to focus on targeting the cardiac neuraxis for AF. Strong evidence from animal models and clinical patients demonstrates that parasympathetic and sympathetic activity within this neuraxis may trigger AF, and the ANS may either induce atrial remodelling or undergo remodelling itself to serve as a substrate for AF. Multiple nexus points within the cardiac neuraxis are therapeutic targets, and neuroablative and neuromodulatory therapies for AF include ganglionated plexus ablation, epicardial botulinum toxin injection, vagal nerve (tragus) stimulation, renal denervation, stellate ganglion block/resection, baroreceptor activation therapy, and spinal cord stimulation. Pre-clinical and clinical studies on these modalities have had promising results and are reviewed here.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: [email protected]

Cardiovasc Res: 15 Jun 2021; 117:1732-1745
Hanna P, Buch E, Stavrakis S, Meyer C, ... Ardell JL, Shivkumar K
Cardiovasc Res: 15 Jun 2021; 117:1732-1745 | PMID: 33989382
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Abstract

Inhibition of myocardial cathepsin-L release during reperfusion following myocardial infarction improves cardiac function and reduces infarct size.

He W, McCarroll CS, Nather K, Ford K, ... Nicklin SA, Loughrey CM
Aims
Identifying novel mediators of lethal myocardial reperfusion injury that can be targeted during primary percutaneous coronary intervention (PPCI) is key to limiting the progression of patients with ST-elevated myocardial infarction (STEMI) to heart failure. Here we show through parallel clinical and integrative preclinical studies the significance of the protease cathepsin-L on cardiac function during reperfusion injury.
Methods and results
We found that direct cardiac release of cathepsin-L in STEMI patients (n = 76) immediately post-PPCI leads to elevated serum cathepsin-L levels and that serum levels of cathepsin-L in the first 24 hour post-reperfusion are associated with reduced cardiac contractile function and increased infarct size. Preclinical studies, demonstrate that inhibition of cathepsin-L release following reperfusion injury with CAA0225 reduces infarct size and improves cardiac contractile function by limiting abnormal cardiomyocyte calcium handling and apoptosis.
Conclusion
Our findings suggest that cathepsin-L is a novel therapeutic target that could be exploited clinically to counteract the deleterious effects of acute reperfusion injury after an acute STEMI.
Translational perspective
New therapeutic targets are urgently required to limit myocardial damage after reperfusion injury. We identified cardiac release of the protease cathepsin-L among patients following primary percutaneous coronary intervention (PPCI). Elevated serum levels of cathepsin-L were associated with reduced contractile function and increased infarct size at 24 hour and 6 months post-PPCI. Work conducted using animal models indicated that cardiac release of cathepsin-L mediated cardiac dysfunction following reperfusion injury. Specific inhibition of cathepsin-L prevented abnormal calcium handling, reduced infarct size and improved contractile function. These novel findings offer the prospect of targeting cathepsin-L-mediated cardiac dysfunction after PPCI.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 15 Jun 2021; epub ahead of print
He W, McCarroll CS, Nather K, Ford K, ... Nicklin SA, Loughrey CM
Cardiovasc Res: 15 Jun 2021; epub ahead of print | PMID: 34132807
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Abstract

Discovery of a first-in-class inhibitor of sulfide:quinone oxidoreductase that protects against adverse cardiac remodeling and heart failure.

Jackson MR, Cox KD, Baugh SDP, Wakeen L, ... Polyak B, Jorns MS
Aims
Hydrogen sulfide (H2S) is a potent signaling molecule that activates diverse cardioprotective pathways by posttranslational modification (persulfidation) of cysteine residues in upstream protein targets. Heart failure patients with reduced ejection fraction (HFrEF) exhibit low levels of H2S. Sulfide: quinone oxidoreductase (SQOR) catalyzes the first irreversible step in the metabolism of H2S and plays a key role in regulating H2S-mediated signaling. Our aim here was to discover a first-in-class inhibitor of human SQOR and evaluate its cardioprotective effect in an animal model of HFrEF.
Methods and results
We identified a potent inhibitor of human SQOR (STI1, IC50 = 29 nM) by high-throughput screening of a small-molecule library, followed by focused medicinal chemistry optimization and structure-based design. STI1 is a competitive inhibitor that binds with high selectivity to the coenzyme Q-binding pocket in SQOR. STI1 exhibited very low cytotoxicity and attenuated the hypertrophic response of neonatal rat ventricular cardiomyocytes and H9c2 cells induced by neurohormonal stressors. A mouse HFrEF model was produced by transverse aortic constriction (TAC). Treatment of TAC mice with STI1 mitigated the development of cardiomegaly, pulmonary congestion, dilatation of the left ventricle, and cardiac fibrosis and decreased the pressure gradient across the aortic constriction. Moreover, STI1 dramatically improved survival, preserved cardiac function, and prevented the progression to HFrEF by impeding the transition from compensated to decompensated left ventricle hypertrophy.
Conclusion
We demonstrate that the coenzyme Q-binding pocket in human SQOR is a druggable target and establish proof of concept for the potential of SQOR inhibitors to provide a novel therapeutic approach for the treatment of HFrEF.
Translational perspective
In HFrEF there is a compelling need for new drugs that mitigate the pathological remodeling induced by injury and improve patient survival. This study identifies SQOR-inhibiting drugs as a promising first-in-class therapy for HFrEF patients. Due to the well-established protective properties of H2S-induced signaling in renal physiology and disease, this novel class of heart failure therapeutics may also address the large unmet need of therapies for approximately 50% of heart failure patients that have coexisting chronic renal dysfunction.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 15 Jun 2021; epub ahead of print
Jackson MR, Cox KD, Baugh SDP, Wakeen L, ... Polyak B, Jorns MS
Cardiovasc Res: 15 Jun 2021; epub ahead of print | PMID: 34132787
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Abstract

Loss of Yap/taz in cardiac fibroblasts attenuates adverse remodeling and improves cardiac function.

Mia MM, Cibi DM, Binte Abdul Ghani SA, Singh A, ... Mao J, Singh MK
Aims
Fibrosis is associated with all forms of adult cardiac diseases including myocardial infarction (MI). In response to MI, the heart undergoes ventricular remodeling that leads to fibrotic scar due to excessive deposition of extracellular matrix mostly produced by myofibroblasts. The structural and mechanical properties of the fibrotic scar are critical determinants of heart function. Yes-associated protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz) are the key effectors of the Hippo signaling pathway and are crucial for cardiomyocyte proliferation during cardiac development and regeneration. However, their role in cardiac fibroblasts, regulating post-MI fibrotic and fibroinflammatory response is not well established.
Methods and results
Using mouse model, we demonstrate that Yap/Taz are activated in cardiac fibroblasts after MI and fibroblasts-specific deletion of Yap/Taz using Col1a2Cre(ER)T mice reduces post-MI fibrotic and fibroinflammatory response and improves cardiac function. Consistently, Yap overexpression elevated post-MI fibrotic response. Gene expression profiling shows significant downregulation of several cytokines involved in post-MI cardiac remodeling. Furthermore, Yap/Taz directly regulate the promoter activity of pro-fibrotic cytokine interleukin-33 (IL33) in cardiac fibroblasts. Blocking of IL33 receptor ST2 using the neutralizing antibody abrogates the Yap-induced pro-fibrotic response in cardiac fibroblasts. We demonstrate that the altered fibroinflammatory program not only affects the nature of cardiac fibroblasts but also the polarization as well as infiltration of macrophages in the infarcted hearts. Furthermore, we demonstrate that Yap/Taz act downstream of both Wnt and TGFβ signaling pathways in regulating cardiac fibroblasts activation and fibroinflammatory response.
Conclusions
We demonstrate that Yap/Taz play an important role in controlling MI-induced cardiac fibrosis by modulating fibroblasts proliferation, transdifferentiation into myofibroblasts, and fibroinflammatory program.
Translational perspective
Cardiac fibroblasts are the most prevalent cell type in the heart and play an important role in regulating post-myocardial infarction (MI) cardiac fibrosis. Excessive cardiac fibrosis causes ventricular stiffness leading to systolic/diastolic cardiac dysfunction and heart failure. Therefore, understanding the molecular mechanism of cardiac fibroblasts activation will help to modulate the post-MI fibrotic response and improve cardiac function. In our study, we show that Yap/Taz play an important role in controlling MI-induced cardiac fibrosis by modulating fibroblasts proliferation, transdifferentiation into myofibroblasts, and fibroinflammatory program.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 15 Jun 2021; epub ahead of print
Mia MM, Cibi DM, Binte Abdul Ghani SA, Singh A, ... Mao J, Singh MK
Cardiovasc Res: 15 Jun 2021; epub ahead of print | PMID: 34132780
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Abstract

The EP300/TP53 pathway, a suppressor of the Hippo and canonical WNT pathways, is activated in human hearts with arrhythmogenic cardiomyopathy in the absence of overt heart failure.

Rouhi L, Fan S, Cheedipudi SM, Braza-Boïls A, ... Zorio E, Marian AJ
Aim
Arrhythmogenic cardiomyopathy (ACM) is a primary myocardial disease that typically manifests with cardiac arrhythmias, progressive heart failure and sudden cardiac death (SCD). ACM is mainly caused by mutations in genes encoding desmosome proteins. Desmosomes are cell-cell adhesion structures and hubs for mechanosensing and mechanotransduction. The objective was to identify the dysregulated molecular and biological pathways in human ACM in the absence of overt heart failure.
Methods and results
Transcriptomes in the right ventricular endomyocardial biopsy samples from three independent individuals carrying truncating mutations in the DSP gene and 5 control samples were analyzed by RNA-Seq (discovery group). These cases presented with cardiac arrhythmias and had a normal right ventricular function. The RNA-Seq analysis identified ∼5,000 differentially expressed genes (DEGs), which predicted suppression of the Hippo and canonical WNT pathways, among others.Dysregulated genes and pathways, identified by RNA-Seq, were tested for validation in the right and left ventricular tissues from 5 independent autopsy-confirmed ACM cases with defined mutations (validation group), who were victims of SCD and had no history of heart failure. Protein levels and nuclear localization of the cWNT and Hippo pathway transcriptional regulators were reduced in the right and left ventricular validation samples. In contrast, levels of acetyltransferase EP300, known to suppress the Hippo and canonical WNT pathways, were increased and its bona fide target TP53 was acetylated. RNA-Seq data identified apical junction, reflective of cell-cell attachment, as the most disrupted biological pathway, which was corroborated by disrupted desmosomes and intermediate filament structures. Moreover, the DEGs also predicted dysregulation of over a dozen canonical signal transduction pathways, including the Tec kinase and integrin signaling pathways. The changes were associated with increased apoptosis and fibro-adipogenesis in the ACM hearts.
Conclusion
Altered apical junction structures is associated with activation of the EP300-TP53 and suppression of the Hippo/cWNT pathways in human ACM caused by defined mutations in the absence of an overt heart failure. The findings implicate altered mechanotransduction in the pathogenesis of ACM.
Translational perspective
The findings suggest that altered mechanosensing at the cell-cell junction instigates a cascade of molecular events through the activation of acetyltransferase EP300/TP53 and suppression of gene expression through the Hippo/canonical WNT pathways in human arrhythmogenic cardiomyopathy (ACM) caused by defined mutations. These molecular changes occur early and in the absence of overt heart failure. Consequently, one may envision cell type-specific interventions to target the dysregulated transcriptional, mechanosensing, and mechanotransduction pathways to prevent the evolving phenotype in human ACM.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 15 Jun 2021; epub ahead of print
Rouhi L, Fan S, Cheedipudi SM, Braza-Boïls A, ... Zorio E, Marian AJ
Cardiovasc Res: 15 Jun 2021; epub ahead of print | PMID: 34132777
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Abstract

Circulating Progenitor Cells Are Associated With Plaque Progression And Long-Term Outcomes In Heart Transplant Patients.

