Therapeutic targeting of inflammation in hypertension: from novel mechanisms to translational perspective.

Murray EC, Nosalski R, MacRitchie N, Tomaszewski M, ... Harrison DG, Guzik TJ
Animal models, human observational and genetic studies have shown that immune and inflammatory mechanisms play a key role in hypertension and its complications. We review the effects of immunomodulatory interventions on blood pressure, target organ damage and cardiovascular risk in humans. In experimental and small clinical studies both non-specific immunomodulatory approaches, such as mycophenolate mofetil and methotrexate, and medications targeting T and B lymphocytes, such as tacrolimus, cyclosporine, everolimus, rituximab, lower blood pressure and reduce organ damage. Mechanistically targeted immune interventions include isolevuglandin (isoLG) scavengers to prevent neo-antigen formation, co-stimulation blockade (abatacept, belatacept), and anti-cytokine therapies (secukinumab, tocilizumab, canakinumab, TNF-α inhibitors). In many studies, trial designs have been complicated by a lack of blood pressure related endpoints, inclusion of largely normotensive study populations, polypharmacy, and established comorbidities. Among a wide range of interventions reviewed, TNF-α inhibitors have provided the most robust evidence of blood pressure lowering. Treatment of periodontitis also appears to deliver non-pharmacological antihypertensive effects. Evidence of immunomodulatory drugs influencing hypertension-mediated organ damage are discussed. Animal model, observational studies, and trial data in humans support the therapeutic potential of immune targeted therapies in blood pressure lowering and in hypertension-mediated organ damage. Targeted studies are now needed to address their effects on blood pressure in hypertensive individuals.

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

Cardiovasc Res: 25 Oct 2021; epub ahead of print

Immune And Inflammatory Mechanisms Mediate Cardiovascular Diseases From Head To Toe.

Libby P, Mallat Z, Weyand C
Practitioners have long recognized the involvement of inflammation in certain acute cardiovascular diseases such as endocarditis, myocarditis, and pericarditis. Attention to the participation of immune and inflammatory mechanisms in chronic cardiovascular diseases has generally lagged. Yet, these pathways contribute to a broad swath of clinically important cardiovascular conditions, both acute and chronic. Understanding of these complex mechanisms can aid specialists in cardiovascular research and practice immeasurably by providing new concepts and illuminating new diagnostic and therapeutic strategies. The collection of essays presented in this focused issue of Cardiovascular Research aims to promote this goal.

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 Oct 2021; epub ahead of print

Pathophysiological pathways in patients with heart failure and atrial fibrillation.

Santema BT, Arita VA, Sama IE, Kloosterman M, ... Rienstra M, Voors AA
Aims
Atrial fibrillation (AF) and heart failure (HF) are two growing epidemics that frequently co-exist. We aimed to gain insights into underlying pathophysiological pathways in HF patients with AF by comparing circulating biomarkers using pathway overrepresentation analyses.
Methods and results
From a panel of 92 biomarkers from different pathophysiological domains available in 1,620 patients with HF, we first tested which biomarkers were dysregulated in patients with HF and AF (n = 648) compared with patients in sinus rhythm (n = 972). Secondly, pathway overrepresentation analyses were performed to identify biological pathways linked to higher plasma concentrations of biomarkers in patients who had HF and AF. Findings were validated in an independent HF cohort (n = 1,219, 38% with AF). Patient with AF and HF were older, less often women, and less often had a history of coronary artery disease compared with those in sinus rhythm. In the index cohort, 24 biomarkers were upregulated in patients with AF and HF. In the validation cohort, 8 biomarkers were upregulated, which all overlapped with the 24 biomarkers found in the index cohort. The strongest up-regulated biomarkers in patients with AF were spondin-1 (fold change 1.18, p = 1.33x10-12), insulin-like growth factor-binding protein-1 (fold change 1.32, p = 1.08x10-8), and insulin-like growth factor-binding protein-7 (fold change 1.33, p = 1.35x10-18). Pathway overrepresentation analyses revealed that the presence of AF was associated with activation amyloid-beta metabolic processes, amyloid-beta formation, and amyloid precursor protein catabolic processes with a remarkable consistency observed in the validation cohort.
Conclusion
In two independent cohorts of patients with HF, the presence of AF was associated with activation of three pathways related to amyloid-beta. These hypothesis-generating results warrant confirmation in future studies.
Translational perspective
Using an unbiased approach, we identified and validated dysregulation of three amyloid-beta related pathways in patients who had heart failure (HF) with concomitant atrial fibrillation (AF). Amyloid-beta depositions are a hallmark of Alzheimer\'s disease, but might also play a role in pathophysiological processes outside the central nervous system. Biopsy studies are needed to confirm the pathophysiological role of amyloid-beta in patients with AF and HF. Diagnostic and therapeutic implications should be investigated in the light of potential pathophysiological overlap between the three aging-related epidemics: Alzheimer\'s disease, AF and HF.

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

Cardiovasc Res: 22 Oct 2021; epub ahead of print

SR-mitochondria crosstalk shapes Ca signaling to impact pathophenotype in disease models marked by dysregulated intracellular Ca release.

Tow BD, Deb A, Neupane S, Patel S, ... Györke S, Liu B
Aims
Diastolic Ca release (DCR) from sarcoplasmic reticulum (SR) Ca release channel ryanodine receptor (RyR2) has been linked to multiple cardiac pathologies, but its exact role in shaping divergent cardiac pathologies remains unclear. We hypothesize that the SR-mitochondria interplay contributes to disease phenotypes by shaping Ca signaling.
Methods and results
A genetic model of catecholaminergic polymorphic ventricular tachycardia (CPVT2 model of CASQ2 knockout) and a pre-diabetic cardiomyopathy model of fructose fed mice (FFD), both marked by DCR, are employed in this study. Mitochondria Ca (mCa) is modulated by pharmacologically targeting mitochondria Ca uniporter (MCU) or permeability transition pore (mPTP), mCa uptake and extrusion mechanisms, respectively. An MCU activator abolished Ca waves in CPVT2 but exacerbated waves in FFD cells. Mechanistically this is ascribed to mitochondria\'s function as a Ca buffer or source of reactive oxygen species (mtROS) to exacerbate RyR2 functionality, respectively. Enhancing mCa uptake reduced and elevated mtROS production in CPVT2 and FFD respectively. In CPVT2, mitochondria took up more Ca in permeabilized cells, and had higher level of mCa content in intact cells vs FFD. Conditional ablation of MCU in the CPVT2 model caused lethality and cardiac remodeling, but reduced arrhythmias in the FFD model. In parallel, CPVT2 mitochondria also employ upregulated mPTP-mediated Ca efflux to avoid mCa overload, as seen by elevated incidence of MitoWinks (an indicator of mPTP-mediated Ca efflux) vs FFD. Both pharmacological and genetic inhibition of mPTP promoted mtROS production and exacerbation of myocyte Ca handling in CPVT2. Further, genetic inhibition of mPTP exacerbated arrhythmias in CPVT2.
Conclusion
In contrast to FFD, which is more susceptible to mtROS-dependent RyR2 leak, in CPVT2 mitochondria buffer SR-derived DCR to mitigate Ca-dependent pathological remodeling and rely on mPTP-mediated Ca efflux to avoid mCa overload. SR-mitochondria interplay contributes to the divergent pathologies by disparately shaping intracellular Ca signaling.
Translational perspective
It is well-established that RyR2 dysfunction is involved in a spectrum of pathological conditions including cardiac arrhythmias. In this study, two disease models marked by RyR2 dysfunction were employed to explore how the interplay between SR and mitochondria contributes to divergent cardiac pathologies. We found mitochondria act as essential Ca buffer to absorb SR-derived Ca to mitigate pathological remodeling in the genetic arrhythmic syndrome CPVT, but they are more susceptible to Ca overload or ROS-related exacerbation of RyR2 dysfunction in pre-diabetic cardiomyopathy. Thus, tailored therapies should be developed to target SR-mitochondria interplay in the aims of treating these 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: 21 Oct 2021; epub ahead of print

Interferon stimulated gene 15 pathway is a novel mediator of endothelial dysfunction and aneurysms development in angiotensin II infused mice through increased oxidative stress.

González-Amor M, García-Redondo AB, Jorge I, Zalba G, ... Salaices M, Briones AM
Aims
Interferon-stimulated gene 15 (ISG15) encodes an ubiquitin-like protein that induces a reversible post-translational modification (ISGylation) and can also be secreted as a free form. ISG15 plays an essential role as host-defense response to microbial infection; however, its contribution to vascular damage associated to hypertension is unknown.
Methods and results
Bioinformatics identified ISG15 as a mediator of hypertension-associated vascular damage. ISG15 expression positively correlated with systolic and diastolic blood pressure and carotid intima-media thickness in human peripheral blood mononuclear cells. Consistently, Isg15 expression was enhanced in aorta from hypertension models and in angiotensinII (AngII)-treated vascular cells and macrophages. Proteomics revealed differential expression of proteins implicated in cardiovascular function, extracellular matrix and remodeling, and vascular redox state in aorta from AngII-infused ISG15-/- mice. Moreover, ISG15-/- mice were protected against AngII-induced hypertension, vascular stiffness, elastin remodeling, endothelial dysfunction, and expression of inflammatory and oxidative stress markers. Conversely, mice with excessive ISGylation (USP18C61A) show enhanced AngII-induced hypertension, vascular fibrosis, inflammation and reactive oxygen species (ROS) generation along with elastin breaks, aortic dilation and rupture. Accordingly, human and murine abdominal aortic aneurysms showed augmented ISG15 expression. Mechanistically, ISG15 induces vascular ROS production, while antioxidant treatment prevented ISG15-induced endothelial dysfunction and vascular remodeling.
Conclusion
ISG15 is a novel mediator of vascular damage in hypertension through oxidative stress and inflammation.
Translational perspective
Recent evidence from randomized clinical trials demonstrate the effectiveness of specific anti-inflammatory treatments in cardiovascular prevention. In this study we have identified a new inflammatory mediator involved in vascular damage in experimental and human hypertension and aneurysms. We found that interferon stimulated gene 15 (ISG15) is increased at the vascular level in animal models of hypertension and aneurysms. More importantly, ISG15 correlates with human hypertension, vascular remodeling, and aneurysms presence. Underlying mechanisms responsible for vascular damage induced by ISG15 include oxidative and inflammation. Our results further support the role of inflammation in vascular damage in different cardiovascular pathologies.

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 Oct 2021; epub ahead of print

Pannexin 1-a novel regulator of acute hypoxic pulmonary vasoconstriction.

Grimmer B, Krauszman A, Hu X, Kabir G, ... Isakson BE, Kuebler WM
Aims
Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to alveolar hypoxia that diverts blood flow from poorly ventilated to better aerated lung areas to optimize ventilation-perfusion matching. Yet, the exact sensory and signaling mechanisms by which hypoxia triggers pulmonary vasoconstriction remain incompletely understood. Recently, ATP release via pannexin 1 (Panx1) and subsequent signaling via purinergic P2Y receptors has been identified as regulator of vasoconstriction in systemic arterioles. Here, we probed for the role of Panx1-mediated ATP release in HPV and chronic hypoxic pulmonary hypertension (PH).
Methods and results
Pharmacological inhibition of Panx1 by probenecid, spironolactone, the Panx1 specific inhibitory peptide (10Panx1) and genetic deletion of Panx1 specifically in smooth muscle attenuated HPV in isolated perfused mouse lungs. In pulmonary artery smooth muscle cells (PASMC), both spironolactone and 10Panx1 attenuated the increase in intracellular Ca2+ concentration ([Ca2+]i) in response to hypoxia. Yet, genetic deletion of Panx1 in either endothelial or smooth muscle cells did not prevent the development of PH in mice. Unexpectedly, ATP release in response to hypoxia was not detectable in PASMC, and inhibition of purinergic receptors or ATP degradation by ATPase failed to attenuate HPV. Rather, transient receptor potential vanilloid 4 (TRPV4) antagonism and Panx1 inhibition inhibited the hypoxia-induced [Ca2+]i increase in PASMC in an additive manner, suggesting that Panx1 regulates [Ca2+]i independently of the ATP-P2Y-TRPV4 pathway. In line with this notion, Panx1 overexpression increased the [Ca2+]i response to hypoxia in HeLa cells.
Conclusion
In the present study we identify Panx1 as novel regulator of HPV. Yet, the role of Panx1 in HPV was not attributable to ATP release and downstream signaling via P2Y receptors or TRPV4 activation, but relates to a role of Panx1 as direct or indirect modulator of the PASMC Ca2+ response to hypoxia. Panx1 did not affect the development of chronic hypoxic PH.
Translational perspective
Hypoxic pulmonary vasoconstriction (HPV) optimizes lung ventilation-perfusion matching, but also contributes to pulmonary pathologies including high altitude pulmonary edema (HAPE) or chronic hypoxic pulmonary hypertension. Here, we demonstrate that pharmaceutical inhibition as well as genetic deletion of the hemichannel pannexin-1 (Panx1) in pulmonary artery smooth muscle cells attenuates the physiological HPV response. Panx1 deficiency did, however, not prevent the development of chronic hypoxic pulmonary hypertension in mice. Panx1 inhibitors such as the mineralocorticoid receptor antagonist spironolactone may thus present a putative strategy for the prevention or treatment of HAPE, yet not for chronic hypoxic lung 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: 19 Oct 2021; epub ahead of print

Ogfod1 deletion increases cardiac beta-alanine levels and protects mice against ischemia-reperfusion injury.

Harris M, Sun J, Keeran K, Aponte A, ... Murphy E, Kennedy LM
Aims
Prolyl hydroxylation is a post-translational modification that regulates protein stability, turnover, and activity. The proteins that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase family of proteins. 2-oxoglutarate- and iron-dependent oxygenase domain-containing protein 1 (Ogfod1), which hydroxylates a proline in ribosomal protein s23 is a newly-described member of this family. The aims of this study were to investigate roles for Ogfod1 in the heart, and in the heart\'s response to stress.
Methods and results
We isolated hearts from wild type (WT) and Ogfod1 knockout (KO) mice and performed quantitative proteomics using Tandem Mass Tag labelling coupled to Liquid Chromatography and tandem Mass Spectrometry (LC-MS/MS) to identify protein changes. Ingenuity Pathway Analysis identified \"Urate Biosynthesis/Inosine 5\'-phosphate Degradation\" and \"Purine Nucleotides Degradation II (Aerobic)\" as the most significantly-enriched pathways. We performed metabolomics analysis and found that both purine and pyrimidine pathways were altered with the purine nucleotide inosine 5\'-monophosphate (IMP) showing a 3.5-fold enrichment in KO hearts (P = 0.011) and the pyrimidine catabolism product beta-alanine showing a 1.7-fold enrichment in KO hearts (P = 0.014). As changes in these pathways have been shown to contribute to cardioprotection, we subjected isolated perfused hearts to ischemia and reperfusion (I/R). KO hearts showed a 41.4% decrease in infarct size and a 34% improvement in cardiac function compared to WT hearts. This protection was also evident in an in vivo I/R model. Additionally, our data show that treating isolated perfused WT hearts with carnosine, a metabolite of beta-alanine, improved protection in the context of I/R injury, whereas treating KO hearts with carnosine had no impact on recovery of function or infarct size.
Conclusions
Taken together, these data show that Ogfod1 deletion alters the myocardial proteome and metabolome to confer protection against I/R injury.
Translational perspective
Heart disease is the leading cause of death in the US. In characterizing the cardiovascular effects of deleting the prolyl hydroxylase Ogfod1 and investigating its role in disease pathology, we found that deleting Ogfod1 protected hearts against ex vivo and in vivo I/R injury. Ogfod1-KO hearts showed significant metabolomic and proteomic changes that supported altered purine and pyrimidine nucleotide synthesis and turnover. Beta-alanine, a precursor of the anti-oxidant carnosine and a product of pyrimidine degradation, accumulated in KO hearts to help confer cardioprotection. Altogether, these data suggest a role for Ogfod1 downregulation as a therapeutic strategy for heart disease.

Published on behalf of the European Society of Cardiology 2021.

Cardiovasc Res: 19 Oct 2021; epub ahead of print

Titin (TTN): from molecule to modifications, mechanics and medical significance.

Loescher CM, Hobbach AJ, Linke WA
The giant sarcomere protein titin is a major determinant of cardiomyocyte stiffness and contributor to cardiac strain sensing. Titin-based forces are highly regulated in health and disease, which aids in the regulation of myocardial function, including cardiac filling and output. Due to the enormous size, complexity, and malleability of the titin molecule, titin properties are also vulnerable to dysregulation, as observed in various cardiac disorders. This review provides an overview of how cardiac titin properties can be changed at a molecular level, including the role isoform diversity and posttranslational modifications (acetylation, oxidation and phosphorylation) play in regulating myocardial stiffness and contractility. We then consider how this regulation becomes unbalanced in heart disease, with an emphasis on changes in titin stiffness and protein quality control. In this context, new insights into the key pathomechanisms of human cardiomyopathy due to a truncation in the titin gene (TTN) are discussed. Along the way, we touch on the potential for titin to be therapeutically targeted to treat acquired or inherited cardiac conditions, such as HFpEF or TTN-truncation cardiomyopathy.

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

Cardiovasc Res: 17 Oct 2021; epub ahead of print

Peripheral blood RNA biomarkers for cardiovascular disease from bench to bedside: A Position Paper from the EU-CardioRNA COST Action CA17129.

