Pathophysiology of Atrial Fibrillation and Chronic Kidney Disease.

Ding WY, Gupta D, Wong CF, Lip GYH

Atrial fibrillation (AF) and chronic kidney disease (CKD) are closely related conditions with shared risk factors. The growing prevalence of both AF and CKD indicates that more patients will suffer from concurrent conditions. There are various complex interlinking mechanisms with important implications for the management of these patients. Furthermore, there is uncertainty regarding the use of oral anticoagulation in AF and CKD that is reflected by a lack of consensus between international guidelines. Therefore, the importance of understanding the implications of co-existing AF and CKD should not be underestimated. In this review, we discuss the pathophysiology and association between AF and CKD, including the underlying mechanisms, risk of thromboembolic and bleeding complications, influence on stroke management, and evidence surrounding the use of oral anticoagulation for stroke prevention.

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

Cardiovasc Res: 31 Aug 2020; epub ahead of print

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

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

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

Cardiovasc Res: 30 Aug 2020; epub ahead of print

Impact of Protein Glycosylation on Lipoprotein Metabolism and Atherosclerosis.

Pirillo A, Svekla M, Catapano AL, Holleboom AG, Norata GD

Protein glycosylation is a post-translational modification consisting of the enzymatic attachment of carbohydrate chains to specific residues of the protein sequence. Several types of glycosylation have been described, with N-glycosylation and O-glycosylation being the most common types impacting on crucial biological processes, such as protein synthesis, trafficking, localization, and function. Genetic defects in genes involved in protein glycosylation may result in altered production and activity of crucial proteins, with a broad range of clinical manifestations, including dyslipidemia and atherosclerosis. A large number of apolipoproteins, lipoprotein receptors, and other proteins involved in lipoprotein metabolism are glycosylated, and alterations in their glycosylation profile are associated with changes in their expression and/or function. Rare genetic diseases and population genetics have provided additional information linking protein glycosylation to the regulation of lipoprotein metabolism.

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

Cardiovasc Res: 03 Sep 2020; epub ahead of print

The effects of liraglutide and dapagliflozin on cardiac function and structure in a multi-hit mouse model of Heart Failure with Preserved Ejection Fraction.

Withaar C, Meems LMG, Markousis-Mavrogenis G, Boogerd CJ, ... Lam CSP, de Boer RA
Aims
Heart failure with preserved ejection fraction (HFpEF) is a multifactorial disease that constitutes several distinct phenotypes, including a common cardiometabolic phenotype with obesity and type 2 diabetes mellitus. Treatment options for HFpEF are limited, and development of novel therapeutics is hindered by the paucity of suitable preclinical HFpEF models that recapitulate the complexity of human HFpEF. Metabolic drugs, like Glucagon Like Peptide Receptor Agonist (GLP-1RA) and Sodium Glucose Transporter 2 inhibitors (SGLT2i), have emerged as promising drugs to restore metabolic perturbations and may have value in the treatment of the cardiometabolic HFpEF phenotype. We aimed to develop a multifactorial HFpEF mouse model that closely resembles the cardiometabolic HFpEF phenotype, and evaluated the GLP-1 RA liraglutide and a SGLT2i dapagliflozin.
Methods & results
Aged (18-22 months old) female C57BL/6J mice were fed a standardized chow (CTRL) or high fat diet (HFD) for 12 weeks. After 8 weeks HFD, Angiotensin-II (ANGII), was administered for 4 weeks via osmotic mini-pumps. HFD+ANGII resulted in a cardiometabolic HFpEF phenotype, including obesity, impaired glucose handling and metabolic dysregulation with inflammation. The multiple-hit resulted in typical clinical HFpEF features, including cardiac hypertrophy and fibrosis with preserved fractional shortening but with impaired myocardial deformation, atrial enlargement lung congestion, and elevated blood pressures. Treatment with liraglutide attenuated the cardiometabolic dysregulation and improved cardiac function, with reduced cardiac hypertrophy, less myocardial fibrosis, and attenuation of atrial weight, natriuretic peptide levels, and lung congestion. Dapagliflozin treatment improved glucose handling, but had mild effects on the HFpEF phenotype.
Conclusions
We developed a mouse model that recapitulates the human HFpEF disease, providing a novel opportunity to study disease pathogenesis and development of enhanced therapeutic approaches. We furthermore show that attenuation of cardiometabolic dysregulation may represent a novel therapeutic target for treatment of HFpEF.
Translational perspective
The failure of many treatment modalities for HFpEF may -at least in part- be explained by the lack of an adequate animal model. The diverse etiology of HFpEF is still largely neglected in pre-clinical research. In this study we developed a murine model that includes advanced age, female sex, in concert with co-morbidities: elevated blood pressure, obesity and T2DM. We demonstrate that this model recapitulates the human cardiometabolic HFpEF phenotype. We showed that contemporary glucose lowering drugs, liraglutide and dapagliflozin, which are both under study for HFpEF, have positive results. Our model may be useful to evaluate novel cardiometabolic, anti-fibrotic, and anti-inflammatory treatments for HFpEF.

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

Cardiovasc Res: 31 Aug 2020; epub ahead of print

B cell-derived anti-beta 2 glycoprotein I antibody contributes to hyperhomocysteinaemia-aggravated abdominal aortic aneurysm.