Ozcan I, Toya T, Corban MT, Ahmad A, ... Kushwaha SS, Lerman A
Aims
Circulating progenitor cells (CPCs) play a role in vascular repair and plaque stability, while osteocalcin (OC) expressing CPCs have been linked to unstable plaque and adverse cardiovascular outcomes. However, their role in cardiac allograft vasculopathy (CAV) has not been elucidated. This cohort study aimed to investigate the contribution of CPCs on CAV progression and cardiovascular events after heart transplantation.
Methods and results
A total of 80 heart transplant patients (mean age 55 ± 14 years, 72% male) undergoing annual intravascular ultrasound (IVUS) had fresh CPCs marked by CD34, CD133, and OC counted in peripheral blood using flow cytometry, on the same day as baseline IVUS. CAV progression was assessed by IVUS as the change (Δ) in plaque volume divided by segment length (PV/SL), adjusted for the time between IVUS measurements (median 3.0, interquartile range (IQR) [2.8, 3.1] years), and was defined as ΔPV/SL that is above the median ΔPV/SL of study population. Major adverse cardiac events (MACE) was defined as any incident of revascularization, myocardial infarction, heart failure admission, re-transplantation, stroke and death. Patients with higher CD34+CD133+ CPCs had a decreased risk of CAV progression (odds ratio 0.58, 95% confidence interval [CI] [0.37, 0.92], p = 0.01) and MACE (hazard ratio [HR] 0.79, 95% CI [0.66, 0.99], p = 0.05) during a median (IQR) follow up of 8.0 years (7.2, 8.3). Contrarily, higher OC+ cell counts were associated with an increased risk of MACE (HR 1.26, 95% CI [1.03, 1.57], p = 0.02).
Conclusions
Lower levels of CD34+CD133+ CPCs are associated with plaque progression and adverse long-term outcomes in patients who underwent allograft heart transplantation. In contrast, higher circulating OC+ levels are associated with adverse long term outcomes. Thus, CPCs might play a role in amelioration of transplant vasculopathy, while OC expression by these cells might play a role in progression.
Translational perspective
The results of the current study suggest lower levels of circulating CD34+CD133+ cell levels are associated with cardiac allograft vasculopathy progression and future adverse cardiovascular events, while higher OC+ cell levels are associated with a greater risk of future cardiovascular events. Further studies confirming our findings might elucidate the role of circulating progenitor cells in the pathophysiology of CAV. Moreover, our findings might support the use of circulating progenitors as biomarkers, as well as the notion of cell therapy as potential treatment option for CAV, a disease with severe burden and limited treatment options.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 15 Jun 2021; epub ahead of print
Ozcan I, Toya T, Corban MT, Ahmad A, ... Kushwaha SS, Lerman A
Cardiovasc Res: 15 Jun 2021; epub ahead of print | PMID: 34132771
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Abstract

COVID-19 related cardiac complications - from clinical evidences to basic mechanisms. Opinion paper of the ESC Working Group on Cellular Biology of the Heart.

Pesce M, Agostoni P, Bøtker HE, Brundel B, ... van Linthout S, Madonna R
The pandemic of Coronavirus disease (COVID)-19 is a global threat, causing high mortality, especially in the elderly. The main symptoms and the primary cause of death are related to interstitial pneumonia. Viral entry also into myocardial cells mainly via the angiotensin converting enzyme type 2 (ACE2) receptor and excessive production of pro-inflammatory cytokines, however, also make the heart susceptible to injury. In addition to the immediate damage caused by the acute inflammatory response, the heart may also suffer from long-term consequences of COVID-19, potentially causing a post-pandemic increase in cardiac complications. Although the main cause of cardiac damage in COVID-19 remains coagulopathy with micro- (and to a lesser extent macro-) vascular occlusion, open questions remain about other possible modalities of cardiac dysfunction, such as direct infection of myocardial cells, effects of cytokines storm, and mechanisms related to enhanced coagulopathy. In this opinion paper, we focus on these lesser appreciated possibilities and propose experimental approaches that could provide a more comprehensive understanding of the cellular and molecular bases of cardiac injury in COVID-19 patients. We first discuss approaches to characterize cardiac damage caused by possible direct viral infection of cardiac cells, followed by formulating hypotheses on how to reproduce and investigate the hyperinflammatory and pro-thrombotic conditions observed in the heart of COVID-19 patients using experimental in vitro systems. Finally, we elaborate on strategies to discover novel pathology biomarkers using omics platforms.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 11 Jun 2021; epub ahead of print
Pesce M, Agostoni P, Bøtker HE, Brundel B, ... van Linthout S, Madonna R
Cardiovasc Res: 11 Jun 2021; epub ahead of print | PMID: 34117887
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Abstract

Adiponectin and cardiometabolic trait and mortality: where do we go?

Jang AY, Scherer PE, Kim JY, Lim S, Koh KK
Adiponectin is an adipocyte-derived cytokine known for its cardioprotective effects in preclinical studies. Early epidemiologic studies replicated these findings and drew great interest. Subsequent large-scale prospective cohorts, however, showed that adiponectin levels seemed not to relate to incident coronary artery disease (CAD). Even more surprisingly, a paradoxical increase of all-cause and cardiovascular (CV) mortality with increased adiponectin levels was reported. The adiponectin-mortality paradox has been explained by some groups asserting that adiponectin secretion is promoted by elevated natriuretic peptides (NP). Other groups have proposed that adiponectin is elevated due to adiponectin resistance in subjects with metabolic syndrome or heart failure (HF). However, there is no unifying theory that can clearly explain this paradox. In patients with HF with reduced ejection fraction (HFrEF), stretched cardiomyocytes secrete NPs, which further promote release of adiponectin from adipose tissue, leading to adiponectin resistance. On the other hand, adiponectin biology may differ in patients with heart failure with preserved ejection fraction (HFpEF), which constitutes 50% of all of HF. Most HFpEF patients are obese, which exerts inflammation and myocardial stiffness, that is likely to prevent myocardial stretch and subsequent NP release. This segment of the patient population may display a different adiponectin biology from its HFrEF counterpart. Dissecting the adiponectin-mortality relation in terms of different HF subtypes may help to comprehensively understand this paradox. Mendelian Randomization (MR) analyses claimed that adiponectin levels are not causally related to CAD or metabolic syndrome. Results from MR studies, however, should be interpreted with great caution because the underlying history of CAD or CHF were not taken into account in these analyses, an issue that may substantially confound the results. Here, we discuss many aspects of adiponectin; cardiometabolic traits, therapeutic interventions, and the ongoing debate about the adiponectin paradox, which were recently described in basic, epidemiologic, and clinical studies.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 11 Jun 2021; epub ahead of print
Jang AY, Scherer PE, Kim JY, Lim S, Koh KK
Cardiovasc Res: 11 Jun 2021; epub ahead of print | PMID: 34117867
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Abstract

AIM2-driven inflammasome activation in heart failure.

Onódi Z, Ruppert M, Kucsera D, Sayour AA, ... Radovits T, Varga ZV
Aims
Interleukin-1β (IL-1β) is an important pathogenic factor in cardiovascular diseases including chronic heart failure (HF). The CANTOS trial highlighted that inflammasomes as primary sources of IL-1 β are promising new therapeutic targets in cardiovascular diseases. Therefore, we aimed to assess inflammasome activation in failing hearts to identify activation patterns of inflammasome subtypes as sources of IL-1β.
Methods and results
Out of the 4 major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human heart failure regardless of the etiology (ischemic or dilated cardiomyopathy) while the NLRP1/NALP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change in HF samples. AIM2 expression was primarily detected in monocytes/macrophages of failing hearts. Translational animal models of HF (pressure or volume overload, and permanent coronary artery ligation in rat, as well as ischemia/reperfusion-induced HF in pigs) demonstrated activation pattern of AIM2 similar to that of observed in end-stages of human HF. In vitro AIM2 inflammasome activation in human THP-1 monocytic cells and human AC16 cells was significantly reduced by pharmacological blockade of pannexin-1 channels by the clinically used uricosuric drug probenecid. Probenecid was also able to reduce pressure overload-induced mortality and restore indices of disease severity in a rat chronic HF model in vivo.
Conclusions
This is the first report showing that AIM2 and NLRC4 inflammasome activation contribute to chronic inflammation in heart failure and that probenecid alleviates chronic HF by reducing inflammasome activation. The present translational study suggests the possibility of repositioning of probenecid for HF indications.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 11 Jun 2021; epub ahead of print
Onódi Z, Ruppert M, Kucsera D, Sayour AA, ... Radovits T, Varga ZV
Cardiovasc Res: 11 Jun 2021; epub ahead of print | PMID: 34117866
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Impact:
Abstract

Progress in cardiac research - from rebooting cardiac regeneration to a complete cell atlas of the heart.

Davidson SM, Padró T, Bollini S, Vilahur G, ... Wojta J, Weber C
We review some of the important discoveries and advances made in basic and translational cardiac research in 2020. For example, in the field of myocardial infarction (MI), new aspects of autophagy and the importance of eosinophils were described. Novel approaches such as a glycocalyx mimetic were used to improve cardiac recovery following MI. The strategy of 3D bio-printing was shown to allow the fabrication of a chambered cardiac organoid. The benefit of combining tissue engineering with paracrine therapy to heal injured myocardium is discussed. We highlight the importance of cell-to cell communication, in particular the relevance of extracellular vesicles such as exosomes, which transport proteins, lipids, non-coding RNAs and mRNAs and actively contribute to angiogenesis and myocardial regeneration. In this rapidly growing field, new strategies were developed to stimulate the release of reparative exosomes in ischaemic myocardium. Single-cell sequencing technology is causing a revolution in the study of transcriptional expression at cellular resolution, revealing unanticipated heterogeneity within cardiomyocytes, pericytes and fibroblasts, and revealing a unique subpopulation of cardiac fibroblasts. Several studies demonstrated that exosome- and non-coding RNA-mediated approaches can enhance human induced pluripotent stem cell (iPSC) viability and differentiation into mature cardiomyocytes. Important details of the mitochondrial Ca2+ uniporter and its relevance were elucidated. Novel aspects of cancer therapeutic-induced cardiotoxicity were described, such as the novel circular RNA circITCH, which may lead to novel treatments. Finally, we provide some insights into the effects of SARS-CoV-2 on the heart.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 10 Jun 2021; epub ahead of print
Davidson SM, Padró T, Bollini S, Vilahur G, ... Wojta J, Weber C
Cardiovasc Res: 10 Jun 2021; epub ahead of print | PMID: 34114614
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Impact:
Abstract

Single-cell transcriptomic analyses of cardiac immune cells reveal that Rel-driven CD72-positive macrophages induce cardiomyocyte injury.

Ni SH, Xu JD, Sun SN, Li Y, ... Wang LJ, Lu L
Aims
The goal of our study was to investigate the heterogeneity of cardiac macrophages (CMφs) in mice with transverse aortic constriction (TAC) via single-cell sequencing and identify a subset of macrophages associated with heart injury.
Methods and results
We selected all CMφs from CD45+ cells using single-cell mRNA sequencing data. Through dimension reduction, clustering and enrichment analyses, CD72hi CMφs were identified as a subset of proinflammatory macrophages. The pseudotime trajectory and ChIP-Seq analyses identified Rel as the key transcription factor that induces CD72hi CMφ differentiation. Rel KD and Rel-/- bone marrow chimera mice subjected to TAC showed features of mitigated cardiac injury, including decreased levels of cytokines and ROS, which prohibited cardiomyocyte death. The transfer of adoptive Rel-overexpressing monocytes and CD72hi CMφ injection directly aggravated heart injury in the TAC model. The CD72hi macrophages also exerted proinflammatory and cardiac injury effects associated with myocardial infarction (MI). In humans, patients with heart failure exhibit increased CD72hi CMφ levels following dilated cardiomyopathy (DCM) and ischemic cardiomyopathy (ICM).
Conclusion
Bone marrow-derived, Rel-mediated CD72hi macrophages play a proinflammatory role, induce cardiac injury and, thus, may serve as a therapeutic target for multiple cardiovascular diseases.
Translational perspective
Heart failure (HF) imposes an enormous clinical and economic burden worldwide and presents limited therapeutic approaches. Given the close association between inflammation and adverse outcomes, proinflammatory immune cells are considered potential therapeutic targets for HF treatment. The present studies identified a specific macrophage subset associated with myocardial injury, which may provide an alternative approach for treating cardiovascular diseases.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 07 Jun 2021; epub ahead of print
Ni SH, Xu JD, Sun SN, Li Y, ... Wang LJ, Lu L
Cardiovasc Res: 07 Jun 2021; epub ahead of print | PMID: 34100920
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Impact:
Abstract

Non-coding RNAs in cardiac inflammation: key drivers in the pathophysiology of heart failure.