Vanhaverbeke M, Attard R, Bartekova M, Ben-Aicha S, ... Devaux Y, EU-CardioRNA COST Action CA17129
Despite significant advances in the diagnosis and treatment of cardiovascular diseases, recent calls have emphasized the unmet need to improve precision-based approaches in cardiovascular disease. Although some studies provide preliminary evidence of the diagnostic and prognostic potential of circulating coding and non-coding RNAs, the complex RNA biology and lack of standardization have hampered the translation of these markers into clinical practice. In this position paper of the CardioRNA COST action CA17129, we provide recommendations to standardize the RNA development process in order to catalyze efforts to investigate novel RNAs for clinical use. We list the unmet clinical needs in cardiovascular disease, such as the identification of high-risk patients with ischemic heart disease or heart failure who require more intensive therapies. The advantages and pitfalls of the different sample types, including RNAs from plasma, extracellular vesicles and whole blood, are discussed in the sample matrix, together with their respective analytical methods. The effect of patient demographics and highly prevalent comorbidities, such as metabolic disorders, on the expression of the candidate RNA is presented and should be reported in biomarker studies. We discuss the statistical and regulatory aspects to translate a candidate RNA from a research-use only assay to an in-vitro diagnostic test for clinical use. Optimal planning of this development track is required, with input from the researcher, statistician, industry and regulatory partners.

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

Cardiovasc Res: 13 Oct 2021; epub ahead of print

Neutrophil extracellular traps: from physiology to pathology.

Hidalgo A, Libby P, Soehnlein O, Aramburu IV, Papayannopoulos V, Silvestre-Roig C
At the frontline of the host defense response, neutrophil antimicrobial functions have adapted to combat infections and injuries of different origins and magnitude. The release of web-like DNA structures named neutrophil extracellular traps (NETs) constitutes an important mechanism by which neutrophils prevent pathogen dissemination or deal with microorganisms of a bigger size. At the same time, nuclear and granule proteins with microbicidal activity bind to these DNA structures promoting the elimination of entrapped pathogens. However, these toxic properties may produce unwanted effects in the host, when neutrophils uncontrollably release NETs upon persistent inflammation. As a consequence, NET accumulation can produce vessel occlusion, tissue damage, and prolonged inflammation associating with the progression and exacerbation of multiple pathologic conditions. This review outlines recent advances in understanding the mechanisms of NET release and functions in sterile disease. We also discuss mechanisms of physiological regulation and the importance of neutrophil heterogeneity in NET formation and composition.

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 Oct 2021; epub ahead of print

Genetic inhibition of Nuclear Factor of Activated T-cell c2 (NFATc2) prevents atrial fibrillation in CREM transgenic mice.

Ni L, Lahiri SK, Nie J, Pan X, ... Dobrev D, Wehrens XHT
Aims
Abnormal intracellular calcium handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signaling pathway contributes to atrial remodeling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models.
Methods and results
Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF. NFATc1 and NFATc2 mRNA levels were elevated in chronic AF (cAF) patients compared with those in sinus rhythm (SR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM: NFATc2-/- mice. Programmed electrical stimulation revealed that CREM: NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with WT controls, which was reversed in CREM: NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM: NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on RyR2 in CREM: RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras And EF-Hand Domain-Containing Protein (RASEF) as a direct target of NFATc2 mediated transcription.
Conclusion
Our findings reveal activation of the NFAT signaling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model.
Translational perspective
Atrial fibrillation (AF) is a progressive disease characterized by electrical and structural remodeling which promotes atrial arrhythmias. This study provides evidence for increased \'nuclear factor of activated T-cell\' (NFAT) signaling in patients with chronic AF. Studies in the CREM transgenic model of progressive AF revealed that the NFATc2 isoform mediates atrial remodeling associated with AF substrate development. Chromatin immunoprecipitation sequencing of atrial biopsies from AF patients identified \'Ras And EF-Hand Domain-Containing Protein\' (RASEF) as a downstream target of NFATc2-mediated transcription, suggesting that targeting these factors might be beneficial for curtailing AF progression.

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 Oct 2021; epub ahead of print

Cardiac SARS-CoV-2 infection is associated with pro-inflammatory transcriptomic alterations within the heart.

Bräuninger H, Stoffers B, Fitzek ADE, Meißner K, ... Westermann D, Lindner D
Aims
Cardiac involvement in COVID-19 is associated with adverse outcome. However, it is unclear whether cell specific consequences are associated with cardiac SARS-CoV-2 infection. Therefore, we investigated heart tissue utilizing in situ hybridization, immunohistochemistry and RNA-sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19.
Methods and results
In this study, 95 SARS-CoV-2-positive autopsy cases were included. A relevant SARS-CoV-2 virus load in the cardiac tissue was detected in 41/95 deceased (43%). MACE-RNA-sequencing was performed to identify molecular pathomechanisms caused by the infection of the heart. A signature matrix was generated based on the single-cell dataset \"Heart Cell Atlas\" and used for digital cytometry on the MACE-RNA-sequencing data. Thus, immune cell fractions were estimated and revealed no difference in immune cell numbers in cases with and without cardiac infection. This result was confirmed by quantitative immunohistological diagnosis.MACE-RNA-sequencing revealed 19 differentially expressed genes (DEGs) with a q-value <0.05 (e.g. up: IFI44L, IFT3, TRIM25; down: NPPB, MB, MYPN). The upregulated DEGs were linked to interferon pathways and originate predominantly from endothelial cells. In contrast, the downregulated DEGs originate predominately from cardiomyocytes. Immunofluorescent staining showed viral protein in cells positive for the endothelial marker ICAM1 but rarely in cardiomyocytes. The GO term analysis revealed that downregulated GO terms were linked to cardiomyocyte structure, whereas upregulated GO terms were linked to anti-virus immune response.
Conclusion
This study reveals, that cardiac infection induced transcriptomic alterations mainly linked to immune response and destruction of cardiomyocytes. While endothelial cells are primarily targeted by the virus, we suggest cardiomyocyte-destruction by paracrine effects. Increased pro-inflammatory gene expression was detected in SARS-CoV-2-infected cardiac tissue but no increased SARS-CoV-2 associated immune cell infiltration was observed.
Translational perspective
Cardiac injury can be documented in COVID-19, regardless the direct cardiac virus infection and is known to be associated with outcome. However, the direct virus infection of the myocardium leads to transcriptomic alterations and might therefore additionally contribute to pathophysiological processes in COVID-19. Therefore, consequences of cardiac virus infection need to be investigated in future studies, since they might also contribute to long-term effects in case of survival.

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

Cardiovasc Res: 13 Oct 2021; epub ahead of print

Temporal relationship between systemic endothelial dysfunction and alterations in erythrocyte function in a murine model of chronic heart failure.

Mohaissen T, Proniewski B, Targosz-Korecka M, Bar A, ... Marzec KM, Chlopicki S
Aims
Endothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterise the relationship between the development of peripheral ED and the occurrence of structural nanomechanical and biochemical changes in red blood cells (RBCs).
Methods and results
Systemic ED was detected in vivo in 8-month-old Tgαq*44 mice, as evidenced by impaired acetylcholine-induced vasodilation in the aorta and increased endothelial permeability in the brachiocephalic artery. ED in the aorta was associated with impaired nitric oxide (NO) production in the aorta and diminished systemic NO bioavailability. ED in the aorta was also characterised by increased superoxide and eicosanoid production. In 4- to 6-month-old Tgαq*44 mice, RBC size and membrane composition displayed alterations that did not result in significant changes in their nanomechanical and functional properties. However, 8-month-old Tgαq*44 mice presented greatly accentuated structural and size changes and increased RBC stiffness. In 12-month-old Tgαq*44 mice, the erythropathy was featured by severely altered RBC shape and elasticity, increased RDW, impaired RBC deformability, and increased oxidative stress (GSH/GSSH ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, co-incubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor (ABH, 2(S)-amino-6-boronohexanoic acid).
Conclusion
In the Tgαq*44 murine model of HF, systemic endothelial dysfunction accelerates erythropathy and, conversely, erythropathy may contribute to endothelial dysfunction. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic endothelial dysfunction in HF. In particular, targeting RBC arginase may represent a novel treatment strategy for systemic endothelial dysfunction in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic endothelial dysfunction in HF.
Translational perspective
Endothelial dysfunction (ED) and red blood cell distribution width (RDW) both have prognostic value for heart failure (HF), but it is not known whether these pathologies are related. We comprehensively characterized endothelial and RBC functional status in a unique murine model of chronic heart failure with a prolonged time course of HF progression. Our results suggest that ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. Accordingly, erythropathy in HF reflects ED and involves various changes (in functional, structural, nanomechanical, and biochemical levels) that could have diagnostic and therapeutic significance for HF.

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

Cardiovasc Res: 06 Oct 2021; epub ahead of print

Studying the role of chromatin organisation in cardiovascular diseases-future perspectives.

Gladka MM, Christoffels VM
Commentary on \'Single-cell measurement of higher-order 3D genome organization with scSPRITE.\'

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 Oct 2021; epub ahead of print

Cardiovascular consequences of viral infections: from COVID to other viral diseases.

Schultheiss HP, Baumeier C, Pietsch H, Bock CT, Poller W, Escher F
Infection of the heart muscle with cardiotropic viruses is one of the major etiologies of myocarditis and acute and chronic inflammatory cardiomyopathy (DCMi). However, viral myocarditis and subsequent dilated cardiomyopathy (DCM) is still a challenging disease to diagnose and to treat and is therefore a significant public health issue globally. Advances in clinical examination and thorough molecular genetic analysis of intramyocardial viruses and their activation status have incrementally improved our understanding of molecular pathogenesis and pathophysiology of viral infections of the heart muscle. To date, several cardiotropic viruses have been implicated as causes of myocarditis and DCMi. These include, among others, classical cardiotropic enteroviruses (coxsackieviruses B), the most commonly detected parvovirus B19, and human herpes virus 6. A newcomer is the respiratory virus that has triggered the worst pandemic in a century, SARS-CoV-2, whose involvement and impact in viral cardiovascular disease is under scrutiny. Despite extensive research into the pathomechanisms of viral infections of the cardiovascular system, our knowledge regarding their treatment and management is still incomplete. Accordingly, in this review we aim to explore and summarize the current knowledge and available evidence on viral infections of the heart. We focus on diagnostics, clinical relevance and cardiovascular consequences, pathophysiology, and current and novel treatment strategies.

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

Cardiovasc Res: 04 Oct 2021; epub ahead of print

Atherosclerosis is a Major Human Killer and Non-resolving Inflammation is a Prime Suspect.

Fredman G, MacNamara KC
The resolution of inflammation (or inflammation-resolution) is an active and highly coordinated process. Inflammation-resolution is governed by several endogenous factors, and specialized pro-resolving mediators (SPMs) are one such class of molecules that have robust biological function. Non-resolving inflammation is associated with a variety of human diseases, including atherosclerosis. Moreover, non-resolving inflammation is a hallmark of aging, an inevitable process associated with increased risk for cardiovascular disease. Uncovering mechanisms as to why inflammation-resolution is impaired in aging and in disease and identifying useful biomarkers for non-resolving inflammation are unmet needs. Recent work has pointed to a critical role for balanced ratios of SPMs and pro-inflammatory lipids (i.e., leukotrienes and/or specific prostaglandins) as a key determinant of timely inflammation resolution. This review will focus on the accumulating findings that support the role of non-resolving inflammation and imbalanced pro-resolving and pro-inflammatory mediators in atherosclerosis. We aim to provide insight as to why these imbalances occur, the importance of aging in disease progression, and how hematopoietic function impacts inflammation-resolution and atherosclerosis. We highlight open questions regarding therapeutic strategies and mechanisms of disease to provide a framework for future studies that aim to tackle this important human 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: 04 Oct 2021; epub ahead of print

Environmental risk factors and cardiovascular diseases: a comprehensive review.

Münzel T, Hahad O, Sørensen M, Lelieveld J, ... Nieuwenhuijsen M, Daiber A
Noncommunicable diseases (NCDs) are fatal for more than 38 million people each year and are thus the main contributors to the global burden of disease accounting for 70% of mortality. The majority of these deaths are caused by cardiovascular disease. The risk of NCDs is strongly associated with exposure to environmental stressors such as pollutants in the air, noise exposure, artificial light at night and climate change, including heat extremes, desert storms and wildfires. In addition to the traditional risk factors for cardiovascular disease such as diabetes, arterial hypertension, smoking, hypercholesterolemia and genetic predisposition, there is a growing body of evidence showing that physicochemical factors in the environment contribute significantly to the high NCD numbers. Furthermore, urbanization is associated with accumulation and intensification of these stressors. This comprehensive expert review will summarize the epidemiology and pathophysiology of environmental stressors with a focus on cardiovascular NCDs. We will also discuss solutions and mitigation measures to lower the impact of environmental risk factors with focus on cardiovascular disease.

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

Cardiovasc Res: 04 Oct 2021; epub ahead of print

Integration of multiple imaging platforms to uncover cardiovascular defects in adult zebrafish.

Bensimon-Brito A, Boezio GLM, Cardeira-da-Silva J, Wietelmann A, ... Mueller C, Stainier DYR
Aims
Mammalian models have been instrumental in investigating adult heart function and human disease. However, electrophysiological differences with human hearts and high costs motivate the need for non-mammalian models. The zebrafish is a well-established genetic model to study cardiovascular development and function; however, analysis of cardiovascular phenotypes in adult specimens is particularly challenging as they are opaque.
Methods and results
Here, we optimized and combined multiple imaging techniques including echocardiography, magnetic resonance imaging, and micro-computed tomography to identify and analyze cardiovascular phenotypes in adult zebrafish. Using alk5a/tgfbr1a mutants as a case study, we observed morphological and functional cardiovascular defects that were undetected with conventional approaches. Correlation analysis of multiple parameters revealed an association between hemodynamic defects and structural alterations of the heart, as observed clinically.
Conclusion
We report a new, comprehensive, and sensitive platform to identify otherwise indiscernible cardiovascular phenotypes in adult zebrafish.
Translational perspective
This study further illustrates the importance of the zebrafish model to investigate cardiovascular phenotypes including morphological and functional alterations as observed in human disease settings.

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

Cardiovasc Res: 04 Oct 2021; epub ahead of print

Remdesivir induces persistent mitochondrial and structural damage in human induced pluripotent stem cell derived cardiomyocytes.

Kwok M, Lee C, Li HS, Deng R, ... Yan BP, Poon EN
Aims
Remdesivir is a prodrug of an adenosine triphosphate analogue and is currently the only drug formally approved for the treatment of hospitalised COVID-19 patients. Nucleoside/nucleotide analogues have been shown to induce mitochondrial damage and cardiotoxicity, and this may be exacerbated by hypoxia, which frequently occurs in severe COVID-19 patients. Although there have been few reports of adverse cardiovascular events associated with remdesivir, clinical data are limited. Here, we investigated whether remdesivir induced cardiotoxicity using an in vitro human cardiac model.
Methods and results
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were exposed to remdesivir under normoxic and hypoxic conditions to simulate mild and severe COVID-19 respectively. Remdesivir induced mitochondrial fragmentation, reduced redox potential and suppressed mitochondrial respiration at levels below the estimated plasma concentration under both normoxic and hypoxic conditions. Non-mitochondrial damage such as electrophysiological alterations and sarcomere disarray were also observed. Importantly, some of these changes persisted after the cessation of treatment, culminating in increased cell death. Mechanistically, we found that inhibition of DRP1, a regulator of mitochondrial fission, ameliorated the cardiotoxic effects of remdesivir, showing that remdesivir-induced cardiotoxicity was preventable and excessive mitochondrial fission might contribute to this phenotype.
Conclusions
Using an in vitro model, we demonstrated that remdesivir can induce cardiotoxicity in hiPSC-CMs at clinically relevant concentrations. These results reveal previously unknown potential side-effects of remdesivir and highlight the importance of further investigations with in vivo animal models and active clinical monitoring to prevent lasting cardiac damage to patients.
Translational perspective
Adult cardiomyocytes have limited ability to regenerate, thus treatment-induced cardiotoxicity can potentially cause irreparable harm. Remdesivir is currently the only FDA approved treatment for COVID-19 but clinical safety data are limited. Using human pluripotent stem cell-derived cardiomyocytes, we revealed that remdesivir induced persistent mitochondrial and structural abnormalities at clinically relevant concentrations. We advise confirmatory experiments in in vivo animal models, investigations of cardioprotective strategies, and closer patient monitoring such that treatment-induced cardiotoxicity does not contribute to the long term sequelae of COVID-19 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: 04 Oct 2021; epub ahead of print

Low molecular weight heparin use in COVID-19 is associated with curtailed viral persistence-a retrospective multicenter observational study.

Pereyra D, Heber S, Schrottmaier WC, Santol J, ... Zoufaly A, Assinger A
Aim
Anticoagulation was associated with improved survival of hospitalized COVID-19 patients in large-scale studies. Yet, the development of COVID-19 associated coagulopathy (CAC) and the mechanism responsible for improved survival of anticoagulated patients with COVID-19 remain largely elusive. This investigation aimed to explore the effects of anticoagulation and low molecular weight heparin (LMWH) in particular on patient outcome, CAC development, thromboinflammation, cell death, and viral persistence.
Methods and results
Data of 586 hospitalized COVID-19 patients from three different regions of Austria were evaluated retrospectively. Of these 419 (71.5%) patients received LMWH and 62 (10.5%) received non-vitamin-K antagonist oral anticoagulants (NOAC) during hospitalization. Plasma was collected at different time points in a subset of 106 patients in order to evaluate markers of thromboinflammation (H3Cit-DNA) and the cell death marker cell-free DNA (cfDNA). Use of LMWH was associated with improved survival upon multivariable Cox-regression (hazard ratio = 0.561, 95% confidence interval: 0.348-0.906). Interestingly, neither LMWH nor NOAC was associated with attenuation of D-dimer increase over time, or thromboinflammation. In contrast, anticoagulation was associated with a decrease in cfDNA during hospitalization and curtailed viral persistence was observed in patients using LMWH leading to a four-day reduction of virus positivity upon quantitative polymerase chain reaction (13 [interquartile range: 6-24] versus 9 [interquartile range: 5-16] days, p = 0.009).
Conclusions
Time courses of hemostatic and thromboinflammatory biomarkers were similar in patients with and without LMWH, indicating either no effects of LMWH on hemostasis or that LMWH reduced hypercoagulability to levels of patients without LMWH. Nonetheless, anticoagulation with LMWH was associated with reduced mortality, improved markers of cell death, and curtailed viral persistence, indicating potential beneficial effects of LMWH beyond hemostasis, which encourages use of LMWH in COVID-19 patients without contraindications.
Translational perspective
The data gathered in this retrospective multicenter observational study could confirm an association of improved survival and anticoagulation and could link LMWH use to improved biomarkers of cell death and curtailed persistence of SARS-CoV-2. This encourages the use of this drug in patients without known contraindications in case this effect can be consolidated in randomized controlled trials. Currently performed prospective trials using LMWH in COVID-19 should focus on viral persistence in order to explore an additional field of application for LMWH during this pandemic.