Shao F, Miao Y, Zhang Y, Han L, ... Feng J, Wang X
Aims
Overactivated B cells secrete pathological antibodies, which in turn accelerate the formation of abdominal aortic aneurysms (AAAs). Hyperhomocysteinaemia (HHcy) aggravates AAA in mice; however, the underlying mechanisms remain largely elusive. In this study, we further investigated whether homocysteine (Hcy)-activated B cells produce antigen-specific antibodies that ultimately contribute to AAA formation.
Methods and results
ELISA assays showed that HHcy induced the secretion of anti-beta 2 glycoprotein I (anti-β2GPI) antibody from B cells both in vitro and in vivo. Mechanistically, Hcy increased the accumulation of various lipid metabolites in B cells tested by liquid chromatography-tandem mass spectrometry, which contributed to elevated anti-β2GPI IgG secretion. By using the toll-like receptor 4 (TLR4)-specific inhibitor TAK-242 or TLR4-deficient macrophages, we found that culture supernatants from Hcy-activated B cells and HHcy plasma IgG polarized inflammatory macrophages in a TLR4-dependent manner. In addition, HHcy markedly increased the incidence of elastase- and CaPO4-induced AAA in male BALB/c mice, which was prevented in μMT mice. To further determine the importance of IgG in HHcy-aggravated AAA formation, we purified plasma IgG from HHcy or control mice and then transferred the IgG into μMT mice, which were subsequently subjected to elastase- or CaPO4-induced AAA. Compared with μMT mice that received plasma IgG from control mice, μMT mice that received HHcy plasma IgG developed significantly exacerbated elastase- or CaPO4-induced AAA accompanied by increased elastin degradation, MMP2/9 expression, and anti-β2GPI IgG deposition in vascular lesions, as shown by immunofluorescence histochemical staining.
Conclusion
Our findings reveal a novel mechanism by which Hcy-induced B cell-derived pathogenic anti-β2GPI IgG might, at least in part, contribute to HHcy-aggravated chronic vascular inflammation and AAA formation.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Aug 2020; 116:1897-1909

Role of Interleukin-23/Interleukin-17 Axis in T-Cell Mediated Actions in Hypertension.

Higaki A, Mahmoud AUM, Paradis P, Schiffrin EL

Current knowledge suggests that hypertension is in part mediated by immune mechanisms. Both IL-23 and IL-17 are upregulated in several experimental hypertensive rodent models, as well as in hypertensive humans in observational studies. Recent preclinical studies have shown that either IL-23 or IL-17A treatment induce blood pressure elevation. However, the IL-23/IL-17 axis has not been a major therapeutic target in hypertension, unlike in other autoimmune diseases. In this review, we summarize current knowledge on the role of these cytokines in immune mechanisms contributing to hypertension, and discuss the potential of IL-23/IL-17-targeted therapy for treatment of hypertension.

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

Cardiovasc Res: 31 Aug 2020; epub ahead of print

Human model of IRX5 mutations reveals key role for this transcription factor in ventricular conduction.

Al Sayed ZR, Canac R, Cimarosti B, Bonnard C, ... Lemarchand P, Gaborit N
Aim
Several inherited arrhythmic diseases have been linked to single gene mutations in cardiac ion channels and interacting proteins. However, the mechanisms underlying most arrhythmias, are thought to involve altered regulation of the expression of multiple effectors. In this study, we aimed to examine the role of a transcription factor belonging to the Iroquois homeobox family, IRX5, in cardiac electrical function.
Methods and results
Using human cardiac tissues, transcriptomic correlative analyses between IRX5 and genes involved in cardiac electrical activity showed that in human ventricular compartment, IRX5 expression strongly correlated to the expression of major actors of cardiac conduction, including the sodium channel, Nav1.5, and Connexin 40 (Cx40). We then generated human induced pluripotent stem cells (hiPSCs) derived from two Hamamy Syndrome-affected patients carrying distinct homozygous loss-of-function mutations in IRX5 gene. Cardiomyocytes derived from these hiPSCs showed impaired cardiac gene expression program, including misregulation in the control of Nav1.5 and Cx40 expression. In accordance with the prolonged QRS interval observed in Hamamy Syndrome patients, a slower ventricular action potential depolarization due to sodium current reduction was observed on electrophysiological analyses performed on patient-derived cardiomyocytes, confirming the functional role of IRX5 in electrical conduction. Finally, a novel cardiac transcription factor complex was identified, composed by IRX5 and GATA4, in which IRX5 potentiated GATA4-induction of SCN5A expression.
Conclusions
Altogether, this work unveils a key role for IRX5 in the regulation of human ventricular depolarization and cardiac electrical conduction, providing therefore new insights into our understanding of cardiac diseases.
Translational perspectives
Inherited cardiac arrhythmias account for about 20% of sudden cardiac deaths, of which a small portion are monogenic familial diseases with mutations in cardiac ion channels. However, pathogeny of inherited cardiac arrhythmias is increasingly thought to result from complex mechanisms involving altered regulation of multiple effectors expression. Taking advantage of cardiomyocytes derived from Hamamy syndrome patients, carrying loss-of-function mutations in IRX5 transcription factor, we uncovered an important role for IRX5 in the regulation of several major players of ventricular depolarization conduction and in arrhythmogenesis. Thus, this study supports systematic screening for genetic variants in IRX5 in inherited cardiac arrhythmias.

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

Cardiovasc Res: 07 Sep 2020; 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 ischemic stroke and cardiac hypertrophy. This project aims at investigating the role of SIRT5 in arterial thrombus formation.
Methods and results
Sirt5 transgenic (Sirt5Tg/0) as well as knock-out (Sirt5-/-) mice underwent photochemically-induced carotid endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) treated with SIRT5 silencing-RNA (si-SIRT5) as well as peripheral blood mononuclear cells (PBMCs) 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 WT 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 (TFPI) 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 expression 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.
Conclusions
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.
Translational perspectives
This study illustrates a novel role for Sirtuin 5 in arterial thrombosis by regulating fibrinolysis through plasminogen activator inhibitor 1 (PAI-1). These results shed new light onto the pathophysiology of arterial thrombus formation which underlies most of the acute atherosclerotic complications. Also, they further affirm the intrinsic relationship between lifespan regulating genes, vascular dysfunction and age-related cardiovascular disease, thus indicating these genes as potential targets for cardiovascular prevention and therapy. Further studies will be needed to assess the predictive ability of SIRT5 in patients with acute cardiovascular or cerebrovascular events. Also, the design of specific SIRT5 inhibitors will allow trials aiming at investigating the efficacy of SIRT5 blockage in the clinical setting.