Sansonetti M, De Windt LJ
Heart failure is among the most progressive diseases and a leading cause of morbidity. Despite several advances in cardiovascular therapies, pharmacological treatments are limited to relieve symptoms without curing cardiac injury. Multiple observations point to the involvement of immune cells as key drivers in the pathophysiology of heart failure. In particular, there is a growing recognition that heart failure is related to a prolonged and insufficiently repressed inflammatory response leading to molecular, cellular, and functional cardiac alterations. Over the last decades, non-coding RNAs are recognized as prominent mediators of the cardiac inflammation, affecting the function of several immune cells. In the current review, we explore the contribution of the diverse immune cells in the progression of heart failure, revealing mechanistic functions for non-coding RNAs in cardiac immune cells as a new and exciting field of investigation.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 06 Jun 2021; epub ahead of print
Sansonetti M, De Windt LJ
Cardiovasc Res: 06 Jun 2021; epub ahead of print | PMID: 34097013
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Impact:
Abstract

PGC-1α deficiency reveals sex-specific links between cardiac energy metabolism and EC-coupling during development of heart failure in mice.

Naumenko N, Mutikainen M, Holappa L, Ruas JL, Tuomainen T, Tavi P
Aims
Biological sex has fundamental effects on mammalian heart physiology and pathogenesis. While it has been established that female sex is a protective factor against most cardiovascular diseases (CVDs), this beneficial effect may involve pathways associated with cardiac energy metabolism. Our aim was to elucidate the role of transcriptional coactivator PGC-1α in sex dimorphism of heart failure development.
Methods and results
Here we show that mice deficient in cardiac expression of the peroxisome proliferator-activated receptor gamma (PPAR-γ) coactivator 1α (PGC-1α) develop dilated heart failure associated with changes in aerobic and anaerobic metabolism, calcium handling, cell structure, electrophysiology as well as gene expression. These cardiac changes occur in both sexes, but female mice develop an earlier and more severe structural and functional phenotype associated with dyssynchronous local calcium release resulting from disruption of t-tubular structures of the cardiomyocytes.
Conclusions
These data reveal that the integrity of the subcellular Ca2+ release and uptake machinery is dependent on energy metabolism and that female hearts are more prone to suffer from contractile dysfunction in conditions with compromised production of cellular energy. Furthermore, these findings suggest that PGC-1α is a central mediator of sex-specific differences in heart function and CVD susceptibility.
Translational perspective
Biological sex is an important variable in clinical medicine, cardiac physiology, and pathogenesis. However, sex-specific clinical practices or therapies are emerging slowly in the absence of deeper understanding of the specific mechanism behind sex dimorphism in cardiac disease progression. Here, we show that energy metabolism has a central role in sex dimorphism of heart failure progression and that a signalling cascade involving PGC-1α might have a role in it. We provide insights into sex specific mechanisms of heart failure development which are necessary to identify sex specific treatment practices for cardiovascular diseases.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 03 Jun 2021; epub ahead of print
Naumenko N, Mutikainen M, Holappa L, Ruas JL, Tuomainen T, Tavi P
Cardiovasc Res: 03 Jun 2021; epub ahead of print | PMID: 34086875
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Impact:
Abstract

Different cardiovascular and pulmonary phenotypes for single- and double-knock-out mice deficient in BMP9 and BMP10.

Bouvard C, Tu L, Rossi M, Desroches-Castan A, ... Guignabert C, Bailly S
Aims
BMP9 and BMP10 mutations were recently identified in patients with pulmonary arterial hypertension (PAH), but their specific roles in the pathogenesis of the disease are still unclear. We aimed to study the roles of BMP9 and BMP10 in cardiovascular homeostasis and pulmonary hypertension using transgenic mouse models deficient in Bmp9 and/or Bmp10.
Methods and results
Single- and double-knockout mice for Bmp9 (constitutive) and/or Bmp10 (tamoxifen inducible) were generated. Single-KO mice developed no obvious age-dependent phenotype when compared with their wild-type littermates. However, combined deficiency in Bmp9 and Bmp10 led to vascular defects resulting in a decrease in peripheral vascular resistance and blood pressure and the progressive development of high-output heart failure (HOHF) and pulmonary hemosiderosis. RNAseq analysis of the lungs of the double-KO mice revealed differential expression of genes involved in inflammation and vascular homeostasis. We next challenged these mice to chronic hypoxia. After three weeks of hypoxic exposure, Bmp10-cKO mice showed an enlarged heart. However, although genetic deletion of Bmp9 in the single and double-KO mice attenuated the muscularization of pulmonary arterioles induced by chronic hypoxia, we observed no differences in Bmp10-cKO mice. Consistent with these results, endothelin-1 levels were significantly reduced in Bmp9 deficient mice but not Bmp10-cKO mice. Furthermore, the effects of BMP9 on vasoconstriction were inhibited by bosentan, an endothelin receptor antagonist, in a chick chorioallantoic membrane assay.
Conclusions
Our data show redundant roles for BMP9 and BMP10 in cardiovascular homeostasis under normoxic conditions (only combined deletion of both Bmp9 and Bmp10 was associated with severe defects) but highlight specific roles under chronic hypoxic conditions. We obtained evidence that BMP9 contributes to chronic hypoxia-induced pulmonary vascular remodeling, whereas BMP10 plays a role in hypoxia-induced cardiac remodeling in mice.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 03 Jun 2021; epub ahead of print
Bouvard C, Tu L, Rossi M, Desroches-Castan A, ... Guignabert C, Bailly S
Cardiovasc Res: 03 Jun 2021; epub ahead of print | PMID: 34086873
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Impact:
Abstract

Pharmacological inhibition of adipose tissue Adipose Triglyceride Lipase (ATGL) by Atglistatin prevents catecholamine-induced myocardial damage.

Thiele A, Luettges K, Ritter D, Beyhoff N, ... Kintscher U, Foryst-Ludwig A
Aims
Heart failure (HF) is characterized by an overactivation of β-adrenergic signaling that directly contributes to impairment of myocardial function. Moreover, β-adrenergic overactivation induces adipose tissue lipolysis, which may further worsen the development of HF. Recently we demonstrated that adipose tissue-specific deletion of adipose triglyceride lipase (ATGL) prevents pressure-mediated HF in mice. In this study, we investigated the cardioprotective effects of a new pharmacological inhibitor of ATGL, Atglistatin, predominantly targeting ATGL in adipose tissue, on catecholamine-induced cardiac damage.
Methods and results
Male 129/Sv mice received repeated injections of isoproterenol (ISO, 25 mg/kg BW) to induce cardiac damage. Five days prior to ISO application, oral Atglistatin (2 mmol/kg diet) or control treatment was started. Two and twelve days after the last ISO injection cardiac function was analyzed by echocardiography. The myocardial deformation was evaluated using speckle-tracking-technique. Twelve days after the last ISO injection, echocardiographic analysis revealed a markedly impaired global longitudinal strain, which was significantly improved by application of Atglistatin. No changes of ejection fraction were observed. Further studies included histological-, WB-, and RT-qPCR-based analysis of cardiac tissue, followed by cell culture experiments and mass spectrometry-based lipidome analysis. ISO application induced subendocardial fibrosis and a profound pro-apoptotic cardiac response, as demonstrated using an apoptosis-specific gene expression-array. Atglistatin treatment led to a dramatic reduction of these pro-fibrotic and pro-apoptotic processes. We then identified a specific set of fatty acids (FAs) liberated from adipocytes under ISO stimulation (palmitic acid, palmitoleic acid and oleic acid), which induced pro-apoptotic effects in cardiomyocytes. Atglistatin significantly blocked this adipocytic FA secretion.
Conclusions
The present study demonstrates cardioprotective effects of Atglistatin in a mouse model of catecholamine-induced cardiac damage/dysfunction, involving anti-apoptotic and anti-fibrotic actions. Notably, beneficial cardioprotective effects of Atglistatin are likely mediated by non-cardiac actions, supporting the concept that pharmacological targeting of adipose tissue may provide an effective way to treat cardiac dysfunction.
Translational perspective
The pharmacological inhibition of ATGL activity in adipose tissue improves heart function in a murine model of catecholamine-induced myocardial damage, via significant reduction of cardiac apoptosis and fibrosis. Our data strongly support the role of an adipose tissue-heart communication in the development of cardiac diseases, associated with increased sympathetic-tone. Atglistatin beneficial actions were only mild, when applied after the catecholamine-induced damage in a therapeutic manner. However, when given prior to the event in a preventive manner, Atglistatin strongly protected against cardiac damage. These data suggest that an Atglistatin-based therapy may be more suitable as a new pharmacological option in cardiovascular prevention.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 31 May 2021; epub ahead of print
Thiele A, Luettges K, Ritter D, Beyhoff N, ... Kintscher U, Foryst-Ludwig A
Cardiovasc Res: 31 May 2021; epub ahead of print | PMID: 34061169
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Impact:
Abstract

Omentin attenuates angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein-E knockout mice.

Fang L, Ohashi K, Otaka N, Ogawa H, ... Murohara T, Ouchi N
Aims
Abdominal aortic aneurysm (AAA) is an increasing and life-threatening disease. Obesity contributes to an increased risk of AAA. Omentin is a circulating adipokine, which is downregulated in obese complications. Here we examined whether omentin could modulate angiotensin (Ang) II-induced AAA formation in apolipoprotein-E knockout (apoE-KO) mice.
Methods and results
ApoE-KO mice were crossed with transgenic mice expressing the human omentin gene in fat tissue (OMT-Tg mice) to generate ApoE-KO/OMT-Tg mice. ApoE-KO/OMT-Tg and apoE-KO mice were subjected to continuous Ang II infusion by using osmotic mini pumps. ApoE-KO/OMT-Tg mice exhibited a lower incidence of AAA formation and a reduced maximal diameter of AAA compared with apo-E KO mice. ApoE-KO/OMT-Tg mice showed attenuated disruption of medial elastic fibers in response to Ang II compared with apo-E KO mice. ApoE-KO/OMT-Tg mice also displayed reduced expression levels of matrix metalloproteinase (MMP) 9, MMP2 and pro-inflammatory genes in aortic walls compared with apo-E KO mice. Furthermore, systemic administration of omentin also attenuated AAA formation and disruption of medial elastic fibers in response to Ang II in apoE-KO mice. Treatment of human monocyte-derived macrophages with omentin protein attenuated expression of MMP9 and pro-inflammatory mediators, and MMP9 activation after stimulation with lipopolysaccharide (LPS). Treatment of human vascular smooth muscle cells with omentin protein reduced expression and activation of MMP2 after stimulation with tumor necrosis factor α. Omentin treatment increased phosphorylation levels of Akt in human macrophages and vascular smooth muscle cells. The suppressive effects of omentin on MMP9 and MMP2 expression were reversed by inhibition of integrin-αVβ3/PI3-kinase/Akt signaling in macrophages and vascular smooth muscle cells, respectively.
Conclusion
These data suggest that omentin acts as an adipokine that can attenuate Ang II-induced development of AAA through suppression of MMP9 and MMP2 expression and inflammatory response in the vascular wall.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 28 May 2021; epub ahead of print
Fang L, Ohashi K, Otaka N, Ogawa H, ... Murohara T, Ouchi N
Cardiovasc Res: 28 May 2021; epub ahead of print | PMID: 34051089
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Impact:
Abstract

The healing myocardium mobilises a distinct B-cell subset through a CXCL13-CXCR5-dependent mechanism.