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

Cardiovasc Res: 04 Oct 2021; epub ahead of print

MMP-2 knockdown blunts age-dependent carotid stiffness by decreasing elastin degradation and augmenting eNOS activation.

Diaz-Canestro C, Puspitasari YM, Liberale L, Guzik TJ, ... Sudano I, Camici GG
Aims
Arterial stiffness is a hallmark of vascular aging that precedes and strongly predicts the development of cardiovascular diseases. Age-dependent stiffening of large elastic arteries is primarily attributed to increased levels of matrix metalloproteinase-2 (MMP-2). However, the mechanistic link between age-dependent arterial stiffness and MMP-2 remains unclear. Thus, we aimed to investigate the efficacy of MMP-2 knockdown using small interfering RNA (siRNA) on age-dependent arterial stiffness.
Methods and results
Pulse wave velocity (PWV) was assessed in right carotid artery of wild type (WT) mice from different age groups. MMP-2 levels in the carotid artery and plasma of young (3 months) and old (20-25 months) WT mice were determined. Carotid PWV as well as vascular and circulating MMP-2 were elevated with increasing age in mice. Old WT mice (18-21-month-old) were treated for 4 weeks with either MMP-2 or scrambled (Scr) siRNA via tail vein injection. Carotid PWV was assessed at baseline, 2 and 4 weeks after start of the treatment. MMP-2 knockdown reduced vascular MMP-2 levels and attenuated age-dependent carotid stiffness. siMMP-2 treated mice showed increased elastin to collagen ratio, lower plasma desmosine (DES), enhanced phosphorylation of endothelial nitric oxide synthase (eNOS) and higher levels of vascular cyclic guanosine monophosphate (cGMP). An age-dependent increase in direct protein-protein interaction between MMP-2 and eNOS was also observed. Lastly, DES, an elastin breakdown product, was measured in a patient cohort (n = 64, 23-86 years old), where carotid-femoral PWV was also assessed; here, plasma levels of DES directly correlated with age and arterial stiffness.
Conclusion
MMP-2 knockdown attenuates age-dependent carotid stiffness by blunting elastin degradation and augmenting eNOS bioavailability. Given the increasing clinical use of siRNA technology, MMP2 knockdown should be investigated further as a possible strategy to mitigate age-dependent arterial stiffness and related CV diseases.
Translational perspective
Arterial stiffness is a hallmark of vascular aging that precedes and strongly predicts the development of cardiovascular diseases. This study provides translational evidence to support a key role for MMP-2 on the development of age-associated arterial stiffness. Silencing of MMP-2 using siRNA technology shows an effect on aged mice where it attenuates age-dependent carotid stiffness by reducing elastin degradation and increasing eNOS bioavailability. Additionally, in humans we show that elastin breakdown increases with age and increased PWV. These findings indicate MMP-2 knockdown as a promising novel strategy to attenuate age-dependent arterial stiffness and 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: 28 Sep 2021; epub ahead of print

Haploinsufficiency of Tmem43 in cardiac myocytes activates the DNA damage response pathway leading to a late-onset senescence-associated pro-fibrotic cardiomyopathy.

Rouhi L, Cheedipudi SM, Chen SN, Fan S, ... Gurha P, Marian AJ
Aims
Arrhythmogenic cardiomyopathy (ACM) encompasses a genetically heterogeneous group of myocardial diseases whose manifestations are sudden cardiac death, cardiac arrhythmias, heart failure, and in a subset fibro-adipogenic infiltration of the myocardium. Mutations in the TMEM43 gene, encoding transmembrane protein 43 (TMEM43) are known to cause ACM. The purpose of the study was to gain insights into the molecular pathogenesis of ACM caused by TMEM43 haploinsufficiency.
Methods and results
The Tmem43 gene was specifically deleted in cardiac myocytes by crossing the Myh6-Cre and floxed Tmem43 mice. Myh6-Cre:Tmem43W/F mice showed an age-dependent phenotype characterized by an increased mortality, cardiac dilatation and dysfunction, myocardial fibrosis, adipogenesis, and apoptosis. Sequencing of cardiac myocyte transcripts prior to and after the onset of cardiac phenotype predicted early activation of the TP53 pathway. Increased TP53 activity was associated with increased levels of markers of DNA damage response (DDR), and a subset of senescence-associated secretary phenotype (SASP). Activation of DDR, TP53, SASP, and their selected downstream effectors, including phospho-SMAD2 and phospho-SMAD3 were validated by alternative methods, including immunoblotting. Expression of SASP was associated with epithelial-mesenchymal transition and age-dependent expression of myocardial fibrosis and apoptosis in the Myh6-Cre:Tmem43W/F mice.
Conclusion
TMEM43 haploinsufficiency is associated with activation of the DDR and the TP53 pathways, which lead to increased expression of SASP and an age-dependent expression of a pro-fibrotic cardiomyopathy. Given that TMEM43 is a nuclear envelope protein and our previous data showing deficiency of another nuclear envelope protein, namely lamin A/C, activates the DDR/TP53 pathway, we surmise that DNA damage is a shared mechanism in the pathogenesis of cardiomyopathies caused by mutations involving nuclear envelope proteins.

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

Cardiovasc Res: 27 Sep 2021; 117:2377-2394

Dietary carbohydrates restriction inhibits the development of cardiac hypertrophy and heart failure.

Nakamura M, Odanovic N, Nakada Y, Dohi S, ... Abdellatif M, Sadoshima J
Aims
A diet with modified components, such as a ketogenic low-carbohydrate (LC) diet, potentially extends longevity and healthspan. However, how an LC diet impacts on cardiac pathology during haemodynamic stress remains elusive. This study evaluated the effects of an LC diet high in either fat (Fat-LC) or protein (Pro-LC) in a mouse model of chronic hypertensive cardiac remodelling.
Methods and results
Wild-type mice were subjected to transverse aortic constriction, followed by feeding with the Fat-LC, the Pro-LC, or a high-carbohydrate control diet. After 4 weeks, echocardiographic, haemodynamic, histological, and biochemical analyses were performed. LC diet consumption after pressure overload inhibited the development of pathological hypertrophy and systolic dysfunction compared to the control diet. An anti-hypertrophic serine/threonine kinase, GSK-3β, was re-activated by both LC diets; however, the Fat-LC, but not the Pro-LC, diet exerted cardioprotection in GSK-3β cardiac-specific knockout mice. β-hydroxybutyrate, a major ketone body in mammals, was increased in the hearts of mice fed the Fat-LC, but not the Pro-LC, diet. In cardiomyocytes, ketone body supplementation inhibited phenylephrine-induced hypertrophy, in part by suppressing mTOR signalling.
Conclusion
Strict carbohydrate restriction suppresses pathological cardiac growth and heart failure after pressure overload through distinct anti-hypertrophic mechanisms elicited by supplemented macronutrients.

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

Cardiovasc Res: 27 Sep 2021; 117:2365-2376

Approaches to treat pulmonary arterial hypertension by targeting BMPR2: from cell membrane to nucleus.

Dunmore BJ, Jones RJ, Toshner MR, Upton PD, Morrell NW
Pulmonary arterial hypertension (PAH) is estimated to affect between 10 and 50 people per million worldwide. The lack of cure and devastating nature of the disease means that treatment is crucial to arrest rapid clinical worsening. Current therapies are limited by their focus on inhibiting residual vasoconstriction rather than targeting key regulators of the cellular pathology. Potential disease-modifying therapies may come from research directed towards causal pathways involved in the cellular and molecular mechanisms of disease. It is widely acknowledged that targeting reduced expression of the critical bone morphogenetic protein type-2 receptor and its associated signalling pathways is a compelling therapeutic avenue to explore. In this review, we highlight the advances that have been made in understanding this pathway and the therapeutics that are being tested in clinical trials and the clinic to treat PAH.

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 Sep 2021; 117:2309-2325

Inducibility, but not stability, of atrial fibrillation is increased by NOX2 overexpression in mice.

Mighiu AS, Recalde A, Ziberna K, Carnicer R, ... Simon JN, Casadei B
Aims
Gp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility.
Methods and results
NOX2-Tg mice showed a 2- to 2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (twofold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1β, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2+ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype.
Conclusion
Together, these data indicate that while atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.

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

Cardiovasc Res: 27 Sep 2021; 117:2354-2364

Truncated YY1 interacts with BASP1 through a 339KLK341 motif in YY1 and suppresses vascular smooth muscle cell growth and intimal hyperplasia after vascular injury.

Santiago FS, Li Y, Zhong L, Raftery MJ, Lins L, Khachigian LM
Aims
In-stent restenosis and late stent thrombosis are complications associated with the use of metallic and drug-coated stents. Strategies that inhibit vascular smooth muscle cell (SMC) proliferation without affecting endothelial cell (EC) growth would be helpful in reducing complications arising from percutaneous interventions. SMC hyperplasia is also a pathologic feature of graft stenosis and fistula failure. Our group previously showed that forced expression of the injury-inducible zinc finger (ZNF) transcription factor, yin yang-1 (YY1), comprising 414 residues inhibits neointima formation in carotid arteries of rabbits and rats. YY1 inhibits SMC proliferation without affecting EC growth in vitro. Identifying a shorter version of YY1 retaining cell-selective inhibition would make it more amenable for potential use as a gene therapeutic agent.
Methods and results
We dissected YY1 into a range of shorter fragments (YY1A-D, YY1Δ) and found that the first two ZNFs in YY1 (construct YY1B, spanning 52 residues) repressed SMC proliferation. Receptor binding domain analysis predicts a three-residue (339KLK341) interaction domain. Mutation of 339KLK341 to 339AAA341 in YY1B (called YY1Bm) abrogated YY1B\'s ability to inhibit SMC but not EC proliferation and migration. Incubation of recombinant GST-YY1B and GST-YY1Bm with SMC lysates followed by precipitation with glutathione-agarose beads and mass spectrometric analysis identified a novel interaction between YY1B and BASP1. Overexpression of BASP1, like YY1, inhibited SMC but not EC proliferation and migration. BASP1 siRNA partially rescued SMC from growth inhibition by YY1B. In the rat carotid balloon injury model, adenoviral overexpression of YY1B, like full-length YY1, reduced neointima formation, whereas YY1Bm had no such effect. CD31+ immunostaining suggested YY1B could increase re-endothelialization in a 339KLK341-dependent manner.
Conclusion
These studies identify a truncated form of YY1 (YY1B) that can interact with BASP1 and inhibit SMC proliferation, migration, and intimal hyperplasia after balloon injury of rat carotid arteries as effectively as full length YY1. We demonstrate the therapeutic potential of YY1B in vascular proliferative 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: 27 Sep 2021; 117:2395-2406

Dynamin-related protein 1 inhibition reduces hepatic PCSK9 secretion.

Rogers MA, Hutcheson JD, Okui T, Goettsch C, ... Aikawa M, Aikawa E
Aims
Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis.
Methods and results
We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages.
Conclusions
We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor.

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 Sep 2021; 117:2340-2353

Vascular smooth muscle cells in atherosclerosis: time for a re-assessment.

Grootaert MOJ, Bennett MR
Vascular smooth muscle cells (VSMCs) are key participants in both early and late-stage atherosclerosis. VSMCs invade the early atherosclerotic lesion from the media, expanding lesions, but also forming a protective fibrous cap rich in extracellular matrix to cover the \'necrotic\' core. Hence, VSMCs have been viewed as plaque-stabilizing, and decreased VSMC plaque content-often measured by expression of contractile markers-associated with increased plaque vulnerability. However, the emergence of lineage-tracing and transcriptomic studies has demonstrated that VSMCs comprise a much larger proportion of atherosclerotic plaques than originally thought, demonstrate multiple different phenotypes in vivo, and have roles that might be detrimental. VSMCs down-regulate contractile markers during atherosclerosis whilst adopting alternative phenotypes, including macrophage-like, foam cell-like, osteochondrogenic-like, myofibroblast-like, and mesenchymal stem cell-like. VSMC phenotypic switching can be studied in tissue culture, but also now in the media, fibrous cap and deep-core region, and markedly affects plaque formation and markers of stability. In this review, we describe the different VSMC plaque phenotypes and their presumed cellular and paracrine functions, the regulatory mechanisms that control VSMC plasticity, and their impact on atherogenesis and plaque stability.

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 Sep 2021; 117:2326-2339

Two-faced Janus: The dual role of macrophages in atherosclerotic calcification.

Waring OJ, Skenteris NT, Biessen EAL, Donners MMPC
Calcification is an independent predictor of atherosclerosis-related cardiovascular events. Microcalcification is linked to inflamed, unstable lesions, in comparison to the fibrotic stable plaque phenotype generally associated with advanced calcification. This paradox relates to recognition that calcification presents in a wide spectrum of manifestations that differentially impact plaque\'s fate. Macrophages, the main inflammatory cells in atherosclerotic plaque, have a multifaceted role in disease progression. They crucially control the mineralisation process, from microcalcification to the osteoid metaplasia of bone-like tissue. It is a bilateral interaction, that weighs heavily on the overall plaque fate, but remains rather unexplored. This review highlights current knowledge about macrophage phenotypic changes in relation to, and interaction with the calcifying environment. On the one hand, macrophage-led inflammation kickstarts microcalcification through a multitude of interlinked mechanisms, which in turn stimulates phenotypic changes in vascular cell types to drive microcalcification. Macrophages may also modulate the expression/activity of calcification inhibitors and inducers, or eliminate hydroxyapatite nucleation points. Contrarily, direct exposure of macrophages to an early calcifying milieu impacts macrophage phenotype, with repercussions for plaque progression and/or stability. Macrophages surrounding macrocalcification deposits show a more reparative phenotype, modulating extracellular matrix, and expressing osteoclast genes. This phenotypic shift favours gradual displacement of the pro-inflammatory hubs; the lipid necrotic core, by macrocalcification. Parallels to bone metabolism may explain many of these changes to macrophage phenotype, with advanced-calcification able to show homeostatic osteoid metaplasia. As the targeted treatment of vascular calcification developing in atherosclerosis is thus far severely lacking, it is crucial to better understand its mechanisms of development.

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

Cardiovasc Res: 21 Sep 2021; epub ahead of print

Gut microbiota, dysbiosis and atrial fibrillation. Arrhythmogenic mechanisms and potential clinical implications.

Gawałko M, Agbaedeng TA, Saljic A, Müller DN, ... Dobrev D, Linz D
Recent preclinical and observational cohort studies have implicated imbalances in gut microbiota composition as a contributor to atrial fibrillation (AF). The gut microbiota is a complex and dynamic ecosystem containing trillions of microorganisms, which produces bioactive metabolites influencing host health and disease development. In addition to host-specific determinants, lifestyle-related factors such as diet and drugs are important determinants of the gut microbiota composition. In this review, we discuss the evidence suggesting a potential bidirectional association between AF and gut microbiota, identifying gut microbiota-derived metabolites as possible regulators of the AF substrate. We summarize the effect of gut microbiota on the development and progression of AF risk-factors, including heart failure, hypertension, obesity and coronary artery disease. We also discuss the potential antiarrhythmic effects of pharmacological and diet-induced modifications of gut microbiota composition, which may modulate and prevent the progression to AF. Finally, we highlight important gaps in knowledge and areas requiring future investigation. Although data supporting a direct relationship between gut microbiota and AF are very limited at the present time, emerging preclinical and clinical research dealing with mechanistic interactions between gut microbiota and AF is important as it may lead to new insights into AF pathophysiology and the discovery of novel 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: 21 Sep 2021; epub ahead of print

Inflammation during the life cycle of the atherosclerotic plaque.

Libby P
Inflammation orchestrates each stage of the life cycle of atherosclerotic plaques. Indeed, inflammatory mediators likely link many traditional and emerging risk factors with atherogenesis. Atheroma initiation involves endothelial activation with recruitment of leukocytes to the arterial intima, where they interact with lipoproteins or their derivatives that have accumulated in this layer. The prolonged and usually clinically silent progression of atherosclerosis involves periods of smoldering inflammation, punctuated by episodes of acute activation that may arise from inflammatory mediators released from sites of extravascular injury or infection or from subclinical disruptions of the plaque. Smooth muscle cells and infiltrating leukocytes can proliferate but also undergo various forms of cell death that typically lead to formation of a lipid-rich \"necrotic\" core within the evolving intimal lesion. Extracellular matrix synthesized by smooth muscle cells can form a fibrous cap that overlies the lesion\'s core. Thus, during progression of atheroma, cells not only procreate but perish. Inflammatory mediators participate in both processes. The ultimate clinical complication of atherosclerotic plaques involves disruption that provokes thrombosis, either by fracture of the plaque\'s fibrous cap or superficial erosion. The consequent clots can cause acute ischemic syndromes if they embarrass perfusion. Incorporation of the thrombi can promote plaque healing and progressive intimal thickening that can aggravate stenosis and further limit downstream blood flow. Inflammatory mediators regulate many aspects of both plaque disruption and the healing process. Thus, inflammatory processes contribute to all phases of the lifecycle of atherosclerotic plaques, and represent ripe targets for mitigating the 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: 21 Sep 2021; epub ahead of print

The adult heart requires baseline expression of the transcription factor Hand2 to withstand RV pressure overload.