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

Cardiovasc Res: 14 Sep 2020; epub ahead of print

Adoptive transfer of CX3CR1 transduced-T regulatory cells improves homing to the atherosclerotic plaques and dampens atherosclerosis progression.

Bonacina F, Martini E, Svecla M, Nour J, ... Kallikourdis M, Norata GD
Aim
Loss of immunosuppressive response supports inflammation during atherosclerosis. We tested whether adoptive cell therapy (ACT) with Tregulatory cells (Tregs) engineered to selectively migrate in the atherosclerotic plaque would dampen the immune-inflammatory response in the arterial wall in animal models of Familial Hypercholesterolemia (FH).
Methods and results
FH patients presented a decreased Tregs suppressive function associated to an increased inflammatory burden. A similar phenotype was observed in Ldlr -/- mice accompanied by a selective increased expression of the chemokine CX3CL1 in the aorta but not in other districts (lymph nodes, spleen and liver). Treg overexpressing CX3CR1 were thus generated (CX3CR1+-Treg) to drive Treg selectively to the plaque. CX3CR1+-Treg were injected (i.v.) in Ldlr -/- fed high-cholesterol diet (WTD) for 8 weeks. CX3CR1+-Treg were detected in the aorta, but not in other tissues, of Ldlr -/- mice 24h after ACT, corroborating the efficacy of this approach. After 4 additional weeks of WTD, ACT with CX3CR1+-Treg resulted in reduced plaque progression and lipid deposition, ameliorated plaque stability by increasing collagen and smooth muscle cells content, while decreasing the number of pro-inflammatory macrophages. Shotgun proteomics of the aorta showed a metabolic rewiring in CX3CR1+-Treg treated Ldlr -/- mice compared to controls that was associated with the improvement of inflammation-resolving pathways and disease progression.
Conclusion
ACT with vasculotropic Treg appears as a promising strategy to selectively target immune activation in the atherosclerotic plaque.
Translational relevance
Improving pro-resolutive inflammatory response represents a promising therapeutic approach to control atherosclerosis progression. Meanwhile, selective immunosuppression at the atherosclerotic plaque looks critical to limit unspecific inhibition of inflammation in other tissues. Our work demonstrates that engineering of immunosuppressive T regulatory cells to be hijacked in the atherosclerotic plaque limits atherosclerosis progression by targeting local inflammation.

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

Cardiovasc Res: 14 Sep 2020; epub ahead of print

Pleiotropic actions of factor Xa inhibition in cardiovascular prevention - mechanistic insights and implications for anti-thrombotic treatment.

Ten Cate H, Guzik TJ, Eikelboom J, Spronk HMH

Atherosclerosis is a chronic inflammatory disease in which atherothrombotic complications lead to cardiovascular morbidity and mortality. At advanced stages, myocardial infarction, ischemic stroke and peripheral artery disease (PAD), including major adverse limb events (MALE), are caused either by acute occlusive atherothrombosis, or by thromboembolism. Endothelial dysfunction, vascular smooth muscle cell activation and vascular inflammation are essential in the development of acute cardiovascular events. Effects of the coagulation system on vascular biology extend beyond thrombosis. Under physiological conditions, coagulation proteases in blood are pivotal in maintaining hemostasis and vascular integrity. Under pathological conditions, including atherosclerosis, the same coagulation proteases (including factor Xa, factor VIIa and thrombin) become drivers of atherothrombosis, working in concert with platelets and vessel wall components. While initially atherothrombosis was attributed primarily to platelets, recent advances indicate the critical role of fibrin clot and plasma coagulation factors. Mechanisms of atherothrombosis and hypercoagulability vary depending on plaque erosion or plaque rupture. In addition to contributing to thrombus formation, factor Xa and thrombin can affect endothelial dysfunction, oxidative stress, vascular smooth muscle cell function as well as immune cell activation and vascular inflammation. By these mechanisms they promote atherosclerosis and contribute to plaque instability. In this review, we first discuss the postulated vasoprotective mechanisms of protease activated receptor (PARs) signaling induced by coagulation enzymes under physiological conditions. Next, we discuss preclinical studies linking coagulation with endothelial cell dysfunction, thromboinflammation and atherogenesis. Understanding these mechanisms is pivotal for the introduction of novel strategies in cardiovascular prevention and therapy. We therefore translate these findings to clinical studies of direct oral anticoagulant (DOAC) drugs and discuss the potential relevance of dual pathway inhibition for atherothrombosis prevention and vascular protection.

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

Cardiovasc Res: 14 Sep 2020; epub ahead of print

Loss of life expectancy from air pollution compared to other risk factors: a worldwide perspective.