Heinrichs M, Ashour D, Siegel J, Büchner L, ... Frantz S, Ramos GC
Aims
Recent studies have revealed that B-cells and antibodies can influence inflammation and remodelling following a myocardial infarction (MI) and culminating in heart failure-but the mechanisms underlying these observations remain elusive. We therefore conducted in mice a deep phenotyping of the post-MI B-cell responses in infarcted hearts and mediastinal lymph nodes, which drain the myocardium. Thereby, we sought to dissect the mechanisms controlling B-cell mobilisation and activity in situ.
Methods and results
Histological, flow cytometry and single-cell RNA-sequencing (scRNA-seq) analyses revealed a rapid accumulation of diverse B-cell subsets in infarcted murine hearts, paralleled by mild clonal expansion of germinal centre B-cells in the mediastinal lymph nodes. The repertoire of cardiac B-cells was largely polyclonal and showed no sign of antigen-driven clonal expansion. Instead, it included a distinct subset exclusively found in the heart, herein termed \"heart-associated B-cells\" (hB) that expressed high levels of Cd69 as an activation marker, C-C-chemokine receptor type 7 (Ccr7), CXC-chemokine receptor type 5 (Cxcr5) and transforming growth factor beta 1 (Tgfb1). This distinct signature was not shared with any other cell population in the healing myocardium. Moreover, we detected a myocardial gradient of CXC-motif chemokine ligand 13 (CXCL13, the ligand of CXCR5) on days 1 and 5 post-MI. When compared to wild type controls, mice treated with a neutralising CXCL13-specific antibody as well as CXCR5-deficient mice showed reduced post-MI infiltration of B-cells and reduced local Tgfb1 expression but no differences in contractile function nor myocardial morphology were observed between groups.
Conclusions
Our study reveals that polyclonal B-cells showing no sign of antigen-specificity readily infiltrate the heart after MI via the CXCL13-CXCR5 axis and contribute to local TGF-ß1 production. The local B-cell responses are paralleled by mild antigen-driven germinal centre reactions in the mediastinal lymph nodes that might ultimately lead to the production of specific antibodies.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 27 May 2021; epub ahead of print
Heinrichs M, Ashour D, Siegel J, Büchner L, ... Frantz S, Ramos GC
Cardiovasc Res: 27 May 2021; epub ahead of print | PMID: 34048536
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Impact:
Abstract

Vascular histopathology and connective tissue ultrastructure in spontaneous coronary artery dissection: pathophysiological and clinical implications.

Margaritis M, Saini F, Baranowska-Clarke AA, Parsons S, ... Sheppard MN, Adlam D
Aims
Spontaneous coronary artery dissection (SCAD) is a cause of acute coronary syndromes and in rare cases sudden cardiac death (SCD). Connective tissue abnormalities, coronary inflammation, increased coronary vasa vasorum density and coronary fibromuscular dysplasia have all been implicated in the pathophysiology of SCAD but have not previously been systematically assessed. We designed a study to investigate the coronary histological and dermal collagen ultrastructural findings in SCAD.
Methods and results
36 autopsy SCAD cases were compared with 359 SCAD survivors. Coronary and myocardial histology and immunohistochemistry were undertaken. Transmission electron microscopy (TEM) of dermal extracellular matrix components (ECM) of n = 31 SCAD survivors and n = 16 healthy volunteers were compared. Autopsy cases were more likely male (19% versus 5%; p = 0.0004) with greater proximal left coronary involvement (56% versus 18%; p < 0.0001) compared to SCAD survivors. N = 24 (66%) of cases showed no myocardial infarction on macro- or microscopic examination consistent with arrhythmogenic death. There was significantly (p < 0.001) higher inflammation in cases with delayed-onset death vs sudden death and significantly more inflammation surrounding the dissected vs. non-dissected vessel segments. N = 17 (47%) cases showed limited intimal fibro-elastic thickening but no features of fibromuscular dysplasia and no endothelial or internal elastic lamina abnormalities. There were no differences in vasa vasorum density between SCAD and control cases. TEM revealed no general ultrastructural differences in ECM components or markers of fibroblast metabolic activity.
Conclusions
Assessment of SCD requires careful exclusion of SCAD, particularly in cases without myocardial necrosis. Peri-coronary inflammation in SCAD is distinct from vasculitides and likely a reaction to, rather than a cause for SCAD. Coronary fibromuscular dysplasia or increased vasa vasorum density do not appear pathophysiologically important. Dermal connective tissue changes are not common in SCAD survivors.
Translational perspective
SCAD, especially of distal coronary territories should be carefully assessed at post mortem in SCD cases, even where there are no signs of myocardial infarction. The immediate cause of SCAD is likely to be the development of a spontaneous intramural haematoma rather than an intimal disruption or \'tear\'. This does not seem to be directly related to increased vasa vasorum density, coronary fibromuscular dysplasia or local inflammation (except as a response to injury). Although SCAD is rarely associated with hereditary connective tissue disorders, there does not seem to be a more generalizable global connective tissue ultrastructural abnormality in most cases.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 27 May 2021; epub ahead of print
Margaritis M, Saini F, Baranowska-Clarke AA, Parsons S, ... Sheppard MN, Adlam D
Cardiovasc Res: 27 May 2021; epub ahead of print | PMID: 34048532
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Abstract

The glycocalyx core protein Glypican 1 protects vessel wall endothelial cells from stiffness-mediated dysfunction and disease.

Mahmoud M, Mayer M, Cancel LM, Bartosch AM, Mathews R, Tarbell JM
Aims
Arterial stiffness is an underlying risk factor and a hallmark of cardiovascular diseases. The endothelial cell (EC) glycocalyx is a glycan rich surface layer that plays a key role in protecting against EC dysfunction and vascular disease. However, the mechanisms by which arterial stiffness promotes EC dysfunction and vascular disease are not fully understood, and whether the mechanism involves the protective endothelial glycocalyx is yet to be determined. We hypothesized that endothelial glycocalyx protects the endothelial cells lining the vascular wall from dysfunction and disease in response to arterial stiffness.
Methods and results
Cells cultured on polyacrylamide (PA) gels of substrate stiffness 10 kPa (mimicking the subendothelial stiffness of aged, unhealthy arteries) showed a significant inhibition of glycocalyx expression compared to cells cultured on softer PA gels (2.5 kPa, mimicking the subendothelial stiffness of young, healthy arteries). Specifically, gene and protein analyses revealed that a glycocalyx core protein Glypican 1 was inhibited in cells cultured on stiff PA gels. These cells had enhanced endothelial cell dysfunction as determined by enhanced cell inflammation (enhanced inflammatory gene expression, monocyte adhesion, and inhibited nitric oxide expression), proliferation, and EndMT. Removal of Glypican 1 using gene-specific silencing with siRNA or gene overexpression using a plasmid revealed that Glypican 1 is required to protect against stiffness-mediated endothelial cell dysfunction. Consistent with this, using a model of age-mediated stiffness, older mice exhibited a reduced expression of Glypican 1 and enhanced endothelial cell dysfunction compared to young mice. Glypican 1 gene deletion in knockout mice (GPC1-/-) exacerbated endothelial dysfunction in young mice, which normally had high endothelial expression, but not in old mice that normally expressed low levels. Endothelial cell dysfunction was exacerbated in young, but not aged, Glypican 1 knockout mice (GPC1-/-).
Conclusion
Arterial stiffness promotes EC dysfunction and vascular disease at least partly through the suppression of the glycocalyx protein Glypican 1. Glypican 1 contributes to the protection against endothelial cell dysfunction and vascular disease in endothelial cells.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 24 May 2021; 117:1592-1605
Mahmoud M, Mayer M, Cancel LM, Bartosch AM, Mathews R, Tarbell JM
Cardiovasc Res: 24 May 2021; 117:1592-1605 | PMID: 32647868
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Impact:
Abstract

VE-PTP inhibition elicits eNOS phosphorylation to blunt endothelial dysfunction and hypertension in diabetes.

Siragusa M, Oliveira Justo AF, Malacarne PF, Strano A, ... Peters KG, Fleming I
Aims
Receptor-type vascular endothelial protein tyrosine phosphatase (VE-PTP) dephosphorylates Tie-2 as well as CD31, VE-cadherin, and vascular endothelial growth factor receptor 2 (VEGFR2). The latter form a signal transduction complex that mediates the endothelial cell response to shear stress, including the activation of the endothelial nitric oxide (NO) synthase (eNOS). As VE-PTP expression is increased in diabetes, we investigated the consequences of VE-PTP inhibition (using AKB-9778) on blood pressure in diabetic patients and the role of VE-PTP in the regulation of eNOS activity and vascular reactivity.
Methods and results
In diabetic patients AKB-9778 significantly lowered systolic and diastolic blood pressure. This could be linked to elevated NO production, as AKB increased NO generation by cultured endothelial cells and elicited the NOS inhibitor-sensitive relaxation of endothelium-intact rings of mouse aorta. At the molecular level, VE-PTP inhibition increased the phosphorylation of eNOS on Tyr81 and Ser1177 (human sequence). The PIEZO1 activator Yoda1, which was used to mimic the response to shear stress, also increased eNOS Tyr81 phosphorylation, an effect that was enhanced by VE-PTP inhibition. Two kinases, i.e. abelson-tyrosine protein kinase (ABL)1 and Src were identified as eNOS Tyr81 kinases as their inhibition and down-regulation significantly reduced the basal and Yoda1-induced tyrosine phosphorylation and activity of eNOS. VE-PTP, on the other hand, formed a complex with eNOS in endothelial cells and directly dephosphorylated eNOS Tyr81 in vitro. Finally, phosphorylation of eNOS on Tyr80 (murine sequence) was found to be reduced in diabetic mice and diabetes-induced endothelial dysfunction (isolated aortic rings) was blunted by VE-PTP inhibition.
Conclusions
VE-PTP inhibition enhances eNOS activity to improve endothelial function and decrease blood pressure indirectly, through the activation of Tie-2 and the CD31/VE-cadherin/VEGFR2 complex, and directly by dephosphorylating eNOS Tyr81. VE-PTP inhibition, therefore, represents an attractive novel therapeutic option for diabetes-induced endothelial dysfunction and hypertension.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 24 May 2021; 117:1546-1556
Siragusa M, Oliveira Justo AF, Malacarne PF, Strano A, ... Peters KG, Fleming I
Cardiovasc Res: 24 May 2021; 117:1546-1556 | PMID: 32653904
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Impact:
Abstract

Angiopoietin-1 enhanced myocyte mitosis, engraftment, and the reparability of hiPSC-CMs for treatment of myocardial infarction.