Videira RF, Koop AMC, Ottaviani L, Poels EM, ... Berger RMF, da Costa Martins P
Aims
Research on the pathophysiology of right ventricular (RV) failure has, in spite of the associated high mortality and morbidity, lagged behind compared to the left ventricle (LV).Previous work from our lab revealed that the embryonic basic helix-loop-helix transcription factor heart and neural crest derivatives expressed-2 (Hand2) is re-expressed in the adult heart and activates a \'fetal gene program\' contributing to pathological cardiac remodeling under conditions of LV pressure overload. As such, ablation of cardiac expression of Hand2 conferred protection to cardiac stress and abrogated the maladaptive effects that were observed upon increased expression levels. In this study, we aimed to understand the contribution of Hand2 to RV remodeling in response to pressure overload induced by pulmonary artery banding (PAB).
Methods and results
In the present study, Hand2F/F and MCM- Hand2F/F mice were treated with tamoxifen (control and knockout, respectively) and subjected to six weeks of RV pressure overload induced by PAB. Echocardiographic- and MRI-derived hemodynamic parameters as well as molecular remodeling were assessed for all experimental groups and compared to sham-operated controls. Six weeks after PAB, levels of Hand2 expression increased in the control banded animals but, as expected, remained absent in the knockout hearts. Despite the dramatic differences in Hand2 expression, pressure overload resulted in impaired cardiac function independently of the genotype. In fact, Hand2 depletion seems to sensitize the RV to pressure overload as these mice develop more hypertrophy and more severe cardiac dysfunction. Higher expression levels of HAND2 were also observed in RV samples of human hearts from patients with pulmonary hypertension. In turn, the LV of RV-pressure overloaded hearts was also dramatically affected as reflected by changes in shape, decreased LV mass and impaired cardiac function. RNA sequencing revealed a distinct set of genes that are dysregulated in the pressure-overloaded RV, compared to the previously described pressure-overloaded LV.
Conclusions
Cardiac-specific depletion of Hand2 is associated with severe cardiac dysfunction in conditions of RV pressure overload. While inhibiting Hand2 expression can prevent cardiac dysfunction in conditions of LV pressure overload, the same does not hold true for conditions of RV pressure overload. This study highlights the need to better understand the molecular mechanisms driving pathological remodeling of the RV in contrast to the LV, in order to better diagnose and treat patients with RV or LV failure.
Translational perspective
RV failure associated with pulmonary hypertension reduces long-term survival rate to 55% within 3 years, suggesting that 3 years after diagnosis almost half of the patients will die. To revert these numbers an adequate RV-specific and, therefore, more efficient treatment is needed. Our work suggests that current therapies and potential mechanisms underlying LV failure may not be suitable for RV failure. While Hand2 deletion is favorable in LV response to stress, it is particularly detrimental in the RV under similar conditions, and thus, highlighting potential severe consequences of not differentiating therapeutic targets or treatment for RV or LV failure.

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

Cardiovasc Res: 21 Sep 2021; epub ahead of print

Role of PDE10A in vascular smooth muscle cell hyperplasia and pathological vascular remodeling.

Luo L, Zhang Y, Hsu C, Korshunov VA, ... Berk BC, Yan C
Aims
Intimal hyperplasia is a common feature of vascular remodeling disorders. Accumulation of synthetic smooth muscle cell (SMC)-like cells is the main underlying cause. Current therapeutic approaches including drug-eluting stents are not perfect due to the toxicity on endothelial cells and novel therapeutic strategies are needed. Our preliminary screening for dysregulated cyclic nucleotide phosphodiesterases (PDEs) in growing SMCs revealed the alteration of PDE10A expression. Herein, we investigated the function of PDE10A in SMC proliferation and intimal hyperplasia both in vitro and in vivo.
Methods and results
RT-qPCR, immunoblot, and in situ proximity ligation assay were performed to determine PDE10A expression in synthetic SMCs and injured vessels. We found that PDE10A mRNA and/or protein levels are up-regulated in cultured SMCs upon growth stimulation, as well as in intimal cells in injured mouse femoral arteries. To determine the cellular functions of PDE10A, we focused on its role in SMC proliferation. The anti-mitogenic effects of PDE10A on SMCs were evaluated via cell counting, BrdU incorporation, and flow cytometry. We found that PDE10A deficiency or inhibition arrested the SMC cell cycle at G1-phase with a reduction of cyclin D1. The anti-mitotic effect of PDE10A inhibition was dependent on cGMP-dependent protein kinase Iα (PKGIα), involving C-natriuretic peptide (CNP) and particulate guanylate cyclase natriuretic peptide receptor 2 (NPR2). In addition, the effects of genetic depletion and pharmacological inhibition of PDE10A on neointimal formation were examined in a mouse model of femoral artery wire injury. Both PDE10A knockout and inhibition decreased injury-induced intimal thickening in femoral arteries by at least 50%. Moreover, PDE10A inhibition decreased ex vivo remodeling of cultured human saphenous vein segments.
Conclusions
Our findings indicate that PDE10A contributes to SMC proliferation and intimal hyperplasia at least partially via antagonizing CNP/NPR2/cGMP/PKG1α signaling, and suggest that PDE10A may be a novel drug target for treating vascular occlusive disease.
Translational perspective
Coronary artery disease is currently the leading cause of death worldwide. SMCs are a major contributor to angioplasty restenosis, graft stenosis, and accelerated atherosclerosis. Current therapeutic approaches including drug-eluting stents targeting cell growth still have limitations. By combining studies on cultured SMCs in vitro, animal surgical models in vivo, and a human organ culture model ex vivo, we revealed an important role of PDE10A in modulating SMC proliferation and injury-induced intimal thickening. Given that PDE10A has been proven to be a safe drug target, its inhibition may represent a novel therapeutic strategy for vascular diseases associated with intimal hyperplasia.

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 Sep 2021; epub ahead of print

Neutrophils in cardiovascular disease: Warmongers, peacemakers or both?

Gopalkrishna S, Johnson J, Jaggers RM, Dahdah A, ... Hanssen NMJ, Nagareddy PR
Neutrophils, the most abundant of all leukocytes and the first cells to arrive at the sites of sterile inflammation/injury act as a double-edged sword. On one hand, they inflict a significant collateral damage to the tissues and on the other hand they help facilitate wound healing by a number of mechanisms. Recent studies have drastically changed the perception of neutrophils from being simple one-dimensional cells with an un-restrained mode of action to a cell type that display maturity and complex behavior. It is now recognized that neutrophils are transcriptionally active and respond to plethora of signals by deploying a wide variety of cargo to influence the activity of other cells in the vicinity. Neutrophils can regulate macrophage behavior, display innate immune memory, and play a major role in the resolution of inflammation in a context-dependent manner. In this review, we provide an update on the factors that regulate neutrophil production and the emerging dichotomous role of neutrophils in the context of cardiovascular diseases, particularly in atherosclerosis and the ensuing complications, myocardial infarction and heart failure. Deciphering the complex behavior of neutrophils during inflammation and resolution may provide novel insights and in turn facilitate the development of potential therapeutic strategies to manage 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: 16 Sep 2021; epub ahead of print

Optical ventricular cardioversion by local optogenetic targeting and LED implantation in a cardiomyopathic rat model.

Nyns ECA, Jin T, Fontes MS, van den Heuvel T, ... de Vries AAF, Pijnappels DA
Aims
Ventricular tachyarrhythmias (VTs) are common in the pathologically remodelled heart. These arrhythmias can be lethal, necessitating acute treatment like electrical cardioversion to restore normal rhythm. Recently, it has been proposed that cardioversion may also be realized via optically controlled generation of bioelectricity by the arrhythmic heart itself through optogenetics and therefore without the need of traumatizing high-voltage shocks. However, crucial mechanistic and translational aspects of this strategy have remained largely unaddressed. Therefore, we investigated optogenetic termination of VTs 1) in the pathologically remodelled heart using a 2) implantable multi-LED device for 3) in vivo closed-chest, local illumination.
Methods and results
In order to mimic a clinically relevant sequence of events, transverse aortic constriction (TAC) was applied to adult male Wistar rats before optogenetic modification. This modification took place three weeks later by intravenous delivery of adeno-associated virus vectors encoding red-activatable channelrhodopsin (ReaChR) or Citrine for control experiments. At 8 to 10 weeks after TAC, VTs were induced ex vivo and in vivo, followed by programmed local illumination of the ventricular apex by a custom-made implanted multi-LED device. This resulted in effective and repetitive VT termination in the remodelled adult rat heart after optogenetic modification, leading to sustained restoration of sinus rhythm in the intact animal. Mechanistically, studies on the single cell and tissue level revealed collectively that, despite the cardiac remodelling, there were no significant differences in bioelectricity generation and subsequent transmembrane voltage responses between diseased and control animals, thereby providing insight into the observed robustness of optogenetic VT termination.
Conclusion
Our results show that implant-based optical cardioversion of VTs is feasible in the pathologically remodelled heart in vivo after local optogenetic targeting because of preserved optical control over bioelectricity generation. These findings add novel mechanistic and translational insight into optical ventricular cardioversion.

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

Cardiovasc Res: 15 Sep 2021; epub ahead of print

Single-cell RNA-seq profiling of mouse endothelial cells in response to pulmonary arterial hypertension.

Rodor J, Chen SH, Scanlon JP, Monteiro JP, ... Brittan M, Baker AH
Aims
Endothelial cell dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterise endothelial cell (EC) dynamics in PAH at single-cell resolution.
Methods and results
We carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/Hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and micro-vasculature.
Conclusions
This study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

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

Cardiovasc Res: 15 Sep 2021; epub ahead of print

Endothelial Poldip2 regulates sepsis-induced lung injury via Rho pathway activation.

Dolmatova EV, Forrester SJ, Wang K, Ou Z, ... Hernandes MS, Griendling KK
Aims
Sepsis-induced lung injury is associated with significant morbidity and mortality. Previously, we showed that heterozygous deletion of polymerase δ-interacting protein 2 (Poldip2) was protective against sepsis-induced lung injury. Since endothelial barrier disruption is thought to be the main mechanism of sepsis-induced lung injury, we sought to determine if the observed protection was specifically due to the effect of reduced endothelial Poldip2.
Methods and results
Endothelial-specific Poldip2 knock-out mice (EC-/-) and their wild type littermates (EC+/+) were injected with saline or lipopolysaccharide (LPS, 18 mg/kg) to model sepsis-induced lung injury. At 18 hours post-injection mice, were euthanized and bronchoalveolar lavage (BAL) fluid and lung tissue were collected to assess leukocyte infiltration. Poldip2 EC-/- mice showed reduced lung leukocyte infiltration in BAL (0.21 ± 0.9x106 vs 1.29 ± 1.8x106 cells/mL) and lung tissue (12.7 ± 1.8 vs 23 ± 3.7% neutrophils of total number of cells) compared to Poldip2 EC+/+ mice. qPCR analysis of the lung tissue revealed a significantly dampened induction of inflammatory gene expression (TNFα 2.23 ± 0.39 vs 4.15 ± 0.5 fold, IκBα 4.32 ± 1.53 vs 8.97 ± 1.59 fold), neutrophil chemoattractant gene expression (CXCL1 68.8 ± 29.6 vs 147 ± 25.7 fold, CXCL2 65 ± 25.6 vs 215 ± 27.3 fold) and a marker of endothelial activation (VCAM1 1.25 ± 0.25 vs 3.8 ± 0.38 fold) in Poldip2 EC-/- compared to Poldip2 EC+/+ lungs. An in vitro model using human pulmonary microvascular endothelial cells was used to assess the effect of Poldip2 knock-down on endothelial activation and permeability. TNFα-induced endothelial permeability and VE-cadherin disruption were significantly reduced with siRNA-mediated knockdown of Poldip2 (5 ± 0.5 vs 17.5 ± 3 fold for permeability, 1.5 ± 0.4 vs 10.9 ± 1.3 fold for proportion of disrupted VE-cadherin). Poldip2 knockdown altered expression of Rho-GTPase-related genes, which correlated with reduced RhoA activation by TNFα (0.94 ± 0.05 vs 1.29 ± 0.01 of relative RhoA activity) accompanied by redistribution of active RhoA staining to the center of the cell.
Conclusion
Poldip2 is a potent regulator of endothelial dysfunction during sepsis-induced lung injury, and its endothelium-specific inhibition may provide clinical benefit.

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 Sep 2021; epub ahead of print

Targeting the Hippo pathway in heart repair.

Meng F, Xie B, Martin JF
The Hippo pathway is an evolutionarily and functionally conserved signaling pathway that controls organ size by regulating cell proliferation, apoptosis, and differentiation. Emerging evidence has shown that the Hippo pathway plays critical roles in cardiac development, homeostasis, disease, and regeneration. Targeting the Hippo pathway has tremendous potential as a therapeutic strategy for treating intractable cardiovascular diseases such as heart failure. In this review, we summarize the function of the Hippo pathway in the heart. Particularly, we highlight the posttranslational modification of Hippo pathway components, including the core kinases LATS1/2 and their downstream effectors YAP/TAZ, in different contexts, which has provided new insights and avenues in cardiac research.

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 Sep 2021; epub ahead of print

Cardiovascular disease and COVID-19: a consensus paper from the ESC Working Group on Coronary Pathophysiology & Microcirculation, ESC Working Group on Thrombosis and the Association for Acute CardioVascular Care (ACVC), in collaboration with the European Heart Rhythm Association (EHRA).

Cenko E, Badimon L, Bugiardini R, Claeys MJ, ... Vilahur G, Tousoulis D
The cardiovascular system is significantly affected in coronavirus disease-19 (COVID-19). Microvascular injury, endothelial dysfunction and thrombosis resulting from viral infection or indirectly related to the intense systemic inflammatory and immune responses are characteristic features of severe COVID-19. Pre-existing cardiovascular disease and viral load are linked to myocardial injury and worse outcomes. The vascular response to cytokine production and the interaction between SARS-CoV-2 and ACE2 receptor may lead to a significant reduction in cardiac contractility and subsequent myocardial dysfunction. In addition, a considerable proportion of patients who have been infected with SARS-CoV-2 do not fully recover and continue to experience a large number of symptoms and post-acute complications in the absence of a detectable viral infection. This conditions often referred to as \"post-acute COVID-19\" may have multiple causes. Viral reservoirs or lingering fragments of viral RNA or proteins contribute to the condition. Systemic inflammatory response to COVID-19 has the potential to increase myocardial fibrosis which in turn may impair cardiac remodelling. Here we summarize the current knowledge of cardiovascular injury and post-acute sequelae of COVID-19. As the pandemic continues and new variants emerge, we can advance our knowledge of the underlying mechanisms only by integrating our understanding of the pathophysiology with the corresponding clinical findings. Identification of new biomarkers of cardiovascular complications, and development of effective treatments for COVID-19 infection are of crucial importance.

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

Cardiovasc Res: 15 Sep 2021; epub ahead of print

Circadian rhythms in ischaemic heart disease. Key aspects for preclinical and translational research: Position paper of the ESC Working Group on Cellular Biology of the Heart.

Lecour S, Du Pré BC, Bøtker HE, Brundel BJJM, ... Young ME, Van Laake LW
Circadian rhythms are internal regulatory processes controlled by molecular clocks present in essentially every mammalian organ that temporally regulate major physiological functions. In the cardiovascular system, the circadian clock governs heart rate, blood pressure, cardiac metabolism, contractility and coagulation. Recent experimental and clinical studies highlight the possible importance of circadian rhythms in the pathophysiology, outcome, or treatment success of cardiovascular disease, including ischaemic heart disease. Disturbances in circadian rhythms are associated with increased cardiovascular risk and worsen outcome. Therefore, it is important to consider circadian rhythms as a key research parameter to better understand cardiac physiology/pathology, and to improve the chances of translation and efficacy of cardiac therapies, including those for ischaemic heart disease. The aim of this Position Paper by the European Society of Cardiology Working Group Cellular Biology of the Heart is to highlight key aspects of circadian rhythms to consider for improvement of preclinical and translational studies related to ischaemic heart disease and cardioprotection. Applying these considerations to future studies may increase the potential for better translation of new treatments into successful clinical outcomes.

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

Cardiovasc Res: 09 Sep 2021; epub ahead of print

Cardiovascular co-morbidities, inflammation and cerebral small vessel disease.

Evans LE, Taylor JL, Smith CJ, Pritchard HAT, Greenstein AS, Allan SM
Cerebral small vessel disease (cSVD) is the most common cause of vascular cognitive impairment and affects all levels of the brain\'s vasculature. Features include diverse structural and functional changes affecting small arteries and capillaries that lead to a decline in cerebral perfusion. Due to an aging population, incidence of cerebral small vessel disease (cSVD) is continually rising. Despite its prevalence and its ability to cause multiple debilitating illnesses, such as stroke and dementia, there are currently no therapeutic strategies for the treatment of cSVD. In the healthy brain, interactions between neuronal, vascular and inflammatory cells are required for normal functioning. When these interactions are disturbed, chronic pathological inflammation can ensue. The interplay between cSVD and inflammation has attracted much recent interest and this review discusses chronic cardiovascular diseases, particularly hypertension, and explores how the associated inflammation may impact on the structure and function of the small arteries of the brain in cSVD. Molecular approaches in animal studies are linked to clinical outcomes in patients and novel hypotheses regarding inflammation and cSVD are proposed that will hopefully stimulate further discussion and study in this important area.

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

Cardiovasc Res: 08 Sep 2021; epub ahead of print

PLX3397, a CSF1 receptor inhibitor, limits allotransplantation-induced vascular remodeling.