Lelieveld J, Pozzer A, Pöschl U, Fnais M, Haines A, Münzel T
Aims
Long-term exposure of humans to air pollution enhances the risk of cardiovascular and respiratory diseases. A novel Global Exposure Mortality Model (GEMM) has been derived from many cohort studies, providing much-improved coverage of the exposure to fine particulate matter (PM2.5). We applied the GEMM to assess excess mortality attributable to ambient air pollution on a global scale and compare to other risk factors.
Methods and results
We used a data-informed atmospheric model to calculate worldwide exposure to PM2.5 and ozone pollution, which was combined with the GEMM to estimate disease-specific excess mortality and loss of life expectancy (LLE) in 2015. Using this model, we investigated the effects of different pollution sources, distinguishing between natural (wildfires, aeolian dust) and anthropogenic emissions, including fossil fuel use. Global excess mortality from all ambient air pollution is estimated at 8.8 (7.11-10.41) million/year, with an LLE of 2.9 (2.3-3.5) years, being a factor of two higher than earlier estimates, and exceeding that of tobacco smoking. The global mean mortality rate of about 120 per 100 000 people/year is much exceeded in East Asia (196 per 100 000/year) and Europe (133 per 100 000/year). Without fossil fuel emissions, the global mean life expectancy would increase by 1.1 (0.9-1.2) years and 1.7 (1.4-2.0) years by removing all potentially controllable anthropogenic emissions. Because aeolian dust and wildfire emission control is impracticable, significant LLE is unavoidable.
Conclusion
Ambient air pollution is one of the main global health risks, causing significant excess mortality and LLE, especially through cardiovascular diseases. It causes an LLE that rivals that of tobacco smoking. The global mean LLE from air pollution strongly exceeds that by violence (all forms together), i.e. by an order of magnitude (LLE being 2.9 and 0.3 years, respectively).

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

Cardiovasc Res: 31 Aug 2020; 116:1910-1917

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 leukocyte 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.
Conclusions
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.
Translational perspective
The significance of endocrine cues, particularly oestrogens, on cardiac physiology and disease pathogenesis has been known for decades. This work highlights basal sex differences in cardiac cellularity and shows that endocrine signalling is necessary to maintain the cellular diversity of the heart. By studying the impact of cardiac cellular diversity on heart physiology and stress responses, and its tractability by factors such as hormones, the cardiac cellular landscape may be modulated for targeted therapeutic approaches in the future.

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

Cardiovasc Res: 16 Sep 2020; epub ahead of print

SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes.

Bojkova D, Wagner JUG, Shumliakivska M, Aslan GS, ... Ciesek S, Dimmeler S
Aims
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as a global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality. It is unclear whether cardiac injury is caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here, we investigate whether cardiomyocytes are permissive for SARS-CoV-2 infection.
Methods and results
Two strains of SARS-CoV-2 infected human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) as demonstrated by detection of intracellular double-stranded viral RNA and viral spike glycoprotein expression. Increasing concentrations of viral RNA are detected in supernatants of infected cardiomyocytes, which induced infections in Caco-2 cell lines, documenting productive infections. SARS-COV-2 infection and induced cytotoxic and proapoptotic effects associated with it abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signalling, apoptosis, and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a 3D cardiosphere tissue model. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Coronavirus particles were further observed in cardiomyocytes of a patient with COVID-19. Infection of iPS-CMs was dependent on cathepsins and angiotensin-converting enzyme 2 (ACE2), and was blocked by remdesivir.
Conclusions
This study demonstrates that SARS-CoV-2 infects cardiomyocytes in vitro in an ACE2- and cathepsin-dependent manner. SARS-CoV-2 infection of cardiomyocytes is inhibited by the antiviral drug remdesivir.
Translational perspective
Although this study cannot address whether cardiac injury and dysfunction in COVID-19 patients is caused by direct infection of cardiomyocytes, the demonstration of direct cardiotoxicity in cardiomyocytes, organ mimics, human heart slices and human hearts warrants the further monitoring of cardiotoxic effects in COVID-19 patients.

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

Cardiovasc Res: 22 Sep 2020; epub ahead of print

The role of angiotensin-converting enzyme 2 in coronaviruses/influenza viruses and cardiovascular disease.

Chen L, Hao G

Angiotensin-converting enzyme 2 (ACE2) has emerged as a key regulator of the renin-angiotensin system in cardiovascular (CV) disease and plays a pivotal role in infections by coronaviruses and influenza viruses. The present review is primarily focused on the findings to indicate the role of ACE2 in the relationship of coronaviruses and influenza viruses to CV disease. It is postulated that the risk of coronavirus or influenza virus infection is high, at least partly due to high ACE2 expression in populations with a high CV risk. Coronavirus and influenza virus vaccine usage in high CV risk populations could be a potential strategy to prevent both CV disease and coronavirus/influenza virus infections.

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

Cardiovasc Res: 30 Sep 2020; 116:1932-1936

Health economic evaluation of rivaroxaban in the treatment of patients with chronic coronary artery disease or peripheral artery disease.

Cowie MR, Lamy A, Levy P, Mealing S, ... Bowrin K, Briere JB
Aims
In the COMPASS trial, rivaroxaban 2.5 mg twice daily (bid) plus acetylsalicylic acid (ASA) 100 mg once daily (od) performed better than ASA 100 mg od alone in reducing the rate of cardiovascular disease, stroke, or myocardial infarction (MI) in patients with coronary artery disease (CAD) and peripheral artery disease (PAD). A Markov model was developed to assess the cost-effectiveness of rivaroxaban plus ASA vs. ASA alone over a lifetime horizon, from the UK National Health System perspective.
Methods and results
The base case analysis assumed that patients entered the model in the event-free health state, with the possibility to experience ≤2 events, transitioning every three-month cycle, through acute and post-acute health states of MI, ischaemic stroke (IS), or intracranial haemorrhage (ICH), and death. Costs, quality-adjusted life-years (QALYs), life years-all discounted at 3.5%-and incremental cost-effectiveness ratios (ICERs) were calculated. Deterministic and probabilistic sensitivity analyses were conducted, as well as scenario analyses. In the model, patients on rivaroxaban plus ASA lived for an average of 14.0 years with no IS/MI/ICH, and gained 9.7 QALYs at a cost of £13 947, while those receiving ASA alone lived for an average of 12.7 years and gained 9.3 QALYs at a cost of £8126. The ICER was £16 360 per QALY. This treatment was cost-effective in 98% of 5000 iterations at a willingness-to-pay threshold of £30 000 per QALY.
Conclusion
This Markov model suggests that rivaroxaban 2.5 mg bid plus ASA is a cost-effective alternative to ASA alone in patients with chronic CAD or PAD.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 31 Aug 2020; 116:1918-1924