Tao Z, Loo S, Su L, Tan S, ... Chen X, Ye L
Aims
To examine whether transient over-expression of angiopoietin-1 (Ang-1) increases the potency of hiPSC-CMs for treatment of heart failure.
Methods and results
Atrial hiPSC-CMs (hiPSC-aCMs) were differentiated from hiPSCs and purified by lactic acid and were transfected with Ang-1 (Ang-1-hiPSC-aCMs) plasmid using lipoSTEM. Ang-1 gene transfection efficiency was characterized in vitro. Gene transfected CMs (1×106) were seeded into a fibrin/thrombin patch and implanted on the rat-infarcted left ventricular (LV) anterior wall after myocardial infarction (MI). Echo function was determined at 1- and 6 weeks post-MI. Immunohistochemistry study was performed at 6 weeks post-MI. Ang-1 (20 and 40 ng/mL) protected hiPSC-aCMs from hypoxia through up-regulating pERK1/2 and inhibiting Bax protein expressions. Ang-1-hiPSC-aCMs transiently secreted Ang-1 protein up to 14 days, with peak level on day-2 post-transfection (24.39 ± 13.02 ng/mL) in vitro. Animal study showed that transplantation of Ang-1-hiPSC-aCM seeded patch more effectively limited rat heart apoptosis at 1 day post-MI as compared with LipoSTEM-Ang-1 or hiPSC-aCMs transplantation. Ang-1-hiPSC-aCMs transplantation induced host (rat) and donor (human) CM mitosis and arteriole formation, improved cell engraftment rate, more effectively limited LV dilation (EDV = 460.7 ± 96.1 μL and ESV = 219.8 ± 72.9 μL) and improved LV global pump function (EF = 53.1 ± 9%) as compared with the MI (EDV = 570.9 ± 91.8 μL, P = 0.033; ESV = 331.6 ± 71.2 μL, P = 0.011; EF = 42.3 ± 4.1%, P = 0.02) or the LipoSTEM-Ang-1 injected (EDV = 491.4 ± 100.4 μL, P = 0.854; ESV = 280.9 ± 71.5 μL, P = 0.287; EF = 43.2 ± 4.6, P = 0.039) or hiPSC-CM transplanted (EDV = 547.9 ± 55.5 μL, P = 0.095; ESV = 300.2 ± 88.4 μL, P = 0.075; EF = 46 ± 10.9%, P = 0.166) animal groups at 6 weeks post-MI and treatment.
Conclusion
Transient over-expression of Ang-1 enhanced hiPSC-aCM mitosis and engraftment and increased the reparability potency of hiPSC-aCMs for treatment of MI.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 24 May 2021; 117:1578-1591
Tao Z, Loo S, Su L, Tan S, ... Chen X, Ye L
Cardiovasc Res: 24 May 2021; 117:1578-1591 | PMID: 32666104
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Impact:
Abstract

A novel method for measuring absolute coronary blood flow and microvascular resistance in patients with ischaemic heart disease.

Morris PD, Gosling R, Zwierzak I, Evans H, ... Narracott AJ, Gunn JP
Aims
Ischaemic heart disease is the reduction of myocardial blood flow, caused by epicardial and/or microvascular disease. Both are common and prognostically important conditions, with distinct guideline-indicated management. Fractional flow reserve (FFR) is the current gold-standard assessment of epicardial coronary disease but is only a surrogate of flow and only predicts percentage flow changes. It cannot assess absolute (volumetric) flow or microvascular disease. The aim of this study was to develop and validate a novel method that predicts absolute coronary blood flow and microvascular resistance (MVR) in the catheter laboratory.
Methods and results
A computational fluid dynamics (CFD) model was used to predict absolute coronary flow (QCFD) and coronary MVR using data from routine invasive angiography and pressure-wire assessment. QCFD was validated in an in vitro flow circuit which incorporated patient-specific, three-dimensional printed coronary arteries; and then in vivo, in patients with coronary disease. In vitro, QCFD agreed closely with the experimental flow over all flow rates [bias +2.08 mL/min; 95% confidence interval (error range) -4.7 to +8.8 mL/min; R2 = 0.999, P < 0.001; variability coefficient <1%]. In vivo, QCFD and MVR were successfully computed in all 40 patients under baseline and hyperaemic conditions, from which coronary flow reserve (CFR) was also calculated. QCFD-derived CFR correlated closely with pressure-derived CFR (R2 = 0.92, P < 0.001). This novel method was significantly more accurate than Doppler-wire-derived flow both in vitro (±6.7 vs. ±34 mL/min) and in vivo (±0.9 vs. ±24.4 mmHg).
Conclusions
Absolute coronary flow and MVR can be determined alongside FFR, in absolute units, during routine catheter laboratory assessment, without the need for additional catheters, wires or drug infusions. Using this novel method, epicardial and microvascular disease can be discriminated and quantified. This comprehensive coronary physiological assessment may enable a new level of patient stratification and management.

© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 24 May 2021; 117:1567-1577
Morris PD, Gosling R, Zwierzak I, Evans H, ... Narracott AJ, Gunn JP
Cardiovasc Res: 24 May 2021; 117:1567-1577 | PMID: 32666101
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Impact:
Abstract

Gene expression profiling of hypertrophic cardiomyocytes identifies new players in pathological remodelling.

Vigil-Garcia M, Demkes CJ, Eding JEC, Versteeg D, ... Boogerd CJ, van Rooij E
Aims
Pathological cardiac remodelling is characterized by cardiomyocyte (CM) hypertrophy and fibroblast activation, which can ultimately lead to maladaptive hypertrophy and heart failure (HF). Genome-wide expression analysis on heart tissue has been instrumental for the identification of molecular mechanisms at play. However, these data were based on signals derived from all cardiac cell types. Here, we aimed for a more detailed view on molecular changes driving maladaptive CM hypertrophy to aid in the development of therapies to reverse pathological remodelling.
Methods and results
Utilizing CM-specific reporter mice exposed to pressure overload by transverse aortic banding and CM isolation by flow cytometry, we obtained gene expression profiles of hypertrophic CMs in the more immediate phase after stress, and CMs showing pathological hypertrophy. We identified subsets of genes differentially regulated and specific for either stage. Among the genes specifically up-regulated in the CMs during the maladaptive phase we found known stress markers, such as Nppb and Myh7, but additionally identified a set of genes with unknown roles in pathological hypertrophy, including the platelet isoform of phosphofructokinase (PFKP). Norepinephrine-angiotensin II treatment of cultured human CMs induced the secretion of N-terminal-pro-B-type natriuretic peptide (NT-pro-BNP) and recapitulated the up-regulation of these genes, indicating conservation of the up-regulation in failing CMs. Moreover, several genes induced during pathological hypertrophy were also found to be increased in human HF, with their expression positively correlating to the known stress markers NPPB and MYH7. Mechanistically, suppression of Pfkp in primary CMs attenuated stress-induced gene expression and hypertrophy, indicating that Pfkp is an important novel player in pathological remodelling of CMs.
Conclusion
Using CM-specific transcriptomic analysis, we identified novel genes induced during pathological hypertrophy that are relevant for human HF, and we show that PFKP is a conserved failure-induced gene that can modulate the CM stress response.

© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 24 May 2021; 117:1532-1545
Vigil-Garcia M, Demkes CJ, Eding JEC, Versteeg D, ... Boogerd CJ, van Rooij E
Cardiovasc Res: 24 May 2021; 117:1532-1545 | PMID: 32717063
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Impact:
Abstract

Human CD16+ monocytes promote a pro-atherosclerotic endothelial cell phenotype via CX3CR1-CX3CL1 interaction.

Roy-Chowdhury E, Brauns N, Helmke A, Nordlohne J, ... Haller H, von Vietinghoff S
Aims
Monocytes are central for atherosclerotic vascular inflammation. The human non-classical, patrolling subtype, which expresses high levels of CD16 and fractalkine receptor CX3CR1, strongly associates with cardiovascular events. This is most marked in renal failure, a condition with excess atherosclerosis morbidity. The underlying mechanism is not understood. This study investigated how human CD16+ monocytes modulate endothelial cell function.
Methods and results
In patients with kidney failure, CD16+ monocyte counts were elevated and dynamically decreased within a year after transplantation, chiefly due to a drop in CD14+CD16+ cells. The CX3CR1 ligand CX3CL1 was similarly elevated in the circulation of humans and mice with renal impairment. CX3CL1 up-regulation was also observed close to macrophage rich human coronary artery plaques. To investigate a mechanistic basis of this association, CD16+CX3CR1HIGH monocytes were co-incubated with primary human endothelium in vitro. Compared to classical CD14+ monocytes or transwell cocultures, CD16+ monocytes enhanced endothelial STAT1 and NF-κB p65 phosphorylation, up-regulated expression of CX3CL1 and interleukin-1β, numerous CCL and CXCL chemokines and molecules promoting leucocyte patrolling and adhesion such as ICAM1 and VCAM1. Genes required for vasodilatation including endothelial nitric oxide synthase decreased while endothelial collagen production increased. Uraemic patients\' monocytes enhanced endothelial CX3CL1 even more markedly. Their receptor CX3CR1 was required for enhanced aortic endothelial stiffness in murine atherosclerosis with renal impairment. CX3CR1 dose-dependently modulated monocyte-contact-dependent gene expression in human endothelium.
Conclusion
By demonstrating endothelial proatherosclerotic gene regulation in direct contact with CD16+ monocytes, in part via cellular CX3CR1-CX3CL1 interaction, our data delineate a mechanism how this celltype can increase cardiovascular risk.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 24 May 2021; 117:1510-1522
Roy-Chowdhury E, Brauns N, Helmke A, Nordlohne J, ... Haller H, von Vietinghoff S
Cardiovasc Res: 24 May 2021; 117:1510-1522 | PMID: 32717023
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Impact:
Abstract

Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis.

Singla B, Lin HP, Chen A, Ahn W, ... Stansfield BK, Csányi G
Aims
Impaired lymphatic drainage of the arterial wall results in intimal lipid accumulation and atherosclerosis. However, the mechanisms regulating lymphangiogenesis in atherosclerotic arteries are not well understood. Our studies identified elevated levels of matrix protein R-spondin 2 (RSPO2) in atherosclerotic arteries. In this study, we investigated the role of RSPO2 in lymphangiogenesis, arterial cholesterol efflux into lesion-draining lymph nodes (LNs) and development of atherosclerosis.
Methods and results
The effect of RSPO2 on lymphangiogenesis was investigated using human lymphatic endothelial cells (LEC) in vitro and implanted Matrigel plugs in vivo. Cellular and molecular approaches, pharmacological agents, and siRNA silencing of RSPO2 receptor LGR4 were used to investigate RSPO2-mediated signalling in LEC. In vivo low-density lipoprotein (LDL) tracking and perivascular blockade of RSPO2-LGR4 signalling using LGR4-extracellular domain (ECD) pluronic gel in hypercholesterolemic mice were utilized to investigate the role of RSPO2 in arterial reverse cholesterol transport and atherosclerosis. Immunoblotting and imaging experiments demonstrated increased RSPO2 expression in human and mouse atherosclerotic arteries compared to non-atherosclerotic controls. RSPO2 treatment inhibited lymphangiogenesis both in vitro and in vivo. LGR4 silencing and inhibition of RSPO2-LGR4 signalling abrogated RSPO2-induced inhibition of lymphangiogenesis. Mechanistically, we found that RSPO2 suppresses PI3K-AKT-endothelial nitric oxide synthase (eNOS) signalling via LGR4 and inhibits activation of the canonical Wnt-β-catenin pathway. ApoE-/- mice treated with LGR4-ECD developed significantly less atherosclerosis compared with control treatment. Finally, increased arterial lymphatic vessel density and improved lymphatic drainage of fluorescently labelled LDL to deep cervical LNs were observed in LGR4-ECD-treated mice.
Conclusion
These findings demonstrate that RSPO2 inhibits lymphangiogenesis via LGR4 and downstream impairment of AKT-eNOS-nitric oxide signalling. These results may also inform new therapeutic strategies to promote lymphangiogenesis and improve cholesterol efflux from atherosclerotic arteries.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 24 May 2021; 117:1489-1509
Singla B, Lin HP, Chen A, Ahn W, ... Stansfield BK, Csányi G
Cardiovasc Res: 24 May 2021; 117:1489-1509 | PMID: 32750106
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Impact:
Abstract

Global variations in the prevalence, treatment, and impact of atrial fibrillation in a multi-national cohort of 153 152 middle-aged individuals.