Almonte VM, Uriyanghai U, Egaña-Gorroño L, Parikh D, ... Riascos-Bernal DF, Sibinga NES
Aims
Graft vascular disease (GVD), a clinically important and highly complex vascular occlusive disease, arises from the interplay of multiple cellular and molecular pathways. While occlusive intimal lesions are composed predominantly of smooth muscle-like cells (SMLCs), the origin of these cells and the stimuli leading to their accumulation in GVD are uncertain. Macrophages have recently been identified as both potential drivers of intimal hyperplasia and as precursors that undergo transdifferentiation to become SMLCs in non-transplant settings. Colony stimulating factor-1 (CSF1) is a well-known regulator of macrophage development and differentiation, and prior preclinical studies have shown that lack of CSF1 limits GVD. We sought to identify the origins of SMLCs and of cells expressing the CSF1 receptor (CSF1R) in GVD, and to test the hypothesis that pharmacologic inhibition of CSF1 signaling would curtail both macrophage and SMLC activities and decrease vascular occlusion.
Methods and results
We used genetically modified mice and a vascular transplant model with minor antigen mismatch to assess cell origins. We found that neointimal SMLCs derive from both donor and recipient, and that transdifferentiation of macrophages to SMLC phenotype is minimal in this model. Cells expressing CSF1R in grafts were identified as recipient-derived myeloid cells of Cx3cr1-lineage, and these cells rarely expressed smooth muscle marker proteins. Blockade of CSF1R activity using the tyrosine kinase inhibitor PLX3397 limited the expression of genes associated with innate immunity and decreased levels of circulating monocytes and intimal macrophages. Importantly, PLX3397 attenuated the development of GVD in arterial allografts.
Conclusion
These studies provide proof of concept for pharmacologic inhibition of the CSF1/CSF1R signaling pathway as a therapeutic strategy in GVD. Further preclinical testing of this pathway in GVD is warranted.
Translational perspective
Graft vascular disease is a major limitation to the long-term success of clinical solid organ transplantation. Currently, there are no effective treatment options to prevent the development of neointimal lesions that obstruct blood flow to the graft. In this study we found that PLX3397, a selective inhibitor of CSF1R signaling, reduced the accumulation of macrophages and ACTA2+ cells within neointimal lesions in a preclinical model of graft vascular disease. Our study highlights a promising role for the pharmacologic targeting of CSF1R signaling to further study the molecular mechanisms that regulate allotransplantation-induced vascular remodeling.

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

Cardiovasc Res: 02 Sep 2021; epub ahead of print

Exercise Effects On Cardiovascular Disease: From Basic Aspects To Clinical Evidence.

Sanchis-Gomar F, Lavie CJ, Marín J, Perez-Quilis C, ... Perez MV, Blair SN
Cardiovascular (CV) disease (CVD) remains the leading cause of major morbidity and CVD- and all-cause mortality in most of the world. It is now clear that regular physical activity (PA) and exercise training (ET) induces a wide range of direct and indirect physiologic adaptations and pleiotropic benefits for human general and CV health. Generally, higher levels of PA, ET, and cardiorespiratory fitness (CRF) are correlated with reduced risk of CVD, including myocardial infarction, CVD-related death, and all-cause mortality. Although exact details regarding the ideal doses of ET, including resistance and, especially, aerobic ET, as well as the potential adverse effects of extreme levels of ET, continue to be investigated, there is no question that most of the world\'s population have insufficient levels of PA/ET, and many also have lower than ideal levels of CRF. Therefore, assessment and promotion of PA, ET, and efforts to improve levels of CRF should be integrated into all health professionals\' practices worldwide. In this state-of-the-art review, we discuss the exercise effects on many areas related to CVD, from basic aspects to clinical practice.

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

Cardiovasc Res: 02 Sep 2021; epub ahead of print

Diseases of the Aorta and Kidney Disease: Conclusions from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference.

Sarafidis P, Martens S, Saratzis A, Kadian-Dodov D, ... Johansen K, for Conference Participants
Chronic kidney disease (CKD) is an independent risk factor for the development of abdominal aortic aneurysm (AAA), as well as for cardiovascular and renal events and all-cause mortality following surgery for AAA or thoracic aortic dissection. In addition, the incidence of acute kidney injury (AKI) after any aortic surgery is particularly high, and this AKI per se is independently associated with future cardiovascular events and mortality. On the other hand, both development of AKI after surgery and the long-term evolution of kidney function differ significantly depending on the type of AAA intervention (open surgery versus the various subtypes of endovascular repair). Current knowledge regarding AAA in the general population may not be always applicable to CKD patients, as they have a high prevalence of co-morbid conditions and elevated risk for periprocedural complications. This summary of a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference group discussion reviews the epidemiology, pathophysiology, diagnosis, and treatment of Diseases of the Aorta in CKD and identifies knowledge gaps, areas of controversy, and priorities for future research.

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

Cardiovasc Res: 31 Aug 2021; epub ahead of print

Cardiovascular disease related circulating biomarkers and cancer incidence and mortality: is there an association?

Jovani M, Liu EE, Paniagua SM, Lau ES, ... Chan AT, Ho JE
Aims
Recent studies suggest an association between cardiovascular disease (CVD) and cancer incidence/mortality, but the pathophysiological mechanisms underlying these associations are unclear. We aimed to examine biomarkers previously associated with CVD and study their association with incident cancer and cancer-related death in a prospective cohort study.
Methods and results
We used a proteomic platform to measure 71 cardiovascular biomarkers among 5,032 participants in the Framingham Heart Study who were free of cancer at baseline. We used multivariable-adjusted Cox models to examine the association of circulating protein biomarkers with risk of cancer incidence and mortality. To account for multiple testing, we set a 2-sided false discovery rate (FDR Q-value) <0.05.Growth differentiation factor-15 (GDF15; also known as macrophage inhibitory cytokine-1 [MIC1])) was associated with increased risk of incident cancer (hazards ratio [HR] per 1 standard deviation increment 1.31, 95% CI 1.17-1.47), incident gastrointestinal cancer (HR 1.85, 95% CI 1.37-2.50), incident colorectal cancer (HR 1.94, 95% CI 1.29-2.91) and cancer-related death (HR 2.15, 95% CI 1.72-2.70). Stromal cell-derived factor-1 (SFD1) showed an inverse association with cancer-related death (HR 0.75, 95% CI 0.65-0.86). Fibroblast growth factor-23 (FGF23) showed an association with colorectal cancer (HR 1.55, 95% CI 1.20-2.00), and granulin (GRN) was associated with hematologic cancer (HR 1.61, 95% CI 1.30-1.99). Other circulating biomarkers of inflammation, immune activation, metabolism, and fibrosis showed suggestive associations with future cancer diagnosis.
Conclusion
We observed several significant associations between circulating CVD biomarkers and cancer, supporting the idea that shared biological pathways underlie both diseases. Further investigations of specific mechanisms that lead to both CVD and cancer are warranted.
Translational perspective
In our prospective cohort study, baseline levels of biomarkers previously associated with CVD were found to be associated with future development of cancer. In particular, GDF15 was associated with increased risk of cancer incidence and mortality, including gastrointestinal and colorectal cancers; SDF1 was inversely associated with cancer-related death, and FGF23 and GRN were associated with increased risk of colorectal and hematologic cancers, respectively. Other biomarkers of inflammation, immune activation, metabolism, and fibrosis showed suggestive associations. These results suggest potential shared biological pathways that underlie both development of cancer and CVD.

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 Aug 2021; epub ahead of print

The conserved long noncoding RNA CARMA regulates cardiomyocyte differentiation.

Kay M, Soltani BM, Nemir M, Aghagolzadeh P, ... Baharvand H, Pedrazzini T
Aims
Production of functional cardiomyocytes from pluripotent stem cells requires tight control of the differentiation process. Long noncoding RNAs (lncRNAs) exert critical regulatory function in cell specification during development. In this study, we designed an integrated approach to identify lncRNAs implicated in cardiogenesis in differentiating human embryonic stem cells (ESCs).
Methods and results
We identified CARMA (CARdiomyocyte Maturation-Associated lncRNA), a conserved lncRNA controlling cardiomyocyte differentiation and maturation in human ESCs. CARMA is located adjacent to MIR-1-1HG, the host gene for two cardiogenic miRNAs: MIR1-1 and MIR-133a2, and transcribed in an antisense orientation. The expression of CARMA and the miRNAs is negatively correlated, and CARMA knockdown increases MIR1-1 and MIR-133a2 expression. In addition, CARMA possesses MIR-133a2 binding sites, suggesting the lncRNA could be also a target of miRNA action. Upon CARMA downregulation, MIR-133a2 target protein-coding genes are coordinately downregulated. Among those, we found RBPJ, the gene encoding the effector of the NOTCH pathway. NOTCH has been shown to control a binary cell fate decision between the mesoderm and the neuroectoderm lineages, and NOTCH inhibition leads to enhanced cardiomyocyte differentiation at the expense of neuroectodermal derivatives. Interestingly, two lncRNAs, linc1230 and linc1335, which are known repressors of neuroectodermal specification, were found upregulated upon Notch1 silencing in ESCs. Forced expression of either linc1230 or linc1335 improved ESC-derived cardiomyocyte production. These two lncRNAs were also found upregulated following CARMA knockdown in ESCs.
Conclusions
Altogether, these data suggest the existence of a network, implicating three newly identified lncRNAs, the two myomirs MIR1-1 and MIR-133a2 and the NOTCH signaling pathway, for the coordinated regulation of cardiogenic differentiation in ESCs.
Translational perspective
Cardiac dysfunction and heart failure develop secondary to a massive loss of cardiomyocytes in the damaged myocardium. Several avenues have been evaluated to promote regeneration following injury. Cell therapy for heart disease envisages the production of functional cardiomyocytes from differentiating pluripotent stem cells prior transfer into the injured heart muscle. Here, we report the functional characterization of CARMA, a lncRNA implicated in cardiogenesis. CARMA knockdown in differentiating human embryonic stem cells (ESCs) promotes cardiogenic commitment and cardiomyocyte differentiation. CARMA represents therefore a novel target for improving human ESC-derived cardiomyocyte production, and cell-based regenerative strategies for heart 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: 29 Aug 2021; epub ahead of print

Sirtuin 5 promotes arterial thrombosis by blunting the fibrinolytic system.

Liberale L, Akhmedov A, Vlachogiannis NI, Bonetti NR, ... Stellos K, Camici GG
Aims
Arterial thrombosis as a result of plaque rupture or erosion is a key event in acute cardiovascular events. Sirtuin 5 (SIRT5) belongs to the lifespan-regulating sirtuin superfamily and has been implicated in acute ischaemic stroke and cardiac hypertrophy. This project aims at investigating the role of SIRT5 in arterial thrombus formation.
Methods and results
Sirt5 transgenic (Sirt5Tg/0) and knock-out (Sirt5-/-) mice underwent photochemically induced carotid endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) were treated with SIRT5 silencing-RNA (si-SIRT5) as well as peripheral blood mononuclear cells from acute coronary syndrome (ACS) patients and non-ACS controls (case-control study, total n = 171) were used to increase the translational relevance of our data. Compared to wild-type controls, Sirt5Tg/0 mice displayed accelerated arterial thrombus formation following endothelial-specific damage. Conversely, in Sirt5-/- mice, arterial thrombosis was blunted. Platelet function was unaltered, as assessed by ex vivo collagen-induced aggregometry. Similarly, activation of the coagulation cascade as assessed by vascular and plasma tissue factor (TF) and TF pathway inhibitor expression was unaltered. Increased thrombus embolization episodes and circulating D-dimer levels suggested augmented activation of the fibrinolytic system in Sirt5-/- mice. Accordingly, Sirt5-/- mice showed reduced plasma and vascular expression of the fibrinolysis inhibitor plasminogen activator inhibitor (PAI)-1. In HAECs, SIRT5-silencing inhibited PAI-1 gene and protein expression in response to TNF-α. This effect was mediated by increased AMPK activation and reduced phosphorylation of the MAP kinase ERK 1/2, but not JNK and p38 as shown both in vivo and in vitro. Lastly, both PAI-1 and SIRT5 gene expressions are increased in ACS patients compared to non-ACS controls after adjustment for cardiovascular risk factors, while PAI-1 expression increased across tertiles of SIRT5.
Conclusion
SIRT5 promotes arterial thrombosis by modulating fibrinolysis through endothelial PAI-1 expression. Hence, SIRT5 may be an interesting therapeutic target in the context of atherothrombotic 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: 28 Aug 2021; 117:2275-2288

Cardiac cellularity is dependent upon biological sex and is regulated by gonadal hormones.

Squiers GT, McLellan MA, Ilinykh A, Branca J, Rosenthal NA, Pinto AR
Aims
Sex differences have been consistently identified in cardiac physiology and incidence of cardiac disease. However, the underlying biological causes for the differences remain unclear. We sought to characterize the cardiac non-myocyte cellular landscape in female and male hearts to determine whether cellular proportion of the heart is sex-dependent and whether endocrine factors modulate the cardiac cell proportions.
Methods and results
Utilizing high-dimensional flow cytometry and immunofluorescence imaging, we found significant sex-specific differences in cellular composition of the heart in adult and juvenile mice, that develops postnatally. Removal of systemic gonadal hormones by gonadectomy results in rapid sex-specific changes in cardiac non-myocyte cellular proportions including alteration in resident mesenchymal cell and leucocyte populations, indicating gonadal hormones and their downstream targets regulate cardiac cellular composition. The ectopic reintroduction of oestrogen and testosterone to female and male mice, respectively, reverses many of these gonadectomy-induced compositional changes.
Conclusion
This work shows that the constituent cell types of the mouse heart are hormone-dependent and that the cardiac cellular landscapes are distinct in females and males, remain plastic, and can be rapidly modulated by endocrine factors. These observations have implications for strategies aiming to therapeutically alter cardiac cellular heterogeneity and underscore the importance of considering biological sex for studies examining cardiac physiology and stress responses.

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

Cardiovasc Res: 28 Aug 2021; 117:2252-2262

Carvedilol induces biased β1 adrenergic receptor-nitric oxide synthase 3-cyclic guanylyl monophosphate signalling to promote cardiac contractility.

Wang Q, Wang Y, West TM, Liu Y, ... Wei W, Xiang YK
Aims
β-blockers are widely used in therapy for heart failure and hypertension. β-blockers are also known to evoke additional diversified pharmacological and physiological effects in patients. We aim to characterize the underlying molecular signalling and effects on cardiac inotropy induced by β-blockers in animal hearts.
Methods and results
Wild-type mice fed high-fat diet (HFD) were treated with carvedilol, metoprolol, or vehicle and echocardiogram analysis was performed. Heart tissues were used for biochemical and histological analyses. Cardiomyocytes were isolated from normal and HFD mice and rats for analysis of adrenergic signalling, calcium handling, contraction, and western blot. Biosensors were used to measure β-blocker-induced cyclic guanosine monophosphate (cGMP) signal and protein kinase A activity in myocytes. Acute stimulation of myocytes with carvedilol promotes β1 adrenergic receptor (β1AR)- and protein kinase G (PKG)-dependent inotropic cardiac contractility with minimal increases in calcium amplitude. Carvedilol acts as a biased ligand to promote β1AR coupling to a Gi-PI3K-Akt-nitric oxide synthase 3 (NOS3) cascade and induces robust β1AR-cGMP-PKG signal. Deletion of NOS3 selectively blocks carvedilol, but not isoproterenol-induced β1AR-dependent cGMP signal and inotropic contractility. Moreover, therapy with carvedilol restores inotropic contractility and sensitizes cardiac adrenergic reserves in diabetic mice with minimal impact in calcium signal, as well as reduced cell apoptosis and hypertrophy in diabetic hearts.
Conclusion
These observations present a novel β1AR-NOS3 signalling pathway to promote cardiac inotropy in the heart, indicating that this signalling paradigm may be targeted in therapy of heart diseases with reduced ejection fraction.

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

Cardiovasc Res: 28 Aug 2021; 117:2237-2251

Oestrogen inhibits salt-dependent hypertension by suppressing GABAergic excitation in magnocellular AVP neurons.

Jin X, Kim WB, Kim MN, Jung WW, ... Kim YB, Kim YI
Aims
Abundant evidence indicates that oestrogen (E2) plays a protective role against hypertension. Yet, the mechanism underlying the antihypertensive effect of E2 is poorly understood. In this study, we sought to determine the mechanism through which E2 inhibits salt-dependent hypertension.
Methods and results
To this end, we performed a series of in vivo and in vitro experiments employing a rat model of hypertension that is produced by deoxycorticosterone acetate (DOCA)-salt treatment after uninephrectomy. We found that E2 prevented DOCA-salt treatment from inducing hypertension, raising plasma arginine-vasopressin (AVP) level, enhancing the depressor effect of the V1a receptor antagonist (Phenylac1,D-Tyr(Et)2,Lys6,Arg8,des-Gly9)-vasopressin, and converting GABAergic inhibition to excitation in hypothalamic magnocellular AVP neurons. Moreover, we obtained results indicating that the E2 modulation of the activity and/or expression of NKCC1 (Cl- importer) and KCC2 (Cl- extruder) underpins the effect of E2 on the transition of GABAergic transmission in AVP neurons. Lastly, we discovered that, in DOCA-salt-treated hypertensive ovariectomized rats, CLP290 (prodrug of the KCC2 activator CLP257, intraperitoneal injections) lowered blood pressure, and plasma AVP level and hyperpolarized GABA equilibrium potential to prevent GABAergic excitation from emerging in the AVP neurons of these animals.
Conclusion
Based on these results, we conclude that E2 inhibits salt-dependent hypertension by suppressing GABAergic excitation to decrease the hormonal output of AVP neurons.

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

Cardiovasc Res: 28 Aug 2021; 117:2263-2274

Co-expression of calcium and hERG potassium channels reduces the incidence of proarrhythmic events.