Carvedilol induces biased β1 adrenergic receptor-Nitric oxide synthase 3-cyclic guanylyl monophosphate signaling 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 signaling 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 signaling, calcium handling, contraction, and western blot. Biosensors were used to measure β-blocker-induced cyclic guanosine monophosphate (cGMP) signal and protein kinase A (PKA) activity in myocytes. Acute stimulation of myocytes with carvedilol promotes β1AR- and 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 signaling pathway to promote cardiac inotropy in the heart, indicating that this signaling paradigm may be targeted in therapy of heart diseases by reduced ejection fraction.
Translational perspective
Carvedilol transduces β1AR coupling to Gi-PI3K-Akt pathway to promote NOS3-cGMP-PKG signals and evokes cardiac inotropy with minimal elevation of calcium signaling. Chronic therapy with carvedilol not only restores cardiac contractility and adrenergic reserves but also leads to reduction of cardiac remodeling with less cell death than metoprolol. These results may explain at least in part that carvedilol had a significant survival advantage over metoprolol in the treatment of HF in clinical studies. The carvedilol-induced β1AR-NOS3 axis represents a novel regulatory paradigm to increase cardiac inotropy, which can be a promising therapeutic target for treating cardiac diseases with depressed cardiac 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: 20 Sep 2020; epub ahead of print

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

Jin X, Kim WB, Kim MN, Jung WW, ... Kim YB, Kim YI
Aim
Abundant evidence indicates that estrogen (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.
Translational perspective
Numerous studies have shown that sex steroids can impact blood pressure (BP) and premenopausal women have a lower incidence of hypertension than age-matched men. Postmenopausal women no longer enjoy this beneficial difference. The causal mechanisms are not clear and hormone replacement therapy with estradiol (E2) does not consistently lower BP in postmenopausal women. In this study we showed that rats treated with CLP290 to control GABAergic excitation in AVP neurons have the same phenotype as E2-supplemented rats in BP and plasma AVP level. This result indicates that CLP290 leverages the same pathway of E2 protective effects against salt-dependent hypertension, and therefore, raises the possibility that classes of drugs like CLP290 may have some utility as alternative antihypertensives in persons resistant to or contraindicated for E2 therapy.

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

Cardiovasc Res: 21 Sep 2020; epub ahead of print

Development, validation and implementation of biomarker testing in cardiovascular medicine state-of-the-art: Proceedings of the European Society of Cardiology - Cardiovascular Round Table.

Elliott P, Cowie MR, Franke J, Ziegler A, ... Twerenbold R, Zannad F

Many biomarkers that could be used to assess ejection fraction, heart failure, or myocardial infarction fail to translate into clinical practice because they lack essential performance characteristics or fail to meet regulatory standards for approval. Despite their potential, new technologies have added to the complexities of successful translation into clinical practice. Biomarker discovery and implementation requires a standardised approach that includes: identification of a clinical need; identification of a valid surrogate biomarker; stepwise assay refinement, demonstration of superiority over current standard-of-care; development and understanding of a clinical pathway; and demonstration of real-world performance. Successful biomarkers should improve efficacy or safety of treatment, while being practical at a realistic cost. Everyone involved in cardiovascular healthcare, including researchers, clinicians, and industry partners, are important stakeholders in facilitating the development and implementation of biomarkers. This paper provides suggestions for a development pathway for new biomarkers, discusses regulatory issues and challenges, and suggestions for accelerating the pathway to improve patient outcomes. Real life examples of successful biomarkers-high sensitivity cardiac troponin (hs-cTn), T2* cardiovascular magnetic resonance (CMR) imaging, and echocardiography-are used to illustrate the value of a standardised development pathway in the translation of concepts into routine clinical practice.

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

Cardiovasc Res: 21 Sep 2020; epub ahead of print

PCSK9 and LRP5 in macrophage lipid internalization and inflammation.

Badimon L, Luquero A, Crespo J, Peña E, Borrell-Pages M
Aims
Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. Low-Density Lipoprotein Receptor (LDLR) play a critical role in regulating blood cholesterol levels by binding to and clearing LDLs from the circulation. The disruption of the interaction between Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) and LDLR reduces blood cholesterol levels. It is not well known whether other members of the LDLR superfamily may be targets of PCSK9. The aim of this work was to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target, and to study the role of PCSK9 and LRP5 in foam cell formation and lipid accumulation.
Methods and results
Primary cultures of human inflammatory cells (monocytes and macrophages) were silenced for LRP5 or PCSK9 and challenged with LDLs. We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages show less intracellular CE accumulation. In macrophages, internalization of LRP5-bound LDL is already highly evident after 5 hours of LDL incubation and lasts up to 24hours; however in the absence of both LRP5 and PCSK9 there is a strong reduction of CE accumulation indicating a role for both proteins in lipid uptake. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally PCSK9 participates in TLR4/NFkB signaling; a decreased TLR4 protein expression levels and a decreased nuclear translocation of NFκB was detected in PCSK9 silenced cells after lipid loading, indicating a down-regulation of the TLR4/NFκB pathway.
Conclusion
Our results show that both LRP5 and PCSK9 participate in lipid uptake in macrophages. In the absence of LRP5 there is a reduced release of PCSK9 indicating that LRP5 also participates in the mechanism of release of soluble PCSK9. Furthermore, PCSK9 up-regulates TLR4/NFκB favoring inflammation.
Translational perspective
We demonstrate that PCSK9 and LRP5 contribute to lipid uptake. We also show that LRP5 participates in PCSK9 transport to the plasma membrane and that PCSK9 inhibition protects against agLDL-induced inflammation associated to the TLR4/NFκB pathway. These results offer new targets to prevent the progression of inflammation and hypercholesterolemia and their increased risk of cardiovascular events.