Joseph PG, Healey JS, Raina P, Connolly SJ, ... Yusuf S, PURE Investigators
Aims
To compare the prevalence of electrocardiogram (ECG)-documented atrial fibrillation (or flutter) (AF) across eight regions of the world, and to examine antithrombotic use and clinical outcomes.
Methods and results
Baseline ECGs were collected in 153 152 middle-aged participants (ages 35-70 years) to document AF in two community-based studies, spanning 20 countries. Medication use and clinical outcome data (mean follow-up of 7.4 years) were available in one cohort. Cross-sectional analyses were performed to document the prevalence of AF and medication use, and associations between AF and clinical events were examined prospectively. Mean age of participants was 52.1 years, and 57.7% were female. Age and sex-standardized prevalence of AF varied 12-fold between regions; with the highest in North America, Europe, China, and Southeast Asia (270-360 cases per 100 000 persons); and lowest in the Middle East, Africa, and South Asia (30-60 cases per 100 000 persons) (P < 0.001). Compared with low-income countries (LICs), AF prevalence was 7-fold higher in middle-income countries (MICs) and 11-fold higher in high-income countries (HICs) (P < 0.001). Differences in AF prevalence remained significant after adjusting for traditional AF risk factors. In LICs/MICs, 24% of participants with AF and a CHADS2 score ≥1 received antithrombotic therapy, compared with 85% in HICs. AF was associated with an increased risk of stroke [hazard ratio (HR) 2.29; 95% confidence interval (CI) 1.49-3.52] and death (HR 2.97; 95% CI 2.25-3.93); with similar rates in different countries grouped by income level.
Conclusions
Large variations in AF prevalence occur in different regions and countries grouped by income level, but this is only partially explained by traditional AF risk factors. Antithrombotic therapy is infrequently used in poorer countries despite the high risk of stroke associated with AF.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 24 May 2021; 117:1523-1531
Joseph PG, Healey JS, Raina P, Connolly SJ, ... Yusuf S, PURE Investigators
Cardiovasc Res: 24 May 2021; 117:1523-1531 | PMID: 32777820
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Abstract

Caloric restriction mimetics for the treatment of cardiovascular diseases.

Sciarretta S, Forte M, Castoldi F, Frati G, ... Kroemer G, Maiuri MC
Caloric restriction mimetics (CRMs) are emerging as potential therapeutic agents for the treatment of cardiovascular diseases. CRMs include natural and synthetic compounds able to inhibit protein acetyltransferases, to interfere with acetyl coenzyme A biosynthesis, or to activate (de)acetyltransferase proteins. These modifications mimic the effects of caloric restriction, which is associated with the activation of autophagy. Previous evidence demonstrated the ability of CRMs to ameliorate cardiac function and reduce cardiac hypertrophy and maladaptive remodelling in animal models of ageing, mechanical overload, chronic myocardial ischaemia, and in genetic and metabolic cardiomyopathies. In addition, CRMs were found to reduce acute ischaemia-reperfusion injury. In many cases, these beneficial effects of CRMs appeared to be mediated by autophagy activation. In the present review, we discuss the relevant literature about the role of different CRMs in animal models of cardiac diseases, emphasizing the molecular mechanisms underlying the beneficial effects of these compounds and their potential future clinical application.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 24 May 2021; 117:1434-1449
Sciarretta S, Forte M, Castoldi F, Frati G, ... Kroemer G, Maiuri MC
Cardiovasc Res: 24 May 2021; 117:1434-1449 | PMID: 33098415
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Abstract

Cardiac fibrosis.

Frangogiannis NG
Myocardial fibrosis, the expansion of the cardiac interstitium through deposition of extracellular matrix proteins, is a common pathophysiologic companion of many different myocardial conditions. Fibrosis may reflect activation of reparative or maladaptive processes. Activated fibroblasts and myofibroblasts are the central cellular effectors in cardiac fibrosis, serving as the main source of matrix proteins. Immune cells, vascular cells and cardiomyocytes may also acquire a fibrogenic phenotype under conditions of stress, activating fibroblast populations. Fibrogenic growth factors (such as transforming growth factor-β and platelet-derived growth factors), cytokines [including tumour necrosis factor-α, interleukin (IL)-1, IL-6, IL-10, and IL-4], and neurohumoral pathways trigger fibrogenic signalling cascades through binding to surface receptors, and activation of downstream signalling cascades. In addition, matricellular macromolecules are deposited in the remodelling myocardium and regulate matrix assembly, while modulating signal transduction cascades and protease or growth factor activity. Cardiac fibroblasts can also sense mechanical stress through mechanosensitive receptors, ion channels and integrins, activating intracellular fibrogenic cascades that contribute to fibrosis in response to pressure overload. Although subpopulations of fibroblast-like cells may exert important protective actions in both reparative and interstitial/perivascular fibrosis, ultimately fibrotic changes perturb systolic and diastolic function, and may play an important role in the pathogenesis of arrhythmias. This review article discusses the molecular mechanisms involved in the pathogenesis of cardiac fibrosis in various myocardial diseases, including myocardial infarction, heart failure with reduced or preserved ejection fraction, genetic cardiomyopathies, and diabetic heart disease. Development of fibrosis-targeting therapies for patients with myocardial diseases will require not only understanding of the functional pluralism of cardiac fibroblasts and dissection of the molecular basis for fibrotic remodelling, but also appreciation of the pathophysiologic heterogeneity of fibrosis-associated myocardial disease.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 24 May 2021; 117:1450-1488
Frangogiannis NG
Cardiovasc Res: 24 May 2021; 117:1450-1488 | PMID: 33135058
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Abstract

Insights into therapeutic products, preclinical research models, and clinical trials in cardiac regenerative and reparative medicine: where are we now and the way ahead. Current opinion paper of the ESC Working Group on Cardiovascular Regenerative and Reparative Medicine.

Grigorian-Shamagian L, Sanz-Ruiz R, Climent A, Badimon L, ... Zimmermann WH, Fernández-Avilés F
Great expectations have been set around the clinical potential of regenerative and reparative medicine in the treatment of cardiovascular diseases [i.e. in particular, heart failure (HF)]. Initial excitement, spurred by encouraging preclinical data, resulted in a rapid translation into clinical research. The sobering outcome of the resulting clinical trials suggests that preclinical testing may have been insufficient to predict clinical outcome. A number of barriers for clinical translation include the inherent variability of the biological products and difficulties to develop potency and quality assays, insufficient rigour of the preclinical research and reproducibility of the results, manufacturing challenges, and scientific irregularities reported in the last years. The failure to achieve clinical success led to an increased scrutiny and scepticism as to the clinical readiness of stem cells and gene therapy products among clinicians, industry stakeholders, and funding bodies. The present impasse has attracted the attention of some of the most active research groups in the field, which were then summoned to analyse the position of the field and tasked to develop a strategy, to re-visit the undoubtedly promising future of cardiovascular regenerative and reparative medicine, based on lessons learned over the past two decades. During the scientific retreat of the ESC Working Group on Cardiovascular Regenerative and Reparative Medicine (CARE) in November 2018, the most relevant and timely research aspects in regenerative and/or reparative medicine were presented and critically discussed, with the aim to lay out a strategy for the future development of the field. We report herein the main ideas and conclusions of that meeting.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: [email protected]

Cardiovasc Res: 24 May 2021; 117:1428-1433
Grigorian-Shamagian L, Sanz-Ruiz R, Climent A, Badimon L, ... Zimmermann WH, Fernández-Avilés F
Cardiovasc Res: 24 May 2021; 117:1428-1433 | PMID: 33258961
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Abstract

Treatment of atrial fibrillation with doxapram: TASK-1 potassium channel inhibition as a novel pharmacological strategy.

Wiedmann F, Beyersdorf C, Zhou XB, Kraft M, ... Katus HA, Schmidt C
Aims
TASK-1 (K2P3.1) two-pore domain potassium channels are atrial-specific and significantly upregulated in atrial fibrillation (AF) patients, contributing to AF-related electrical remodelling. Inhibition of TASK-1 in cardiomyocytes of AF patients was shown to counteract AF-related action potential duration shortening. Doxapram was identified as a potent inhibitor of the TASK-1 channel. In the present study, we investigated the antiarrhythmic efficacy of doxapram in a porcine model of AF.
Methods and results
Doxapram successfully cardioverted pigs with artificially induced episodes of AF. We established a porcine model of persistent AF in domestic pigs via intermittent atrial burst stimulation using implanted pacemakers. All pigs underwent catheter-based electrophysiological investigations prior to and after 14 d of doxapram treatment. Pigs in the treatment group received intravenous administration of doxapram once per day. In doxapram-treated AF pigs, the AF burden was significantly reduced. After 14 d of treatment with doxapram, TASK-1 currents were still similar to values of sinus rhythm animals. Doxapram significantly suppressed AF episodes and normalized cellular electrophysiology by inhibition of the TASK-1 channel. Patch-clamp experiments on human atrial cardiomyocytes, isolated from patients with and without AF could reproduce the TASK-1 inhibitory effect of doxapram.
Conclusions
Repurposing doxapram might yield a promising new antiarrhythmic drug to treat AF in patients.
Translational perspective
Pharmacological suppression of atrial TASK 1 potassium currents prolongs atrial refractoriness with no effects on ventricular repolarization, resulting in atrial-specific class III antiarrhythmic effects. In our preclinical pilot study the respiratory stimulant doxapram was successfully administered for cardioversion of acute AF as well as rhythm control of persistent AF in a clinically relevant porcine animal model.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 23 May 2021; epub ahead of print
Wiedmann F, Beyersdorf C, Zhou XB, Kraft M, ... Katus HA, Schmidt C
Cardiovasc Res: 23 May 2021; epub ahead of print | PMID: 34028533
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Abstract

Heterogeneous subpopulations of adventitial progenitor cells regulate vascular homeostasis and pathological vascular remodeling.

Jolly AJ, Lu S, Strand KA, Dubner AM, ... Moulton KS, Weiser-Evans MCM
Cardiovascular diseases are characterized by chronic vascular dysfunction and provoke pathological remodeling events such as neointima formation, atherosclerotic lesion development, and adventitial fibrosis. While lineage-tracing studies have shown that phenotypically modulated smooth muscle cells (SMCs) are the major cellular component of neointimal lesions, the cellular origins and microenvironmental signaling mechanisms that underlie remodeling along the adventitial vascular layer are not fully understood. However, a growing body of evidence supports a unique population of adventitial lineage-restricted progenitor cells expressing the stem cell marker, stem cell antigen-1 (Sca1; AdvSca1 cells) as important effectors of adventitial remodeling and suggests that they are at least partially responsible for subsequent pathological changes that occur in the media and intima. AdvSca1 cells are being studied in murine models of atherosclerosis, perivascular fibrosis, and neointima formation in response to acute vascular injury. Depending on the experimental conditions, AdvSca1 cells exhibit the capacity to differentiate into SMCs, endothelial cells, chondrocytes, adipocytes, and pro-remodeling cells such as myofibroblasts and macrophages. These data indicate that AdvSca1 cells may be a targetable cell population to influence the outcomes of pathologic vascular remodeling. Important questions remain regarding the origins of AdvSca1 cells and the essential signaling mechanisms and microenvironmental factors that regulate both maintenance of their stem-like, progenitor phenotype and their differentiation into lineage-specified cell types. Adding complexity to the story, recent data indicate that the collective population of adventitial progenitor cells is likely composed of several smaller, lineage-restricted subpopulations which are not fully defined by their transcriptomic profile and differentiation capabilities. The aim of this review is to outline the heterogeneity of Sca1+ adventitial progenitor cells, summarize their role in vascular homeostasis and remodeling, and comment on their translational relevance in humans.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 13 May 2021; epub ahead of print
Jolly AJ, Lu S, Strand KA, Dubner AM, ... Moulton KS, Weiser-Evans MCM
Cardiovasc Res: 13 May 2021; epub ahead of print | PMID: 33989378
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Abstract

Primary cilia control endothelial permeability by regulating expression and location of junction proteins.