Ballouz S, Mangala MM, Perry MD, Heitmann S, ... Hill AP, Vandenberg JI
Aims
Cardiac electrical activity is extraordinarily robust. However, when it goes wrong it can have fatal consequences. Electrical activity in the heart is controlled by the carefully orchestrated activity of more than a dozen different ion conductances. While there is considerable variability in cardiac ion channel expression levels between individuals, studies in rodents have indicated that there are modules of ion channels whose expression co-vary. The aim of this study was to investigate whether meta-analytic co-expression analysis of large-scale gene expression datasets could identify modules of co-expressed cardiac ion channel genes in human hearts that are of functional importance.
Methods and results
Meta-analysis of 3653 public human RNA-seq datasets identified a strong correlation between expression of CACNA1C (L-type calcium current, ICaL) and KCNH2 (rapid delayed rectifier K+ current, IKr), which was also observed in human adult heart tissue samples. In silico modelling suggested that co-expression of CACNA1C and KCNH2 would limit the variability in action potential duration seen with variations in expression of ion channel genes and reduce susceptibility to early afterdepolarizations, a surrogate marker for proarrhythmia. We also found that levels of KCNH2 and CACNA1C expression are correlated in human-induced pluripotent stem cell-derived cardiac myocytes and the levels of CACNA1C and KCNH2 expression were inversely correlated with the magnitude of changes in repolarization duration following inhibition of IKr.
Conclusion
Meta-analytic approaches of multiple independent human gene expression datasets can be used to identify gene modules that are important for regulating heart function. Specifically, we have verified that there is co-expression of CACNA1C and KCNH2 ion channel genes in human heart tissue, and in silico analyses suggest that CACNA1C-KCNH2 co-expression increases the robustness of cardiac electrical activity.

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

Cardiovasc Res: 28 Aug 2021; 117:2216-2227

Differences in biomarkers and molecular pathways according to age for patients with HFrEF.

Ferreira JP, Ouwerkerk W, Santema BT, van Veldhuisen DJ, ... Voors AA, Zannad F
Aims 
Elderly patients with heart failure with reduced ejection fraction (HFrEF) have worse prognosis and less often receive guideline-recommended therapies. We aim to better understand the underlying pathophysiological processes associated with ageing in HFrEF potentially leading to targeted therapies in this vulnerable population.
Methods and results 
From a panel of 363 cardiovascular biomarkers available in 1611 patients with HFrEF in the BIOSTAT-CHF index cohort and cross-validated in 823 patients in the BIOSTAT-CHF validation cohort, we tested which biomarkers were dysregulated in patients aged >75 vs. <65 years. Second, pathway overrepresentation analyses were performed to identify biological pathways linked to higher plasma concentrations of biomarkers in elderly vs. younger patients. After adjustment, multiple test correction [false discovery rate (FDR) 1%], and cross-validation, 27/363 biomarkers were associated with older age, 22 positively and 5 negatively. The biomarkers that were positively associated with older age were associated with tumour cell regulation, extra-cellular matrix organization, and inflammatory processes, whereas biomarkers negatively associated with older age were associated with pathways that may point to cell proliferation and tumourigenesis. Among the 27 biomarkers, WFDC2 (WAP four-disulphide core domain protein 2)-that broadly functions as a protease inhibitor-was associated with older age and had the strongest association with all outcomes. No protein-by-sex interaction was observed.
Conclusions 
In elderly HFrEF patients, pathways associated with extra-cellular matrix organization, inflammatory processes, and tumour cell regulation were activated, while pathways associated with tumour proliferation functions were down-regulated. These findings may help in a better understanding of the ageing processes in HFrEF and identify potential therapeutic targets.

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

Cardiovasc Res: 28 Aug 2021; 117:2228-2236

SPEG: a key regulator of cardiac calcium homeostasis.

Campbell H, Aguilar-Sanchez Y, Quick AP, Dobrev D, Wehrens XHT
Proper cardiac Ca2+ homeostasis is essential for normal excitation-contraction coupling. Perturbations in cardiac Ca2+ handling through altered kinase activity has been implicated in altered cardiac contractility and arrhythmogenesis. Thus, a better understanding of cardiac Ca2+ handling regulation is vital for a better understanding of various human disease processes. \'Striated muscle preferentially expressed protein kinase\' (SPEG) is a member of the myosin light chain kinase family that is key for normal cardiac function. Work within the last 5 years has revealed that SPEG has a crucial role in maintaining normal cardiac Ca2+ handling through maintenance of transverse tubule formation and phosphorylation of junctional membrane complex proteins. Additionally, SPEG has been causally impacted in human genetic diseases such as centronuclear myopathy and dilated cardiomyopathy as well as in common acquired cardiovascular disease such as heart failure and atrial fibrillation. Given the rapidly emerging role of SPEG as a key cardiac Ca2+ regulator, we here present this review in order to summarize recent findings regarding the mechanisms of SPEG regulation of cardiac excitation-contraction coupling in both physiology and human disease. A better understanding of the roles of SPEG will be important for a more complete comprehension of cardiac Ca2+ regulation in physiology and 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: 28 Aug 2021; 117:2175-2185

Network medicine in Cardiovascular Research.

Lee LY, Pandey AK, Maron BA, Loscalzo J
The ability to generate multi-omics data coupled with deeply characterizing the clinical phenotype of individual patients promises to improve understanding of complex cardiovascular pathobiology. There remains an important disconnection between the magnitude and granularity of these data and our ability to improve phenotype-genotype correlations for complex cardiovascular diseases. This shortcoming may be due to limitations associated with traditional reductionist analytical methods, which tend to emphasize a single molecular event in the pathogenesis of diseases more aptly characterized by crosstalk between overlapping molecular pathways. Network medicine is a rapidly growing discipline that considers diseases as the consequences of perturbed interactions between multiple interconnected biological components. This powerful integrative approach has enabled a number of important discoveries in complex disease mechanisms. In this review, we introduce the basic concepts of network medicine and highlight specific examples by which this approach has accelerated cardiovascular research. We also review how network medicine is well-positioned to promote rational drug design for patients with cardiovascular diseases, with particular emphasis on advancing precision medicine.

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

Cardiovasc Res: 28 Aug 2021; 117:2186-2202

Cardiac troponin and defining myocardial infarction.

Kaier TE, Alaour B, Marber M
The 4th Universal Definition of Myocardial Infarction has stimulated considerable debate since its publication in 2018. The intention was to define the types of myocardial injury through the lens of their underpinning pathophysiology. In this review, we discuss how the 4th Universal Definition of Myocardial Infarction defines infarction and injury and the necessary pragmatic adjustments that appear in clinical guidelines to maximize triage of real-world patients.

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

Cardiovasc Res: 28 Aug 2021; 117:2203-2215

Standardised measurement of coronary inflammation using cardiovascular CT: integration in clinical care as a prognostic medical device.

Oikonomou EK, Antonopoulos AS, Schottlander D, Marwan M, ... Achenbach S, Antoniades C
Aims
Coronary CT angiography (CCTA) is a first-line modality in the investigation of suspected coronary artery disease (CAD). Mapping of perivascular Fat Attenuation Index (FAI) on routine CCTA enables the non-invasive detection of coronary artery inflammation by quantifying spatial changes in perivascular fat composition. We now report the performance of a new medical device, CaRi-Heart®, which integrates standardised FAI mapping together with clinical risk factors and plaque metrics to provide individualised cardiovascular risk prediction.
Methods and results
The study included 3912 consecutive patients undergoing CCTA as part of clinical care in the United States (n = 2040) and Europe (n = 1872). These cohorts were used to generate age-specific nomograms and percentile curves as reference maps for the standardised interpretation of FAI. The first output of CaRi-Heart® is the FAI-Score of each coronary artery, which provides a measure of coronary inflammation adjusted for technical, biological and anatomical characteristics. FAI-Score is then incorporated into a risk prediction algorithm together with clinical risk factors and CCTA-derived coronary plaque metrics to generate the CaRi-Heart® Risk that predicts the likelihood of a fatal cardiac event at 8 years. CaRi-Heart® Risk was trained in the US population and its performance was validated externally in the European population. It improved risk discrimination over a clinical risk factor-based model (Δ[C-statistic] of 0.085, P = 0.01 in the US Cohort and 0.149, P < 0.001 in the European cohort) and had a consistent net clinical benefit on decision curve analysis above a baseline traditional risk factor-based model across the spectrum of cardiac risk.
Conclusion
CaRi-Heart® reliably improves cardiovascular risk prediction by incorporating traditional cardiovascular risk factors along with comprehensive CCTA coronary plaque and perivascular adipose tissue phenotyping. This integration advances the prognostic utility of CCTA for individual patients and paves the way for its use as a screening tool among patients referred for CCTA.
Translational perspective
Mapping of perivascular Fat Attenuation Index (FAI) on coronary computed tomography angiography (CCTA) enables the non-invasive detection of coronary artery inflammation by quantifying spatial changes in perivascular fat composition. We now report the performance of a new medical device, CaRi-Heart®, which integrates standardised FAI mapping together with clinical risk factors and plaque metrics to provide age-standardised reference maps and individualised cardiovascular risk prediction. This integration advances the prognostic value of CCTA and paves the way for its use as a screening tool among patients referred for CCTA.

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

Cardiovasc Res: 26 Aug 2021; epub ahead of print

Humoral immunity in atherosclerosis and myocardial infarction: from B cells to antibodies.

Porsch F, Mallat Z, Binder CJ
Immune mechanisms are critically involved in the pathogenesis of atherosclerosis and its clinical manifestations. Associations of specific antibody levels and defined B cell subsets with cardiovascular disease activity in humans as well as mounting evidence from preclinical models demonstrate a role of B cells and humoral immunity in atherosclerotic cardiovascular disease. These include all aspects of B cell immunity, the generation of antigen-specific antibodies, antigen presentation and co-stimulation of T cells, as well as production of cytokines. Through their impact on adaptive and innate immune responses and the regulation of many other immune cells, B cells mediate both protective and detrimental effects in cardiovascular disease. Several antigens derived from (oxidised) lipoproteins, the vascular wall and classical autoantigens have been identified. The unique antibody responses they trigger and their relationship with atherosclerotic cardiovascular disease are reviewed. In particular, we focus on the different effector functions of specific IgM, IgG, and IgE antibodies and the cellular responses they trigger and highlight potential strategies to target B cell functions for therapy.

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

Cardiovasc Res: 26 Aug 2021; epub ahead of print

Innate and adaptative immunity: The understudied driving force of heart valve disease.

Bartoli-Leonard F, Zimmer J, Aikawa E
Calcific aortic valve disease (CAVD) and its clinical manifestation; calcific aortic valve stenosis, is the leading cause for valve disease within the developed world, with no current pharmacological treatment available to delay or halt its progression. Characterized by progressive fibrotic remodeling and subsequent pathogenic mineralization of the valve leaflets, valve disease affects 2.5% of the western population, thus highlighting the need for urgent intervention. Whilst the pathobiology of valve disease is complex, involving genetic factors, lipid infiltration and oxidative damage, the immune system is now being accepted to play a crucial role in pathogenesis and disease continuation. No longer considered a passive degenerative disease, CAVD is understood to be an active inflammatory process, involving a multitude of proinflammatory mechanisms, with both the adaptive and the innate immune system underpinning these complex mechanisms. Within the valve, 15% of cells evolve from haemopoietic origin, and this number greatly expands following inflammation, as macrophages, T lymphocytes, B lymphocytes and innate immune cells infiltrate the valve, promoting further inflammation. Whether chronic immune infiltration or pathogenic clonal expansion of immune cells within the valve or a combination of the two is responsible for disease progression, it is clear that greater understanding of the immune systems role in valve disease is required to inform future treatment strategies for control of CAVD development.

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

Cardiovasc Res: 24 Aug 2021; epub ahead of print

Downregulation of AMPK/PPARδ Signaling promotes endoplasmic reticulum stress-induced endothelial dysfunction in adult rat offspring exposed to maternal diabetes.

Luo H, Lan C, Fan C, Gong X, ... Xu Z, Zeng C
Aims
Exposure to maternal diabetes is associated with increased prevalence of hypertension in the offspring. The mechanisms underlying the prenatal programming of hypertension remain unclear. Because endoplasmic reticulum (ER) stress plays a key role in vascular endothelial dysfunction in hypertension, we investigated whether aberrant ER stress causes endothelial dysfunction and high blood pressure in the offspring of dams with diabetes.
Methods and results
Pregnant Sprague-Dawley rats were intraperitoneally injected with streptozotocin (35 mg/kg) or citrate buffer at day 0 of gestation. Compared with control mother offspring (CMO), the diabetic mother offspring (DMO) had higher blood pressure and impaired endothelium-dependent relaxation (EDR) in mesenteric arteries, accompanied by decreased AMPK phosphorylation and PPARδ expression, increased ER stress markers and reactive oxygen species (ROS) levels. The inhibition of ER stress reversed these aberrant changes in DMO. Ex vivo treatment of mesenteric arteries with an AMPK agonist (A769662) or a PPARδ agonist (GW1516) improved the impaired EDR in DMO and reversed the tunicamycin-induced ER stress, ROS production, and EDR impairment in mesenteric arteries from CMO. The effects of A769662 were abolished by co-treatment with GSK0660 (PPARδ antagonist), whereas the effects of GW1516 were unaffected by Compound C (AMPK inhibitor).
Conclusions
These results suggest an abnormal fetal programming of vascular endothelial function in offspring of rats with maternal diabetes that is associated with increased ER stress, which can be ascribed to down-regulation of AMPK/PPARδ signaling cascade.
Translational perspective
Increasing evidence indicates that fetal programming is an important factor that contributes to the development of cardiovascular disease including hypertension and atherosclerosis later in life. This study explains the roles of AMPK/PPARδ/ER stress signaling cascade and endothelial dysfunction in maternal diabetes-programed adult hypertension in the offspring and provides a potential target for the prevention and therapy of this 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: 19 Aug 2021; epub ahead of print

Lymphatic Contractile Function: A Comprehensive Review of Drug Effects and Potential Clinical Application.

Russell PS, Hong J, Trevaskis NL, Windsor JA, Martin ND, Phillips ARJ
Background
The lymphatic system and the cardiovascular system work together to maintain body fluid homeostasis. Despite that, the lymphatic system has been relatively neglected as a potential drug target and a source of adverse effects from cardiovascular drugs. Like the heart, the lymphatic vessels undergo phasic contractions to promote lymph flow against a pressure gradient. Dysfunction or failure of the lymphatic pump results in fluid imbalance and tissue oedema. While this can due to drug effects, it is also a feature of breast cancer-associated lymphoedema, chronic venous insufficiency, congestive heart failure and acute systemic inflammation. There are currently no specific drug treatments for lymphatic pump dysfunction in clinical use despite the wealth of data from pre-clinical studies.
Aim
To identify (1) drugs with direct effects on lymphatic tonic and phasic contractions with potential for clinical application, and (2) drugs in current clinical use that have a positive or negative side effect on lymphatic function.
Methods
We comprehensively reviewed all studies that tested the direct effect of a drug on the contractile function of lymphatic vessels.
Results
Of the 208 drugs identified from 193 studies, about a quarter had only stimulatory effects on lymphatic tone, contraction frequency and/or contraction amplitude. Of FDA-approved drugs, there were 14 that increased lymphatic phasic contractile function. The most frequently used class of drug with inhibitory effects on lymphatic pump function were the calcium channels blockers.
Conclusion
This review highlights the opportunity for specific drug treatments of lymphatic dysfunction in various disease states and for avoiding adverse drug effects on lymphatic contractile function.

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

Cardiovasc Res: 19 Aug 2021; epub ahead of print

Ischemic tissue released microvesicles induce monocyte reprogramming and increase tissue repair by a tissue factor-dependent mechanism.

Arderiu G, Peña E, Badimon L
Aims
Despite increasing evidence that monocytes may acquire endothelial features, it remains unclear how monocytes participate in angiogenesis after ischemic damage. We investigated whether ischemic cells can release microvesicles (MVs) and promote neovascularisation in a model of peripheral artery disease (PAD).
Methods and results
To model PAD we used an in vivo experimental model of hind limb ischemia (HLI) in mice. MVs were isolated from the ischemic muscle and from peripheral blood at different times after unilateral femoral artery ligation. MVs were phenotypically characterized to identify cell origin. HLI in mice induced the release of MVs with a much higher content of tissue factor (TF) than non-HLI control mice both in the MVs isolated from the affected limb muscle area and from blood. MVs were mainly released from endothelial cells (ECs) and induced Mo differentiation to endothelial cell-like (ECL) cells. Differentiation to ECL cells encompassed highly strict hierarchycal transcription factor activation, initiated by ETS1 activation. MVs secreted by microvascular ECs overexpressing TF (upTF-EMVs), were injected in the ischemic hind limb in parallel with control EMVs (from random siRNA-treated cells) or EMVs released by silenced TF endothelial cells (siTF-EMVs). In animals treated with upTF-EMVs in the ischemic zone there was a highly significant increase in functional new vessels formation (seen by magnetic resonance angiography), a concomitant increase in the pool of circulating Ly6Clow Mo expressing vascular endothelial cell markers, and a significantly higher number of Mo/Macrophages surrounding and integrating the newly formed collaterals.
Conclusion
Ischemia-activated ECs release EMVs rich in TF that induce monocyte differentiation into ECL cells and the formation of new vessels in the ischemic zone. TF by this mechanism of formation of new blood microvessels can contribute to ischemic tissue repair.
Translational perspective
Neovascularization is the cornerstone of limb preservation in peripheral artery disease. Neovessel formation occurring during postnatal development is usually connected with inflammation. Advanced studies in the field of vascular biology have reported that monocytes can acquire endothelial features under angiogenic stimulation. We report that after ischemia affected endothelial cells release microvesicles rich in tissue factor that act as endogenous triggers by interacting with monocytes in an autocrine fashion, coaxing the cells to differentiate into functional endothelial cells. These differentiated cells have the ability to increase blood flow into ischemic tissue. The present study depicts a new concept in the mechanisms governing vessel formation in ischemic tissue.

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 Aug 2021; epub ahead of print

Recommendations for Nomenclature and Definition Of Cell Products Intended for Human Cardiovascular Use.

Taylor DA, Chacon-Alberty L, Sampaio LC, Del Hierro MG, ... Bolli R, Cardiovascular Cell Therapy Research Network (CCTRN)
Exogenous cell-based therapy has emerged as a promising new strategy to facilitate repair of hearts damaged by acute or chronic injury. However, the field of cell-based therapy is handicapped by the lack of standardized definitions and terminology, making comparisons across studies challenging. Even the term \"stem cell therapy\" is misleading because only a small percentage of cells derived from adult bone marrow, peripheral blood, or adipose tissue meets the accepted hematopoietic or developmental definition of stem cells. Furthermore, cells (stem or otherwise) are dynamic biological products, meaning that their surface marker expression, phenotypic and functional characteristics, and the products they secrete in response to their microenvironment can change. It is also important to point out that most surface markers are seldom specific for a cell type. In this article, we discuss the lack of consistency in the descriptive terminology used in cell-based therapies and offer guidelines aimed at standardizing nomenclature and definitions to improve communication among investigators and the general public.