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

Cardiovasc Res: 28 Sep 2020; epub ahead of print

Recent Advances in the Role of the Adenosinergic System in Coronary Artery Disease.

Paganelli F, Gaudry M, Ruf J, Guieu R

Adenosine is an endogenous nucleoside that plays a major role in the physiology and physiopathology of the coronary artery system, mainly by activating its A2A receptors (A2AR). Adenosine is released by myocardial, endothelial and immune cells during hypoxia, ischaemia or inflammation, each condition being present in coronary artery disease (CAD). While activation of A2AR improves coronary blood circulation and leads to anti-inflammatory effects, downregulation of A2AR has many deleterious effects during CAD. A decrease in the level and/or activity of A2AR leads to: i) lack of vasodilation, which decreases blood flow, leading to a decrease in myocardial oxygenation and tissue hypoxia; ii) an increase in the immune response, favouring inflammation; and iii) platelet aggregation, which therefore participates, in part, in the formation of a fibrin-platelet thrombus after the rupture or erosion of the plaque, leading to the occurrence of acute coronary syndrome. Inflammation contributes to the development of atherosclerosis, leading to myocardial ischaemia, which in turn leads to tissue hypoxia. Therefore, a vicious circle is created that maintains and aggravates CAD. In some cases, studying the adenosinergic profile can help assess the severity of CAD. In fact, inducible ischaemia in CAD patients, as assessed by exercise stress test or fractional flow reserve, is associated with the presence of a reserve of A2AR called spare receptors. The purpose of this review is to present emerging experimental evidence supporting the existence of this adaptive adenosinergic response to ischaemia or inflammation in CAD. We believe that we have achieved a breakthrough in the understanding and modeling of spare A2AR, based upon a new concept allowing for a new and non-invasive CAD management.

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 Sep 2020; epub ahead of print

3-Hydroxyanthralinic acid metabolism controls the hepatic SREBP/lipoprotein axis, inhibits inflammasome activation in macrophages, and decreases atherosclerosis in Ldlr-/- mice.

Berg M, Polyzos KA, Agardh H, Baumgartner R, ... Hansson GK, Ketelhuth DFJ
Aims
Atherosclerosis is a chronic inflammatory disease involving immunological and metabolic processes. Metabolism of tryptophan (Trp) via the kynurenine pathway has shown immunomodulatory properties and the ability to modulate atherosclerosis. We identified 3-hydroxyanthranilic acid (3-HAA) as a key metabolite of Trp modulating vascular inflammation and lipid metabolism. The molecular mechanisms driven by 3-HAA in atherosclerosis have not been completely elucidated. In this study, we investigated whether two major signalling pathways, activation of SREBPs and inflammasome, are associated with the 3-HAA-dependent regulation of lipoprotein synthesis and inflammation in the atherogenesis process. Moreover, we examined whether inhibition of endogenous 3-HAA degradation affects hyperlipidaemia and plaque formation.
Methods and results
In vitro, we showed that 3-HAA reduces SREBP-2 expression and nuclear translocation and apolipoprotein B secretion in HepG2 cell cultures, and inhibits inflammasome activation and IL-1β production by macrophages. Using Ldlr-/- mice, we showed that inhibition of 3-HAA 3,4-dioxygenase (HAAO), which increases the endogenous levels of 3-HAA, decreases plasma lipids and atherosclerosis. Notably, HAAO inhibition led to decreased hepatic SREBP-2 mRNA levels and lipid accumulation, and improved liver pathology scores.
Conclusions
We show that the activity of SREBP-2 and the inflammasome can be regulated by 3-HAA metabolism. Moreover, our study highlights that targeting HAAO is a promising strategy to prevent and treat hypercholesterolaemia and atherosclerosis.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 30 Sep 2020; 116:1948-1957

Mechanistic definition of the cardiovascular mPGES-1/COX-2/ADMA axis.

Kirkby NS, Raouf J, Ahmetaj-Shala B, Liu B, ... Jakobsson PJ, Mitchell JA
Aims
Cardiovascular side effects caused by non-steroidal anti-inflammatory drugs (NSAIDs), which all inhibit cyclooxygenase (COX)-2, have prevented development of new drugs that target prostaglandins to treat inflammation and cancer. Microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors have efficacy in the NSAID arena but their cardiovascular safety is not known. Our previous work identified asymmetric dimethylarginine (ADMA), an inhibitor of endothelial nitric oxide synthase, as a potential biomarker of cardiovascular toxicity associated with blockade of COX-2. Here, we have used pharmacological tools and genetically modified mice to delineate mPGES-1 and COX-2 in the regulation of ADMA.
Methods and results
Inhibition of COX-2 but not mPGES-1 deletion resulted in increased plasma ADMA levels. mPGES-1 deletion but not COX-2 inhibition resulted in increased plasma prostacyclin levels. These differences were explained by distinct compartmentalization of COX-2 and mPGES-1 in the kidney. Data from prostanoid synthase/receptor knockout mice showed that the COX-2/ADMA axis is controlled by prostacyclin receptors (IP and PPARβ/δ) and the inhibitory PGE2 receptor EP4, but not other PGE2 receptors.
Conclusion
These data demonstrate that inhibition of mPGES-1 spares the renal COX-2/ADMA pathway and define mechanistically how COX-2 regulates ADMA.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 30 Sep 2020; 116:1972-1980

Abnormal neurovascular coupling as a cause of excess cerebral vasodilation in familial migraine.