Diagbouga MR, Morel S, Cayron AF, Haemmerli J, ... Bijlenga P, Kwak BR
Aims
Wall shear stress (WSS) determines intracranial aneurysm (IA) development. Polycystic kidney disease (PKD) patients have a high IA incidence and risk of rupture. Dysfunction/absence of primary cilia in PKD endothelial cells (ECs) may impair mechano-transduction of WSS and favour vascular disorders. The molecular links between primary cilia dysfunction and IAs are unknown.
Methods and results
Wild-type and primary cilia-deficient Tg737orpk/orpk arterial ECs were submitted to physiological (30 dynes/cm2) or aneurysmal (2 dynes/cm2) WSS and unbiased transcriptomics were performed. Tg737orpk/orpk ECs displayed a 5-fold increase in the number of WSS-responsive genes compared to wild-type cells. Moreover, we observed a lower trans-endothelial resistance and a higher endothelial permeability, which correlated with disorganized intercellular junctions in Tg737orpk/orpk cells. We identified ZO-1 as a central regulator of primary cilia-dependent endothelial junction integrity. Finally, clinical and histological characteristics of IAs from non-PKD and PKD patients were analysed. IAs in PKD patients were more frequently located in the middle cerebral artery (MCA) territory than in non-PKD patients. IA domes from the MCA of PKD patients appeared thinner with less collagen and reduced endothelial ZO-1 compared with IA domes from non-PKD patients.
Conclusion
Primary cilia dampen the endothelial response to aneurysmal low WSS. In absence of primary cilia, ZO-1 expression levels are reduced, which disorganizes intercellular junctions resulting in increased endothelial permeability. This altered endothelial function may not only contribute to the severity of IA disease observed in PKD patients, but may also serve as a potential diagnostic tool to determine the vulnerability of IAs.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 10 May 2021; epub ahead of print
Diagbouga MR, Morel S, Cayron AF, Haemmerli J, ... Bijlenga P, Kwak BR
Cardiovasc Res: 10 May 2021; epub ahead of print | PMID: 33974072
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Abstract

CNP regulates cardiac contractility and increases cGMP near both SERCA and TnI - difference from BNP visualized by targeted cGMP biosensors.

Manfra O, Calamera G, Froese A, Arunthavarajah D, ... Levy FO, Andressen KW
Aims
Guanylyl cyclase-B (GC-B; natriuretic peptide receptor-B, NPR-B) stimulation by C-type natriuretic peptide (CNP) increases cGMP and causes a lusitropic and negative inotropic response in adult myocardium. These effects are not mimicked by NPR-A (GC-A) stimulation by brain natriuretic peptide (BNP), despite similar cGMP increase. More refined methods are needed to better understand the mechanisms of the differential cGMP signaling and compartmentation. The aim of this work was to measure cGMP near proteins involved in regulating contractility to understand compartmentation of cGMP signaling in adult cardiomyocytes.
Methods and results
We constructed several fluorescence resonance energy transfer (FRET)-based biosensors for cGMP subcellularly targeted to phospholamban (PLB) and troponin I (TnI). CNP stimulation of adult rat cardiomyocytes increased cGMP near PLB and TnI, whereas BNP stimulation increased cGMP near PLB, but not TnI. The phosphodiesterases PDE2 and PDE3 constrained cGMP in both compartments. Local receptor stimulation aided by scanning ion conductance microscopy (SICM) combined with FRET revealed that CNP stimulation both in the t-tubules and on the cell crests increases cGMP similarly near both TnI and PLB. In ventricular strips, CNP stimulation, but not BNP, induced a lusitropic response, enhanced by inhibition of either PDE2 or PDE3, and a negative inotropic response. In cardiomyocytes from heart failure rats, CNP increased cGMP near PLB and TnI more pronounced than in cells from sham-operated animals.
Conclusions
These targeted biosensors demonstrate that CNP, but not BNP, increases cGMP near TnI in addition to PLB, explaining how CNP, but not BNP is able to induce lusitropic and negative inotropic responses.
Translational perspective
Although best known as heart failure biomarkers, natriuretic peptides (ANP, BNP and CNP) are important signaling molecules in the heart and other organs through increasing cyclic GMP (cGMP). Treatment preventing their degradation improves heart failure prognosis. To better understand their cardiac signaling, we employed fluorescent cGMP biosensors targeted to troponin I and phospholamban and found that BNP and CNP increase cGMP differently around these proteins in both normal and failing cardiomyocytes. This may explain the different effects of BNP and CNP on cardiac contractility and relaxation, with possible implications for understanding and treatment of heart failure.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 09 May 2021; epub ahead of print
Manfra O, Calamera G, Froese A, Arunthavarajah D, ... Levy FO, Andressen KW
Cardiovasc Res: 09 May 2021; epub ahead of print | PMID: 33970224
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Abstract

Plasma HDL cholesterol and risk of dementia - observational and genetic studies.

Kjeldsen EW, Thomassen JQ, Juul Rasmussen I, Nordestgaard BG, Tybjærg-Hansen A, Frikke-Schmidt R
Aims
The association of plasma high-density lipoprotein (HDL) cholesterol with risk of dementia is unclear. We therefore tested the hypothesis that high levels of plasma HDL cholesterol are associated with increased risk of dementia and whether a potential association is of a causal nature.
Methods and results
In two prospective population-based studies, the Copenhagen General Population Study and the Copenhagen City Heart Study (N = 111,984 individuals), we first tested whether high plasma HDL cholesterol is associated with increased risk of any dementia and its subtypes. These analyses in men and women separately were adjusted multifactorially for other risk factors including apolipoprotein E (APOE) genotype. Second, taking advantage of two-sample Mendelian randomization, we tested whether genetically elevated HDL cholesterol was causally associated with Alzheimer\'s disease using publicly available consortia data on 643,836 individuals. Observationally, multifactorially adjusted Cox regression restricted cubic spline models showed that both men and women with extreme high HDL cholesterol concentrations had increased risk of any dementia and of Alzheimer\'s disease. Men in the 96th-99th and 100th versus the 41st-60th percentiles of HDL cholesterol had multifactorially including APOE genotype adjusted hazard ratios of 1.66 (95% confidence interval 1.30-2.11) and 2.00 (1.35-2.98) for any dementia and 1.59 (1.16-2.20) and 1.87 (1.11-3.16) for Alzheimer\'s disease. Corresponding estimates for women were 0.94 (0.74-1.18) and 1.45 (1.03-2.05) for any dementia and 0.94 (0.70-1.26) and 1.69 (1.13-2.53) for Alzheimer\'s disease. Genetically, the two-sample Mendelian randomization odds ratio for Alzheimer\'s disease per 1 standard deviation increase in HDL cholesterol was 0.92 (0.74-1.10) in the IGAP2019 consortium and 0.98 (0.95-1.00) in the ADSP/IGAP/PGC-ALZ/UKB consortium. Similar estimates were observed in sex stratified analyses.
Conclusion
High plasma HDL cholesterol was observationally associated with increased risk of any dementia and Alzheimer\'s disease, suggesting that HDL cholesterol can be used as an easily accessible plasma biomarker for individual risk assessment.
Translational perspective
The present study identifies very high plasma HDL cholesterol levels as an independent risk factor for any dementia and Alzheimer\'s disease in both men and women of the general population. Two-sample Mendelian randomization studies do not support that this association is of a causal nature, indicating HDL cholesterol as a non-causal risk factor for Alzheimer\'s disease. Our findings suggest that very high HDL cholesterol can be used as an easily accessible plasma biomarker to evaluate increased risk of dementia and potential identification of high-risk individuals for early targeted prevention - an area highly recommended to direct attention towards.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 07 May 2021; epub ahead of print
Kjeldsen EW, Thomassen JQ, Juul Rasmussen I, Nordestgaard BG, Tybjærg-Hansen A, Frikke-Schmidt R
Cardiovasc Res: 07 May 2021; epub ahead of print | PMID: 33964140
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Abstract

Arginase II protein regulates Parkin-dependent p32 degradation that contributes to Ca2+-dependent eNOS activation in endothelial cells.

Koo BH, Won MH, Kim YM, Ryoo S
Aims
Arginase II (ArgII) plays a key role in the regulation of Ca2+ between the cytosol and mitochondria in a p32-dependent manner. p32 contributes to endothelial nitric oxide synthase (eNOS) activation through the Ca2+/CaMKII/AMPK/p38MAPK/Akt signaling cascade. Therefore, we investigated a novel function of ArgII in the regulation of p32 stability.
Methods and results
mRNA levels were measured by qRT-PCR, and protein levels and activation were confirmed by western blot analysis. Ca2+ concentrations were measured by FACS analysis and a vascular tension assay was performed. ArgII bound to p32, and ArgII protein knockdown using siArgII facilitated the ubiquitin-dependent proteasomal degradation of p32. β-lactone, a proteasome inhibitor, inhibited the p32 degradation associated with endothelial dysfunction in a Ca2+-dependent manner. The amino acids Lys154, Lys 180, and Lys220 of the p32 protein were identified as putative ubiquitination sites. When these sites were mutated, p32 was resistant to degradation in the presence of siArgII, and endothelial function was impaired. Knockdown of Pink/Parkin as an E3-ubiquitin ligase with siRNAs resulted in increased p32, decreased [Ca2+]c, and attenuated CaMKII-dependent eNOS activation by siArgII. SiArgII-dependent Parkin activation was attenuated by KN93, a CaMKII inhibitor. Knockdown of ArgII mRNA and its gene, but not inhibition of its activity, accelerated the interaction between p32 and Parkin and reduced p32 levels. In aortas of ArgII-/- mice, p32 levels were reduced by activated Parkin and inhibition of CaMKII attenuated Parkin-dependent p32 lysis. SiParkin blunted the phosphorylation of the activated CaMKII/AMPK/p38MAPK/Akt/eNOS signaling cascade. However, ApoE-/- mice fed a high-cholesterol diet had greater ArgII activity, significantly attenuated phosphorylation of Parkin, and increased p32 levels. Incubation with siArgII augmented p32 ubiquitination through Parkin activation, and induced signaling cascade activation.
Conclusion
The results suggest a novel function for ArgII protein in Parkin-dependent ubiquitination of p32 that is associated with Ca2+-mediated eNOS activation in endothelial cells.
Translational perspective
In many vascular disorders, the downregulation of ArgII has been shown to be beneficial. This enzyme plays a crucial role in the regulation Ca2+ concentrations in a p32-dependent manner and activates the eNOS activation signaling cascade. In this study, we discovered that ArgII downregulation, inhibition of its activity, and gene knockout/down, induced the activation of Parkin (an E3-ubiquitin ligase) through a CaMKII-dependent mechanism. ArgII protein, as a p32 binding partner, prevented Parkin-dependent p32 ubiquitination, but inhibition of ArgII activity had no effect on ubiquitination. These novel findings have the potential to be translated into future therapeutic strategies to treat vascular diseases.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 07 May 2021; epub ahead of print
Koo BH, Won MH, Kim YM, Ryoo S
Cardiovasc Res: 07 May 2021; epub ahead of print | PMID: 33964139
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Abstract

Senescence mechanisms and targets in the heart.

Chen MS, Lee RT, Garbern JC
Cellular senescence is a state of irreversible cell cycle arrest associated with ageing. Senescence of different cardiac cell types can direct the pathophysiology of cardiovascular diseases such as atherosclerosis, myocardial infarction, and cardiac fibrosis. While age-related telomere shortening represents a major cause of replicative senescence, the senescent state can also be induced by oxidative stress, metabolic dysfunction, and epigenetic regulation, among other stressors. It is critical that we understand the molecular pathways that lead to cellular senescence and the consequences of cellular senescence in order to develop new therapeutic approaches to treat cardiovascular disease. In this review, we discuss molecular mechanisms of cellular senescence, explore how cellular senescence of different cardiac cell types (including cardiomyocytes, cardiac endothelial cells, cardiac fibroblasts, vascular smooth muscle cells, valve interstitial cells) can lead to cardiovascular disease, and highlight potential therapeutic approaches that target molecular mechanisms of cellular senescence to prevent or treat cardiovascular disease.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 06 May 2021; epub ahead of print
Chen MS, Lee RT, Garbern JC
Cardiovasc Res: 06 May 2021; epub ahead of print | PMID: 33963378
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Abstract

Patient-specific iPSC-derived cardiomyocytes reveal abnormal regulation of FGF16 in a familial atrial septal defect.