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

Cardiovasc Res: 12 Aug 2021; epub ahead of print

CARDIOVASCULAR EFFECTS OF NON-INSULIN GLUCOSE-LOWERING AGENTS: A COMPREHENSIVE REVIEW OF TRIAL EVIDENCE AND POTENTIAL CARDIOPROTECTIVE MECHANISMS.

Savarese G, Butler J, Lund LH, Bhatt DL, Anker SD
Type 2 diabetes mellitus (T2DM) is highly prevalent and associated with a 2-fold increased mortality, mostly explained by cardiovascular diseases. Trial evidence on older glucose-lowering agents such as metformin and sulfonylureas is limited in terms of cardiovascular efficacy. Since 2008, after rosiglitazone was observed to increase the risk of myocardial infarction and heart failure (HF), cardiovascular outcome trials (CVOT) have been required by regulators for licensing new glucose-lowering agents. In the following CVOTs, dipeptidyl peptidase 4 inhibitors (DPP4i) have been shown to be safe but not to improve morbidity/mortality, except for saxagliptin which increased the risk of HF. Several glucagon-like peptide-1 receptor agonists (GLP1-Ra) and sodium-glucose cotransporter-2 inhibitors (SGLT2i) have been demonstrated to reduce the risk of cardiovascular morbidity and mortality. SGLT2i have shown a class effect for the reduction in risk of HF events in patients with T2DM, leading to trials testing their efficacy/safety in HF regardless of T2DM. In the DAPA-HF and the EMPEROR-Reduced trials dapagliflozin and empagliflozin, respectively, improved cardiovascular mortality/morbidity in patients with HF and reduced ejection fraction (HFrEF), with and without T2DM. Therefore, these drugs are now key part of HFrEF pharmacotherapy. In the SOLOIST-WHF, sotagliflozin reduced cardiovascular mortality/morbidity in patients with T2DM and a recent acute episode of HF regardless of EF. The DELIVER and the EMPEROR-Preserved are testing dapagliflozin and empagliflozin, respectively, in patients with HF with mildly reduced and preserved EF. A strong renal protective role of SGLT2i has also emerged in trials enrolling patients with and without T2DM.

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

Cardiovasc Res: 12 Aug 2021; epub ahead of print

Impairment of mitophagy and autophagy accompanies calcific aortic valve stenosis favoring cell death and the severity of disease.

Morciano G, Patergnani S, Pedriali G, Cimaglia P, ... Ferrari R, Pinton P
Aims
In the last 15 years, some observations tried to shed light on the dysregulation of the cellular self-digestion process in calcific aortic valve stenosis (CAVS), but the results obtained remain still controversial. This work is aimed to definitively establish the trend of autophagy in patients affected by CAVS, to analyze the putative involvement of other determinants which impact on the mitochondrial quality control mechanisms and to explore possible avenues for pharmacological interventions in the treatment of CAVS.
Methods and results
This observational study, performed exclusively in ex vivo human samples (cells and serum), by using biochemical approaches and correlations with clinical data, describes new biological features of the calcified valve in terms of mitochondrial dysfunctions. In detail, we unveiled a significant deficiency in mitochondrial respiration and in ATP production coupled to increased production of lactates. In addition, mitochondrial population in the pathologic group is aged with significant alterations in biogenesis and mitophagy pathways. We are also reporting an updated view about autophagy accompanying the calcification process and advanced stages of the disease. We provided evidence for a rapamycin-based therapeutic strategy to revert the calcified phenotype to the wild type one.
Conclusions
Our data suggest that the Calcific Aortic Valve Stenosis phenotype is featured by defects in mitochondrial quality control mechanisms and that autophagy is not activated enough to counteract cell death and sustain cell functions. Thus, boosting autophagy and mitophagy from short to long-term revert quite all pathological phenotypes.
Translational perspective
The findings from this study provide evidence for new molecular targets involving mitochondrial quality control mechanisms becoming dysregulated in CAVS. These pathways should be considered as amenable for a combination of new therapies in humans for three reasons: i) no pharmacological treatments are still available to slow down the development of advanced CAVS, ii) being calcification a recurring pathway and iii) the targets proposed are druggable by existing drugs used in the clinic for different purposes. This work also suggests a serum biomarker to be highly related to the stage of disease and the calcification grade of the valve.

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

Cardiovasc Res: 09 Aug 2021; epub ahead of print

EDIL3 deficiency ameliorates adverse cardiac remodeling by neutrophil extracellular traps (NET)-mediated macrophage polarization.

Wei X, Zou S, Xie Z, Wang Z, ... Cui K, Li J
Aims
After myocardial infarction (MI), injured cardiomyocytes recruit neutrophils and monocytes/macrophages to myocardium, which in turn initiates inflammatory and reparative cascades, respectively. Either insufficient or excessive inflammation impairs cardiac healing. As an endogenous inhibitor of neutrophil adhesion, EDIL3 plays a crucial role in inflammatory regulation. However, the role of EDIL3 in MI remains obscure. We aimed to define the role of EDIL3 in cardiac remodeling after MI.
Methods and results
Serum EDIL3 levels in MI patients were negatively associated with MI biomarkers. Consistently, WT mice after MI showed low levels of cardiac EDIL3. Compared with WT mice, Edil3-/- mice showed improvement of post-MI adverse remodeling, as they exhibited lower mortality, better cardiac function, shorter scar length and smaller LV cavity. Accordingly, infarcted hearts of Edil3-/- mice contained fewer cellular debris and lower amounts of fibrosis content, with decreased collagen I/III expression and the percentage of α-smooth muscle actin (α-SMA) myofibroblasts. Mechanistically, EDIL3 deficiency did not affect the recruitment of monocytes or T cells, but enhanced neutrophil recruitment and following expansion of pro-inflammatory Mertk-MHC-IIlo-int (myeloid-epithelial-reproductive tyrosine kinase/major histocompatibility complex II) macrophages. The injection of neutrophil-specific C-X-C motif chemokine receptor 2 (CXCR2) antagonist eliminated the differences in macrophage polarization and cardiac function between WT and Edil3-/- mice after MI. Neutrophil extracellular traps (NETs), which were more abundant in the hearts of Edil3-/- mice, contributed to Mertk-MHC-IIlo-int polarization via toll-like receptor 9 pathway. The inhibition of NET formation by treatment of neutrophil elastase inhibitor or DNase I impaired macrophage polarization, increased cellular debris and aggravated cardiac adverse remodeling, thus removed the differences of cardiac function between WT and Edil3-/- mice. Totally, EDIL3 plays an important role in NET-primed macrophage polarization and cardiac remodeling during MI.
Conclusion
We not only reveal that EDIL3 deficiency ameliorates adverse cardiac healing via NET-mediated pro-inflammatory macrophage polarization but also discover a new crosstalk between neutrophil and macrophage after MI.
Translational perspective
We established EDIL3 as a critical regulator of neutrophil recruitment and macrophage polarization during post-MI cardiac remodeling. EDIL3 may be a candidate prognostic biomarker and drug target for cardiovascular diseases. The novel pathways and mechanisms revealed in this study has renewed our understanding of the role of leukocyte adhesion inhibitors in cardiovascular disease. Meanwhile, our study reaffirmed the indispensable role of inflammation in the healing process, thereby prompting the reevaluation of post-MI anti-inflammatory treatments.

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

Cardiovasc Res: 09 Aug 2021; epub ahead of print

MicroRNA-27b-3p downregulates FGF1 and aggravates pathological cardiac remodelling.

Li G, Shao Y, Guo HC, Zhi Y, ... Du J, Li Y
Aims
The heart undergoes pathological remodelling under increased stress and neuronal imbalance. MicroRNAs (miRNAs) are involved in post-transcriptional regulation of genes in cardiac physiology and pathology. However, the mechanisms underlying miRNA-mediated regulation of pathological cardiac remodelling remain to be studied. This study aimed to explore the function of endogenous microRNA-27b-3p (miR-27b-3p) in pathological cardiac remodelling.
Methods and results
miR-27b-3p expression was elevated in the heart of a transverse aortic constriction (TAC)-induced cardiac hypertrophy mouse model. MiR-27b-knockout mice showed significantly attenuated cardiac hypertrophy, fibrosis, and inflammation induced by two independent pathological cardiac hypertrophy models, TAC and Angiotensin II (Ang II) perfusion. Transcriptome sequencing analysis revealed that miR-27b deletion significantly downregulated TAC-induced cardiac hypertrophy, fibrosis, and inflammatory genes. We identified fibroblast growth factor 1 (FGF1) as a miR-27b-3p target gene in the heart and was upregulated in miR-27b-null mice. We found that both recombinant FGF1 (rFGF1) and inhibition of miR-27b-3p enhanced mitochondrial oxidative phosphorylation (OXPHOS) and inhibited cardiomyocyte hypertrophy. Importantly, rFGF1 administration inhibited cardiac hypertrophy and fibrosis in TAC or Ang II-induced models, and enhanced OXPHOS by activating PGC1α/β.
Conclusions
Our study demonstrated that miR-27b-3p induces pathological cardiac remodelling and suggests that inhibition of endogenous miR-27b-3p or administration of FGF1 might have the potential to suppress cardiac remodelling in a clinical setting.
Translational perspective
MicroRNAs (miRNAs) are involved in post-transcriptional regulation of genes in cardiac physiology and pathology. However, the mechanisms underlying miRNA-mediated regulation of pathological cardiac remodelling remain to be studied. We show for the first time that miR-27b deletion attenuates cardiac hypertrophy, fibrosis, and inflammation and that rFGF1 administration inhibits cardiac hypertrophy and fibrosis in TAC- or Ang II-induced models, and enhances OXPHOS by activating PGC1α/β. Our findings suggest that miR-27b-3p and FGF1 may be potential therapeutic targets to treat conditions characterised by pathological cardiac remodelling.

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

Cardiovasc Res: 05 Aug 2021; epub ahead of print

Risk factors profile of young and older patients with Myocardial Infarction.

Sagris M, Antonopoulos AS, Theofilis P, Oikonomou E, ... Kaski JC, Tousoulis D
Myocardial infarction (MI) among young adults (< 45 years) represents a considerable proportion of the total heart attack incidents. The underlying pathophysiologic characteristics, atherosclerotic plaque features and risk factors profile differ between young and older patients with MI. This review article discusses the main differences between the younger and elderly MI patients as well as the different pathogenic mechanisms underlying the development of MI in the younger. Young patients with MI often have eccentric atherosclerotic plaques with inflammatory features but fewer lesions, and are more likely to be smokers, obese, and have poor lifestyle, such as inactivity and alcohol intake. Compared to older MI patients, younger are more likely to be men, have familial-combined hyperlipidemia and increased levels of lipoprotein-a. In addition, MI in younger patients may be related to use of cannabis, cocaine use and androgenic anabolic steroids. Genomic differences especially in the pathways of coagulation and lipid metabolism have also been identified between young and older patients with MI. Better understanding of the risk factors and the anatomic and pathophysiologic processes in young adults can improve MI prevention and treatment strategies in this patient group. Awareness could help identify young subjects at increased risk and guide primary prevention strategies. Additional studies focusing on gene pathways related to lipid metabolism, inflammation and coagulation are needed.

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 Aug 2021; epub ahead of print

Using multimarker screening to identify biomarkers associated with cardiovascular death in patients with atrial fibrillation.

Pol T, Hijazi Z, Lindbäck J, Oldgren J, ... Siegbahn A, Wallentin L
Aims
Atrial fibrillation (AF) is associated with higher mortality. Biomarkers may improve the understanding of key pathophysiologic processes in AF that lead to death. Using a new multiplex analytic technique, we explored the association between 268 biomarkers and cardiovascular (CV) death in anticoagulated patients with AF.
Methods and results
A case-cohort design with 1.8 -1.9 years follow-up. The identification cohort included 517 cases and 4057 randomly selected patients from ARISTOTLE. The validation cohort included 277 cases and 1042 randomly selected controls from RE-LY. Plasma collected at randomization was analyzed with conventional immunoassays and the OLINK proximity extension assay-panels; CVDII, CVDIII, and Inflammation. Association between biomarkers and CV-death was evaluated using Random Survival Forest, Boruta and adjusted Cox-regression analyses.The biomarkers most strongly and consistently associated with CV-death were (hazard ratio for inter-quartile comparison [95% CI]): N-terminal pro-B-type natriuretic peptide (NT-proBNP; 1.63 [1.37-1.93]), cardiac troponin T (cTnT-hs; 1.60[1.35-1.88]), interleukin-6 (IL-6; 1.29[1.13-1.47]), growth differentiation factor-15 (GDF-15; 1.30[1.10-1.53]) fibroblast growth factor 23 (FGF-23; 1.21[1.10-1.33]), urokinase receptor (uPAR; 1.38[1.16-1.64]), trefoil factor 3 (TFF3; 1.27[1.10-1.46]), tumor necrosis factor receptor 1 (TNFR1; 1.21[1.01-1.45]), TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2; 1.18[1.04-1.34]) and cathepsin L1 (CTSL1; 1.22[1.07-1.39]).
Conclusion
In this comprehensive screening of 268 biomarkers in anticoagulated patients with AF the underlying mechanisms most strongly associated with CV-death were cardiorenal dysfunction (NT-proBNP, cTnT-hs, CTSL1, TFF3), oxidative stress (GDF-15), inflammation (IL-6, GDF-15), calcium balance, vascular and renal dysfunction (FGF-23), fibrinolysis (suPAR) and apoptosis (TNFR1, TRAILR2). These findings provide novel insights into pathophysiologic aspects associated with CV-death in AF.
Translational perspective
In patients with AF there is an unmet need for better understanding of the pathophysiological processes involved with CV-death. Using a targeted proteomic approach, 10 biomarkers were identified as having a strong association with CV-death. The identified biomarkers reflect several biological pathways involved with CV-death in AF. The present study provides valuable insights into important processes involved with CV-death in patients with AF and may facilitate the identification of important risk factors for death, thus allowing for earlier intervention and possibly even for targeted therapy to reduce AF-related mortality.
Clinicaltrials.gov identifier
NCT00412984 and NCT00262600.

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

Cardiovasc Res: 05 Aug 2021; epub ahead of print

Functional and structural adaptations of the coronary macro- and micro-vasculature to regular aerobic exercise by activation of physiological, cellular and molecular mechanisms: Esc Working Group on Coronary Pathophysiology & Microcirculation Position Paper.

Koller A, Laughlin MH, Cenko E, de Wit C, ... Gielen S, Duncker DJ
Regular aerobic exercise (RAEX) elicits several positive adaptations in all organs and tissues of the body, culminating in improved health and well-being. Indeed, in over half a century, many studies have shown the benefit of RAEX on cardiovascular outcome in terms of morbidity and mortality. RAEX elicits a wide range of functional and structural adaptations in the heart and its coronary circulation, all of which are to maintain optimal myocardial oxygen and nutritional supply during increased demand. Although there is no evidence suggesting that oxidative metabolism is limited by coronary blood flow (CBF) rate in the normal heart even during maximal exercise, increased CBF and capillary exchange capacities have been reported. Adaptations of coronary macro- and microvessels include outward remodeling of epicardial coronary arteries, increased coronary arteriolar size and density, and increased capillary surface area. In addition, there are adjustments in the neural and endothelial regulation of coronary macrovascular tone. Similarly, there are several adaptations at the level of microcirculation, including enhanced smooth muscle dependent pressure-induced myogenic constriction and upregulated endothelium-dependent flow-/shear-stress-induced dilation, increasing the range of diameter change. Alterations in the signaling interaction between coronary vessels and cardiac metabolism have also been described. At the molecular and cellular level, ion channels are key players in the local coronary vascular adaptations to RAEX, with enhanced activation of influx of Ca2+ contributing to the increased myogenic tone (via voltage gated Ca2+ channels) as well as the enhanced endothelium-dependent dilation (via TRPV4 channels). Finally, RAEX elicits a number of beneficial effects on several hemorheological variables that may further improve CBF and myocardial oxygen delivery and nutrient exchange in the microcirculation by stabilizing and extending the range and further optimizing the regulation of myocardial blood flow during exercise. These adaptations also act to prevent and/or delay the development of coronary and cardiac 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: 05 Aug 2021; epub ahead of print

Vascular endothelial Tissue Factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis.