Staehr C, Rajanathan R, Postnov DD, Hangaard L, ... Aalkjaer C, Matchkov VV
Aims
Acute migraine attack in familial hemiplegic migraine type 2 (FHM2) patients is characterized by sequential hypo- and hyperperfusion. FHM2 is associated with mutations in the Na, K-ATPase α2 isoform. Heterozygous mice bearing one of these mutations (α2+/G301R mice) were shown to have elevated cerebrovascular tone and, thus, hypoperfusion that might lead to elevated concentrations of local metabolites. We hypothesize that these α2+/G301R mice also have increased cerebrovascular hyperaemic responses to these local metabolites leading to hyperperfusion in the affected part of the brain.
Methods and results
Neurovascular coupling was compared in α2+/G301R and matching wild-type (WT) mice using Laser Speckle Contrast Imaging. In brain slices, parenchymal arteriole diameter and intracellular calcium changes in neuronal tissue, astrocytic endfeet, and smooth muscle cells in response to neuronal excitation were assessed. Wall tension and smooth muscle membrane potential were measured in isolated middle cerebral arteries. Quantitative polymerase chain reaction, western blot, and immunohistochemistry were used to assess the molecular background underlying the functional changes. Whisker stimulation induced larger increase in blood perfusion, i.e. hyperaemic response, of the somatosensory cortex of α2+/G301R than WT mice. Neuronal excitation was associated with larger parenchymal arteriole dilation in brain slices from α2+/G301R than WT mice. These hyperaemic responses in vivo and ex vivo were inhibited by BaCl2, suggesting involvement of inward-rectifying K+ channels (Kir). Relaxation to elevated bath K+ was larger in arteries from α2+/G301R compared to WT mice. This difference was endothelium-dependent. Endothelial Kir2.1 channel expression was higher in arteries from α2+/G301R mice. No sex difference in functional responses and Kir2.1 expression was found.
Conclusion
This study suggests that an abnormally high cerebrovascular hyperaemic response in α2+/G301R mice is a result of increased endothelial Kir2.1 channel expression. This may be initiated by vasospasm-induced accumulation of local metabolites and underlie the hyperperfusion seen in FHM2 patients during migraine attack.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 30 Sep 2020; 116:2009-2020

Interstitial macrophage-derived thrombospondin-1 contributes to hypoxia-induced pulmonary hypertension.

Kumar R, Mickael C, Kassa B, Sanders L, ... Tuder RM, Graham BB
Aims
Transforming growth factor-β (TGF-β) signalling is required for chronic hypoxia-induced pulmonary hypertension (PH). The activation of TGF-β by thrombospondin-1 (TSP-1) contributes to the pathogenesis of hypoxia-induced PH. However, neither the cellular source of pathologic TSP-1 nor the downstream signalling pathway that link activated TGF-β to PH have been determined. In this study, we hypothesized that circulating monocytes, which are recruited to become interstitial macrophages (IMs), are the major source of TSP-1 in hypoxia-exposed mice, and TSP-1 activates TGF-β with increased Rho-kinase signalling, causing vasoconstriction.
Methods and results
Flow cytometry revealed that a specific subset of IMs is the major source of pathologic TSP-1 in hypoxia. Intravenous depletion and parabiosis experiments demonstrated that these cells are circulating prior to recruitment into the interstitium. Rho-kinase-mediated vasoconstriction was a major downstream target of active TGF-β. Thbs1 deficient bone marrow (BM) protected against hypoxic-PH by blocking TGF-β activation and Rho-kinase-mediated vasoconstriction.
Conclusion
In hypoxia-challenged mice, BM derived and circulating monocytes are recruited to become IMs which express TSP-1, resulting in TGF-β activation and Rho-kinase-mediated vasoconstriction.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 30 Sep 2020; 116:2021-2030

Nitric oxide modulates cardiomyocyte pH control through a biphasic effect on sodium/hydrogen exchanger-1.

Richards MA, Simon JN, Ma R, Loonat AA, ... Fliegel L, Swietach P
Aims
When activated, Na+/H+ exchanger-1 (NHE1) produces some of the largest ionic fluxes in the heart. NHE1-dependent H+ extrusion and Na+ entry strongly modulate cardiac physiology through the direct effects of pH on proteins and by influencing intracellular Ca2+ handling. To attain an appropriate level of activation, cardiac NHE1 must respond to myocyte-derived cues. Among physiologically important cues is nitric oxide (NO), which regulates a myriad of cardiac functions, but its actions on NHE1 are unclear.
Methods and results
NHE1 activity was measured using pH-sensitive cSNARF1 fluorescence after acid-loading adult ventricular myocytes by an ammonium prepulse solution manoeuvre. NO signalling was manipulated by knockout of its major constitutive synthase nNOS, adenoviral nNOS gene delivery, nNOS inhibition, and application of NO-donors. NHE1 flux was found to be activated by low [NO], but inhibited at high [NO]. These responses involved cGMP-dependent signalling, rather than S-nitros(yl)ation. Stronger cGMP signals, that can inhibit phosphodiesterase enzymes, allowed [cAMP] to rise, as demonstrated by a FRET-based sensor. Inferring from the actions of membrane-permeant analogues, cGMP was determined to activate NHE1, whereas cAMP was inhibitory, which explains the biphasic regulation by NO. Activation of NHE1-dependent Na+ influx by low [NO] also increased the frequency of spontaneous Ca2+ waves, whereas high [NO] suppressed these aberrant forms of Ca2+ signalling.
Conclusions
Physiological levels of NO stimulation increase NHE1 activity, which boosts pH control during acid-disturbances and results in Na+-driven cellular Ca2+ loading. These responses are positively inotropic but also increase the likelihood of aberrant Ca2+ signals, and hence arrhythmia. Stronger NO signals inhibit NHE1, leading to a reversal of the aforementioned effects, ostensibly as a potential cardioprotective intervention to curtail NHE1 overdrive.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 30 Sep 2020; 116:1958-1971