Ye L, Yu Y, Zhao ZA, Zhao D, ... Lei W, Hu S
Aims
Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the GATA4 gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human induced pluripotent stem cells (iPSCs) from a family cohort with ASD.
Methods and results
Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers in vitro, thus presenting a promising approach for disease modeling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4T280M) from a family cohort carrying a hereditary ASD mutation in GATA4 gene (T280M), as well as a human embryonic stem cell line (ESC-G4T280M) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4T280M-mutant iPSCs (iPSC-G4T280M-CMs) and ESCs (ESC-G4T280M-CMs), while the impaired proliferation ability of iPSC-G4T280M-CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that FGF16 is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the FGF16 promoter region, leading to impaired activation of FGF16 transcription. Overexpression of FGF16 in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4T280M and ASD was demonstrated in a human iPSC model for the first time.
Conclusions
In summary, our study revealed the molecular mechanism of the GATA4T280M mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 05 May 2021; epub ahead of print
Ye L, Yu Y, Zhao ZA, Zhao D, ... Lei W, Hu S
Cardiovasc Res: 05 May 2021; epub ahead of print | PMID: 33956078
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Abstract

The role of autophagy in cardiovascular pathology.

Gatica D, Chiong M, Lavandero S, Klionsky DJ
Macroautophagy/autophagy is a conserved catabolic recycling pathway in which cytoplasmic components are sequestered, degraded, and recycled to survive various stress conditions. Autophagy dysregulation has been observed and linked with the development and progression of several pathologies, including cardiovascular diseases, the leading cause of death in the developed world. In this review, we aim to provide a broad understanding of the different molecular factors that govern autophagy regulation and how these mechanisms are involved in the development of specific cardiovascular pathologies, including ischemic and reperfusion injury, myocardial infarction, cardiac hypertrophy, cardiac remodeling, and heart failure.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 05 May 2021; epub ahead of print
Gatica D, Chiong M, Lavandero S, Klionsky DJ
Cardiovasc Res: 05 May 2021; epub ahead of print | PMID: 33956077
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Impact:
Abstract

mRNA modifications in cardiovascular biology and disease: with a focus on m6A modification.

Kumari R, Ranjan P, Suleiman ZG, Goswami SK, ... Prasad R, Verma SK
Among several known RNA modifications, N6-methyladenosine (m6A) is the most studied RNA epitranscriptomic modification and controls multiple cellular functions during development, differentiation, and disease. Current research advancements have made it possible to examine the regulatory mechanisms associated with RNA methylation and reveal its functional consequences in the pathobiology of many diseases, including heart failure. m6A methylation has been described both on coding (mRNA) and non-coding RNA species including rRNA, tRNA, small nuclear RNA and circular RNAs. The protein components which catalyze the m6A methylation are termed methyltransferase or \"m6A writers.\" The family of proteins that recognize this methylation are termed \"m6A readers\" and finally the enzymes involved in the removal of a methyl group from RNA are known as demethylases or \"m6A erasers.\" At the cellular level, different components of methylation machinery are tightly regulated by many factors to maintain the m6A methylation dynamics. The m6A methylation process impacts different stages of mRNA metabolism and the biogenesis of long non-coding RNA and miRNA. Although, mRNA methylation was initially described in the 1970s, its regulatory roles in various diseases, including cardiovascular diseases are broadly unexplored. Recent investigations suggest the important role of m6A mRNA methylation in both hypertrophic and ischemic heart diseases. In the present review, we evaluate the significance of m6A methylation in the cardiovascular system, in cardiac homeostasis and disease, all of which may help to improve therapeutic intervention for the treatment of heart failure.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 05 May 2021; epub ahead of print
Kumari R, Ranjan P, Suleiman ZG, Goswami SK, ... Prasad R, Verma SK
Cardiovasc Res: 05 May 2021; epub ahead of print | PMID: 33956076
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Abstract

Neuronal nitric oxide synthase regulates regional brain perfusion in healthy humans.

O\'Gallagher K, Puledda F, O\'Daly O, Ryan M, ... Goadsby PJ, Shah AM
Aims
Neuronal nitric oxide synthase (nNOS) is highly expressed within the cardiovascular and nervous systems. Studies in genetically modified mice suggest roles in brain blood flow regulation while dysfunctional nNOS signalling is implicated in cerebrovascular ischaemia and migraine. Previous human studies have investigated the effects of non-selective NOS inhibition but there has been no direct investigation of the role of nNOS in human cerebrovascular regulation. We hypothesized that inhibition of the tonic effects of nNOS would result in global or localized changes in cerebral blood flow (CBF), as well as changes in functional brain connectivity.
Methods and results
We investigated the acute effects of a selective nNOS inhibitor, S-methyl-L-thiocitrulline (SMTC), on CBF and brain functional connectivity in healthy human volunteers (n = 19). We performed a randomized, placebo-controlled, crossover study with either intravenous SMTC or placebo, using magnetic resonance imaging protocols with arterial spin labelling and functional resting state neuroimaging. SMTC infusion induced an ∼4% decrease in resting global CBF [-2.3 (-0.3, -4.2) mL/100g/min, mean (95% confidence interval, CI), P = 0.02]. In a whole-brain voxel-wise factorial-design comparison of CBF maps, we identified a localized decrease in regional blood flow in the right hippocampus and parahippocampal gyrus following SMTC vs. placebo (2921 voxels; T = 7.0; x = 36; y = -32; z = -12; P < 0.001). This was accompanied by a decrease in functional connectivity to the left superior parietal lobule vs. placebo (484 voxels; T = 5.02; x = -14; y = -56; z = 74; P = 0.009). These analyses adjusted for the modest changes in mean arterial blood pressure induced by SMTC as compared to placebo [+8.7 mmHg (+1.8, +15.6), mean (95% CI), P = 0.009].
Conclusions
These data suggest a fundamental physiological role of nNOS in regulating regional CBF and functional connectivity in the human hippocampus. Our findings have relevance to the role of nNOS in the regulation of cerebral perfusion in health and disease.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 29 Apr 2021; epub ahead of print
O'Gallagher K, Puledda F, O'Daly O, Ryan M, ... Goadsby PJ, Shah AM
Cardiovasc Res: 29 Apr 2021; epub ahead of print | PMID: 34120160
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Abstract

Drebrin attenuates atherosclerosis by limiting smooth muscle cell transdifferentiation.

Wu JH, Zhang L, Nepliouev I, Brian L, ... Freedman NJ, Stiber JA
Aims
The F-actin-binding protein Drebrin inhibits smooth muscle cell (SMC) migration, proliferation and pro-inflammatory signaling. Therefore, we tested the hypothesis that Drebrin constrains atherosclerosis.
Methods and results
SM22-Cre+/Dbnflox/flox/Ldlr-/- (SMC-Dbn-/-/Ldlr-/-) and control mice (SM22-Cre+/Ldlr-/-, Dbnflox/flox/Ldlr-/-, and Ldlr-/-) were fed a Western diet for 14-20 weeks. Brachiocephalic arteries of SMC-Dbn-/-/Ldlr-/- mice exhibited 1.5- or 1.8-fold greater cross-sectional lesion area than control mice at 14 or 20 wk, respectively. Aortic atherosclerotic lesion surface area was 1.2-fold greater in SMC-Dbn-/-/Ldlr-/- mice. SMC-Dbn-/-/Ldlr-/- lesions comprised necrotic cores that were two-fold greater in size than those of control mice. Consistent with their bigger necrotic core size, lesions in SMC-Dbn-/- arteries also showed more transdifferentiation of SMCs to macrophage-like cells: 1.5- to 2.5-fold greater, assessed with BODIPY or with CD68, respectively. In vitro data were concordant: Dbn-/- SMCs had 1.7-fold higher levels of KLF4 and transdifferentiated to macrophage-like cells more readily than Dbnflox/flox SMCs upon cholesterol loading, as evidenced by greater up-regulation of CD68 and galectin-3. Adenovirally mediated Drebrin rescue produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs. During early atherogenesis, SMC-Dbn-/-/Ldlr-/- aortas demonstrated 1.6-fold higher levels of reactive oxygen species than control mouse aortas. The 1.8-fold higher levels of Nox1 in Dbn-/- SMCs was reduced to WT levels with KLF4 silencing. Inhibition of Nox1 chemically or with siRNA produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs.
Conclusions
We conclude that SMC Drebrin limits atherosclerosis by constraining SMC Nox1 activity and SMC transdifferentiation to macrophage-like cells.
Translational perspective
Drebrin is abundantly expressed in vascular smooth muscle cells (SMCs) and is up-regulated in human atherosclerosis. A hallmark of atherosclerosis is the accumulation of foam cells that secrete pro-inflammatory cytokines and contribute to plaque instability. A large proportion of these foam cells in humans derive from SMCs. We found that SMC Drebrin limits atherosclerosis by reducing SMC transdifferentiation to macrophage-like foam cells in a manner dependent on Nox1 and KLF4. For this reason, strategies aimed at augmenting SMC Drebrin expression in atherosclerotic plaques may limit atherosclerosis progression and enhance plaque stability by bridling SMC-to-foam-cell transdifferentiation.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions please email: [email protected]

Cardiovasc Res: 28 Apr 2021; epub ahead of print
Wu JH, Zhang L, Nepliouev I, Brian L, ... Freedman NJ, Stiber JA
Cardiovasc Res: 28 Apr 2021; epub ahead of print | PMID: 33914863
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Abstract

Endothelial Zeb2 preserves the hepatic angioarchitecture and protects against liver fibrosis.

de Haan W, Dheedene W, Apelt K, Décombas-Deschamps S, ... Huylebroeck D, Luttun A
Aims
Hepatic capillaries are lined with specialised liver sinusoidal endothelial cells (LSECs) which support macromolecule passage to hepatocytes and prevent fibrosis by keeping hepatic stellate cells (HSCs) quiescent. LSEC specialisation is co-determined by transcription factors. The Zinc-Finger E-Box-binding Homeobox (Zeb)2 transcription factor is enriched in LSECs. Here, we aimed to elucidate the endothelium-specific role of Zeb2 during maintenance of the liver and in liver fibrosis.
Methods and results
To study the role of Zeb2 in liver endothelium we generated EC-specific Zeb2 knock-out (ECKO) mice. Sequencing of liver EC RNA revealed that deficiency of Zeb2 results in prominent expression changes in angiogenesis-related genes. Accordingly, the vascular area was expanded and the presence of pillars inside ECKO liver vessels indicated that this was likely due to increased intussusceptive angiogenesis. LSEC marker expression was not profoundly affected and fenestrations were preserved upon Zeb2 deficiency. However, an increase in continuous EC markers suggested that Zeb2-deficient LSECs are more prone to dedifferentiation, a process called \'capillarisation\'. Changes in the endothelial expression of ligands that may be involved in HSC quiescence together with significant changes in the expression profile of HSCs showed that Zeb2 regulates LSEC-HSC communication and HSC activation. Accordingly, upon exposure to the hepatotoxin carbon tetrachloride (CCl4), livers of ECKO mice showed increased capillarisation, HSC activation and fibrosis compared to livers from wild-type littermates. The vascular maintenance and anti-fibrotic role of endothelial Zeb2 was confirmed in mice with EC-specific overexpression of Zeb2, as the latter resulted in reduced vascularity and attenuated CCl4-induced liver fibrosis.
Conclusion
Endothelial Zeb2 preserves liver angioarchitecture and protects against liver fibrosis. Zeb2 and Zeb2-dependent genes in liver ECs may be exploited to design novel therapeutic strategies to attenuate hepatic fibrosis.

© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 27 Apr 2021; epub ahead of print
de Haan W, Dheedene W, Apelt K, Décombas-Deschamps S, ... Huylebroeck D, Luttun A
Cardiovasc Res: 27 Apr 2021; epub ahead of print | PMID: 33909875
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This program is still in alpha version.