Witkowski M, Witkowski M, Friebel J, Buffa JA, ... Rauch U, Hazen SL
Aims
Gut microbiota and their generated metabolites impact the host vascular phenotype. The metaorganismal metabolite trimethylamine N-oxide (TMAO) is both associated with adverse clinical thromboembolic events, and enhances platelet responsiveness in subjects. The impact of TMAO on vascular tissue factor (TF) in vivo is unknown. Here, we explore whether TMAO-enhanced thrombosis potential extends beyond TMAO effects on platelets, and is linked to TF. We also further explore the links between gut microbiota and vascular endothelial TF expression in vivo.
Methods and results
In initial exploratory clinical studies, we observed that among sequential stable subjects (n = 2,989) on anti-platelet therapy undergoing elective diagnostic cardiovascular evaluation at a single-site referral center, TMAO levels were associated with an increased incident (3 yr) risk for major adverse cardiovascular events (MACE, myocardial infarction, stroke or death) [4th quartile(Q4) versus Q1 adjusted hazard ratio(95% confidence interval) HR(95%CI), 1.73(1.25-2.38)]. Similar results were observed within subjects on aspirin mono-therapy during follow-up [adjusted HR(95%CI) 1.75(1.25-2.44), n = 2,793). Leveraging access to a second higher risk cohort with previously reported TMAO data and monitoring of anti-platelet medication use, we also observed a strong association between TMAO and incident (1 yr) MACE risk in the multi-site Swiss Acute Coronary Syndromes (ACS) Cohort, focusing on the subset (n = 1,469) on chronic dual anti-platelet therapy during follow-up [adjusted HR(95% CI) 1.70(1.08-2.69)]. These collective clinical data suggest that the thrombosis-associated effects of TMAO may be mediated by cells/factors that are not inhibited by anti-platelet therapy. To test this, we first observed in human microvascular endothelial cells that TMAO dose-dependently induced expression of TF and vascular cell adhesion molecule (VCAM)1. In mouse studies, we observed that TMAO enhanced aortic TF and VCAM1 mRNA and protein expression, which upon immunolocalization studies, was shown to co-localize with vascular endothelial cells. Finally, in arterial injury mouse models, TMAO-dependent enhancement of in vivo TF expression and thrombogenicity were abrogated by either a TF-inhibitory antibody or a mechanism-based microbial choline TMA lyase inhibitor (fluoromethylcholine, FMC).
Conclusions
Endothelial TF contributes to TMAO-related arterial thrombosis potential, and can be specifically blocked by targeted non-lethal inhibition of gut microbial choline TMA lyase.
Translational perspective
The pro-thrombotic effects of the gut microbial TMAO pathway are shown to extend beyond enhancement of platelet responsiveness and include heightened vascular Tissue Factor(TF). In clinical studies, TMAO is shown to predict event risk in patients in the presence of anti-platelet drugs. In animal studies, TMAO elevation is shown to promote vascular endothelial TF expression and a TF-dependent pro-thrombotic effect. Pharmacological targeting of gut microbial choline TMA lyase reduced host TMAO, vascular TF and abrogated the pro-thrombotic TMAO-associated phenotype. These studies suggest inhibiting the TMAO pathway may be a rational target for reducing residual risk in patients on antiplatelet 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: 04 Aug 2021; epub ahead of print

Trimethylamine -N-oxide induces osteogenic responses in human aortic valve interstitial cells in vitro and aggravates aortic valve lesions in mice.

Li J, Zeng Q, Xiong Z, Xian G, ... Ren H, Xu D
Aims
Recent studies have shown that the choline-derived metabolite trimethylamine N-oxide (TMAO) is a biomarker that promotes cardiovascular disease through the induction of inflammation and stress. Inflammatory responses and stress are involved in the progression of calcified aortic valve disease (CAVD). Here, we examined whether TMAO induces the osteogenic differentiation of aortic valve interstitial cells (AVICs) through endoplasmic reticulum (ER) and mitochondrial stress pathways in vitro and in vivo.
Methods and results
Plasma TMAO levels were higher in patients with CAVD (n = 69) than in humans without CAVD (n = 263), as examined by liquid chromatography-tandem mass spectrometry. Western blot and staining probes showed that TMAO- induced an osteogenic response in human AVICs. Moreover, TMAO promoted ER stress, mitochondrial stress and NF-κB activation in vitro. Notably, the TMAO- mediated effects were reversed by the use of ER stress, mitochondrial stress and NF-κB activation inhibitors and siRNA. Mice treated with supplemental choline in a high fat diet had markedly increased TMAO levels and aortic valve thicknesses, which were reduced by 3,3-dimethyl-1-butanol (DMB, an inhibitor of trimethylamine formation) treatment.
Conclusions
Choline-derived TMAO promotes osteogenic differentiation through ER and mitochondrial stress pathways in vitro and aortic valve lesions in vivo.
Translational perspective
Trimethylamine-N-oxide (TMAO), a gut microbiota-generated metabolite, is associated with cardiovascular diseases. Here, we show that patients with calcified aortic valve disease (CAVD) have elevated circulating TMAO levels. TMAO induces osteogenic responses in human aortic valve interstitial cells via endoplasmic reticulum-mitochondrial stress in vitro and aggravates aortic valve lesions in mice. This may provide clues to the pathogenesis of CAVD and attractive potential targets for the prevention, diagnosis and treatment of this disease.

Published on behalf of the European Society of Cardiology 2021.

Cardiovasc Res: 04 Aug 2021; epub ahead of print

Cardiac Allograft Vasculopathy: Current Review and Future Research Directions.

Pober JS, Chih S, Kobashigawa J, Madsen JC, Tellides G
Cardiac allograft vasculopathy (CAV) is a pathologic immune-mediated remodeling of the vasculature in transplanted hearts and, by impairing perfusion, is the major cause of late graft loss. Although best understood following cardiac transplantation, similar forms of allograft vasculopathy occur in other vascularized organ grafts and some features of CAV may be shared with other immune-mediated vasculopathies. Here we describe the incidence and diagnosis, the nature of the vascular remodeling, immune and non-immune contributions to pathogenesis, current therapies and future areas of research in CAV.

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

Cardiovasc Res: 02 Aug 2021; epub ahead of print

Heterogeneity of immune cells in human atherosclerosis revealed by scRNA-Seq.

Vallejo J, Cochain C, Zernecke A, Ley K
Immune cells in atherosclerosis include T, B, natural killer (NK) and NKT cells, macrophages, monocytes, dendritic cells (DCs), neutrophils and mast cells. Advances in single cell RNA sequencing (sRNA-Seq) have refined our understanding of immune cell subsets. Four recent studies have used scRNA-Seq of immune cells in human atherosclerotic lesions and peripheral blood mononuclear cells (PBMCs), some including cell surface phenotypes revealed by oligonucleotide-tagged antibodies, which confirmed known and identified new immune cell subsets and identified genes significantly upregulated in PBMCs from HIV+ subjects with atherosclerosis compared to PBMCs from matched HIV+ subjects without atherosclerosis. The ability of scRNA-Seq to identify cell types is greatly augmented by adding cell surface phenotype using antibody sequencing. In this review we summarize the latest data obtained by scRNA-Seq on plaques and human PBMCs in human subjects with atherosclerosis.

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

Cardiovasc Res: 02 Aug 2021; epub ahead of print

Cardiac natriuretic peptide deficiency sensitizes the heart to stress induced ventricular arrhythmias via impaired CREB signaling.

Hall EJ, Pal S, Glennon MS, Shridhar P, ... Lal H, Becker JR
Aims
The cardiac natriuretic peptides (atrial natriuretic peptide [ANP] and B-type natriuretic peptide [BNP]) are important regulators of cardiovascular physiology, with reduced natriuretic peptide (NP) activity linked to multiple human cardiovascular diseases. We hypothesized that deficiency of either ANP or BNP would lead to similar changes in left ventricular structure and function given their shared receptor affinities.
Methods and results
We directly compared murine models deficient of ANP or BNP in the same genetic backgrounds (C57BL6/J) and environments. We evaluated control, ANP deficient (Nppa-/-) or BNP deficient (Nppb-/-) mice under unstressed conditions and multiple forms of pathological myocardial stress. Survival, myocardial structure, function and electrophysiology, tissue histology, and biochemical analyses were evaluated in the groups. In vitro validation of our findings was performed using human derived induced pluripotent stem cell cardiomyocytes (iPS-CM). In the unstressed state, both ANP and BNP deficient mice displayed mild ventricular hypertrophy which did not increase up to 1 year of life. NP-deficient mice exposed to acute myocardial stress secondary to thoracic aortic constriction (TAC) had similar pathological myocardial remodeling but a significant increase in sudden death. We discovered that the NP-deficient mice are more susceptible to stress induced ventricular arrhythmias using both in vivo and ex vivo models. Mechanistically, deficiency of either ANP or BNP led to reduced myocardial cGMP levels and reduced phosphorylation of the cAMP response element-binding protein (CREBS133) transcriptional regulator. Selective CREB inhibition sensitized wild type hearts to stress induced ventricular arrhythmias. ANP and BNP regulate cardiomyocyte CREBS133 phosphorylation through a cGMP-dependent protein kinase 1 (PKG1) and p38 mitogen activated protein kinase (p38 MAPK) signaling cascade.
Conclusions
Our data show that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels to regulate cardiomyocyte p38 MAPK and CREB activity. Cardiac natriuretic peptide deficiency leads to a reduction in CREB signaling which sensitizes the heart to stress induced ventricular arrhythmias.
Translational perspective
Our study found that ANP or BNP deficiency leads to increased sudden death and ventricular arrhythmias after acute myocardial stress in murine models. We discovered that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels and uncovered a unique role for these peptides in regulating cardiomyocyte p38 MAPK and CREB signaling through a cGMP-PKG1 pathway. Importantly, this signaling pathway was conserved in human cardiomyocytes. This study provides mechanistic insight into how modulating natriuretic peptide levels in human heart failure patients reduces sudden death and mortality.

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

Cardiovasc Res: 29 Jul 2021; epub ahead of print

Resistin deletion protects against heart failure injury by targeting DNA damage response.

Zhao B, Bouchareb R, Lebeche D
Aims
Increased Resistin (Retn) levels are associated with development of cardiovascular diseases. However, the role of Retn in heart failure (HF) is still unclear. Here we probed the functional and molecular mechanism underlying the beneficial effect of Retn deletion in HF.
Methods and results
Wildtype (WT) and adipose tissue-specific Retn-knockout (RKO) mice were subjected to transverse aortic constriction (TAC) induced HF. Cardiac function and hemodynamic changes were measured by echocardiography and left ventricular catheterization. Adipose tissue Retn deletion attenuated while Retn cardiac-selective overexpression, via a recombinant adeno-associated virus-9 vector, exacerbated TAC-induced hypertrophy, cardiac dysfunction and myocardial fibrosis in WT and RKO mice. Mechanistically, we showed that Gadd45α was significantly increased in RKO HF mice while cardiac overexpression of Retn led to its down-regulation. miR148b-3p directly targets Gadd45α and inhibits its expression. Retn overexpression upregulated miR148b-3p expression and triggered DNA damage response (DDR) in RKO-HF mice. Inhibition of miR148b-3p in vivo normalized Gadd45α expression, decreased DDR and reversed cardiac dysfunction and fibrosis. In vitro Retn overexpression in adult mouse cardiomyocytes activated miR148b-3p and reduced Gadd45α expression. Gadd45α overexpression in H9C2-cardiomyoblasts protected against hydrogen peroxide- and Retn-induced DDR.
Conclusion
These findings reveal that diminution in circulating Retn reduced myocardial fibrosis and apoptosis, and improved heart function in a mouse model of HF, at least in part, through attenuation of miR148b-3p and DDR. The results of this study indicate that controlling Retn levels may provide a potential therapeutic approach for treating pressure overload-induced 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: 28 Jul 2021; epub ahead of print

Nitro-oleic acid (NO2-OA) reduces thoracic aortic aneurysm progression in a mouse model of Marfan syndrome.

Nettersheim FS, Lemties J, Braumann S, Geißen S, ... Mollenhauer M, Adam M
Aims
Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the Fibrillin-1 gene. It is associated with formation of thoracic aortic aneurysms that can potentially be a life-threatening condition due to aortic rupture or dissection. Excessive non-canonical transforming growth factor beta signalling, mediated by activation of extracellular-signal regulated kinases 1/2 (ERK1/2), as well as inducible nitric oxide synthase (NOS2)-dependent nitric oxide production have been identified to drive aortic pathology in MFS through induction of elastin fragmentation and smooth muscle cell apoptosis. Despite promising results in animal studies, specific pharmacological interventions approved for clinical use in patients with MFS-related aortic disease are rare. Nitro-oleic acid (NO2-OA) is an endogenously generated signalling modulator, which is available as an oral compound and has been shown to inhibit ERK1/2 activation and NOS2 expression in different disease models, thereby exerting promising therapeutic effects. In this study, we investigated whether NO2-OA decreases aortic dilation in MFS.
Methods and results
Eight-week-old MFS (Fbn1C1041G/+) mice were treated with NO2-OA or vehicle for four weeks via subcutaneously implanted osmotic minipumps. Echocardiography indicated progressive ascending aortic dilation and wall stiffening in MFS mice, which was significantly attenuated by NO2-OA treatment. This protective effect was mediated by inhibition of aortic ERK1/2, Smad2 as well as nuclear factor kappa B overactivation and consequent attenuation of elastin fragmentation by matrix metalloproteinase 2, apoptosis and collagen deposition. Critically, the therapeutic efficacy of NO2-OA in MFS was further emphasized by demonstrating its capability to reduce lethal aortic complications in Fbn1C1041G/+mice challenged with Angiotensin II.
Conclusion
NO2-OA distinctly attenuates progression of aortic dilation in MFS via modulation of well-established disease-mediating pathways, thereby meriting further investigation into its application as a therapeutic agent for the treatment of this condition.
Translational perspective
Thoracic aortic aneurysm formation is the major life-threatening complication of Marfan syndrome, a relatively common genetic connective tissue disorder. Although various potential therapeutic targets have been identified, specific pharmacological treatment options are still unavailable. In this study, we demonstrate that Nitro-oleic acid reduces ascending aortic elastin fragmentation, apoptosis, and fibrotic remodelling in Marfan syndrome through inhibition of extracellular-signal regulated kinases 1/2, Smad2 as well as nuclear factor kappa B overactivation and thereby mitigates aneurysm formation. Thus, Nitro-oleic acid, which has been developed as an oral compound, emerges as a potential treatment option for Marfan-related aortic 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: 28 Jul 2021; epub ahead of print

Acute coronary syndrome is associated with a substantial change in the platelet lipidome.

Harm T, Bild A, Dittrich K, Goldschmied A, ... LäMmerhofer M, Gawaz M
Aims
Platelets play a key role in the pathophysiology of coronary artery disease (CAD) and patients with enhanced platelet activation are at increased risk to develop adverse cardiovascular events. Beyond reliable cardiovascular risk factors such as dyslipoproteinaemia, significant changes of platelet lipids occur in patients with CAD. In this study, we investigate the platelet lipidome by untargeted liquid chromatography-mass spectrometry, highlighting significant changes between acute coronary syndrome (ACS) and chronic coronary syndrome (CCS) patients. Additionally, we classify the platelet lipidome, spotlighting specific glycerophospholipids as key players in ACS patients. Furthermore, we examine the impact of significantly altered lipids in ACS on platelet-dependent thrombus formation and aggregation.
Methods and results
In this consecutive study, we characterized the platelet lipidome in a CAD cohort (n = 139) and showed significant changes of lipids between patients with ACS and CCS. We found that among 928 lipids, 7 platelet glycerophospholipids were significantly up-regulated in ACS, whereas 25 lipids were down-regulated compared to CCS. The most prominent up-regulated lipid in ACS, PC18:0 (PC 10:0-8:0), promoted platelet activation and ex vivo platelet-dependent thrombus formation.
Conclusions
Our results reveal that the platelet lipidome is altered in ACS and up-regulated lipids embody primarily glycerophospholipids. Alterations of the platelet lipidome, especially of medium chain lipids, may play a role in the pathophysiology of ACS.

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 Jul 2021; epub ahead of print

Central role of c-Src in NOX5- mediated redox signaling in vascular smooth muscle cells in human hypertension.

Camargo LL, Montezano AC, Hussain M, Wang Y, ... Hartley RC, Touyz RM
Aims
NOX-derived reactive oxygen species (ROS) are mediators of signaling pathways implicated in vascular smooth muscle cell (VSMC) dysfunction in hypertension. Among the numerous redox-sensitive kinases important in VSMC regulation is c-Src. However, mechanisms linking NOX/ROS to c-Src are unclear, especially in the context of oxidative stress in hypertension. Here we investigated the role of NOX-induced oxidative stress in VSMCs in human hypertension focusing on NOX5, and explored c-Src, as a putative intermediate connecting NOX5-ROS to downstream effector targets underlying VSMC dysfunction.
Methods and results
VSMC from arteries from normotensive (NT) and hypertensive (HT) subjects were studied. NOX1,2,4,5 expression, ROS generation, oxidation/phosphorylation of signaling molecules, actin polymerization and migration were assessed in the absence and presence of NOX5 (melittin) and Src (PP2) inhibitors. NOX5 and p22phox-dependent NOXs (NOX1-4) were downregulated using NOX5 siRNA and p22phox-siRNA approaches. As proof of concept in intact vessels, vascular function was assessed by myography in transgenic mice expressing human NOX5 in a VSMC-specific manner. In HT VSMCs NOX5 was upregulated, with associated oxidative stress, hyperoxidation (c-Src, peroxiredoxin, DJ-1) and hyperphosphorylation (PKC, ERK1/2, MLC20) of signaling molecules. NOX5 siRNA reduced ROS generation in NT and HT subjects. NOX5 siRNA, but not p22phox-siRNA, blunted c-Src phosphorylation in HT VSMCs. NOX5 siRNA reduced phosphorylation of MLC20 and FAK in NT and HT. In p22phox- silenced HT VSMCs, Ang II-induced phosphorylation of MLC20 was increased, effects blocked by melittin and PP2. NOX5 and c-Src inhibition attenuated actin polymerization and migration in HT VSMCs. In NOX5 transgenic mice, vascular hypercontractilty was decreased by melittin and PP2.
Conclusions
We define NOX5/ROS/c-Src as a novel feedforward signaling network in human VSMCs. Amplification of this system in hypertension contributes to VSMC dysfunction. Dampening the NOX5/ROS/c-Src pathway may ameliorate hypertension-associated vascular injury.
Translational perspective
Oxidative stress is a major factor contributing to vascular damage in hypertension. We corroborate experimental evidence that NOX-derived ROS generation is increased in human vascular smooth muscle cells (VSMC) and demonstrate that in human hypertension NOX5 upregulation is a major trigger of VSMC dysfunction. We uncover new regulatory molecular mechanisms of NOX5 and define NOX5/ROS/c-Src as a novel signaling pathway in human VSMCs. This system is augmented in hypertension contributing to abnormal VSMC redox signaling, cytoskeletal disorganization and vascular dysfunction. Modulating the NOX5/ROS/c-Src pathway may have therapeutic potential by targeting redox signaling pathways involved in vascular dysfunction associated with hypertension.

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

Cardiovasc Res: 27 Jul 2021; epub ahead of print