Mas receptor is translocated to the nucleus upon agonist stimulation in brainstem neurons from spontaneously hypertensive rats but not normotensive rats.

Cerniello FM, Silva MG, Carretero OA, Gironacci MM
Aims 
Activation of the angiotensin (Ang)-(1-7)/Mas receptor (R) axis protects from sympathetic overactivity. Endocytic trafficking is an essential process that regulates receptor (R) function and its ultimate cellular responses. We investigated whether the blunted responses to Ang-(1-7) in hypertensive rats are associated to an alteration in MasR trafficking.
Methods and results 
Brainstem neurons from Wistar-Kyoto (WKY) or spontaneously hypertensive rats (SHRs) were investigated for (i) Ang-(1-7) levels and binding and MasR expression, (ii) Ang-(1-7) responses (arachidonic acid and nitric oxide release and Akt and ERK1/2 phosphorylation), and (iii) MasR trafficking. Ang-(1-7) was determined by radioimmunoassay. MasR expression and functionality were evaluated by western blot and binding assays. MasR trafficking was evaluated by immunofluorescence. Ang-(1-7) treatment induced an increase in nitric oxide and arachidonic acid release and ERK1/2 and Akt phosphorylation in WKY neurons but did not have an effect in SHR neurons. Although SHR neurons showed greater MasR expression, Ang-(1-7)-elicited responses were substantially diminished presumably due to decreased Ang-(1-7) endogenous levels concomitant with impaired binding to its receptor. Through immunocolocalization studies, we evidenced that upon Ang-(1-7) stimulation MasRs were internalized through clathrin-coated pits and caveolae into early endosomes and slowly recycled back to the plasma membrane. However, the fraction of internalized MasRs into early endosomes was larger and the fraction of MasRs recycled back to the plasma membrane was smaller in SHR than in WKY neurons. Surprisingly, in SHR neurons but not in WKY neurons, Ang-(1-7) induced MasR translocation to the nucleus. Nuclear MasR expression and Ang-(1-7) levels were significantly greater in the nuclei of Ang-(1-7)-stimulated SHR neurons, indicating that the MasR is translocated with its ligand bound to it.
Conclusion 
MasRs display differential trafficking in brainstem neurons from SHRs, which may contribute to the impaired responses to Ang-(1-7).

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 30 Sep 2020; 116:1995-2008

The LINC00961 transcript and its encoded micropeptide, small regulatory polypeptide of amino acid response, regulate endothelial cell function.

Spencer HL, Sanders R, Boulberdaa M, Meloni M, ... Rodor J, Baker AH
Aims
Long non-coding RNAs (lncRNAs) play functional roles in physiology and disease, yet understanding of their contribution to endothelial cell (EC) function is incomplete. We identified lncRNAs regulated during EC differentiation and investigated the role of LINC00961 and its encoded micropeptide, small regulatory polypeptide of amino acid response (SPAAR), in EC function.
Methods and results
Deep sequencing of human embryonic stem cell differentiation to ECs was combined with Encyclopedia of DNA Elements (ENCODE) RNA-seq data from vascular cells, identifying 278 endothelial enriched genes, including 6 lncRNAs. Expression of LINC00961, first annotated as an lncRNA but reassigned as a protein-coding gene for the SPAAR micropeptide, was increased during the differentiation and was EC enriched. LINC00961 transcript depletion significantly reduced EC adhesion, tube formation, migration, proliferation, and barrier integrity in primary ECs. Overexpression of the SPAAR open reading frame increased tubule formation; however, overexpression of the full-length transcript did not, despite production of SPAAR. Furthermore, overexpression of an ATG mutant of the full-length transcript reduced network formation, suggesting a bona fide non-coding RNA function of the transcript with opposing effects to SPAAR. As the LINC00961 locus is conserved in mouse, we generated an LINC00961 locus knockout (KO) mouse that underwent hind limb ischaemia (HLI) to investigate the angiogenic role of this locus in vivo. In agreement with in vitro data, KO animals had a reduced capillary density in the ischaemic adductor muscle after 7 days. Finally, to characterize LINC00961 and SPAAR independent functions in ECs, we performed pull-downs of both molecules and identified protein-binding partners. LINC00961 RNA binds the G-actin sequestering protein thymosin beta-4x (Tβ4) and Tβ4 depletion phenocopied the overexpression of the ATG mutant. SPAAR binding partners included the actin-binding protein, SYNE1.
Conclusion
The LINC00961 locus regulates EC function in vitro and in vivo. The gene produces two molecules with opposing effects on angiogenesis: SPAAR and LINC00961.

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

Cardiovasc Res: 30 Sep 2020; 116:1981-1994

Pathobiology of pulmonary artery hypertension: role of long non-coding RNAs.

Zahid KR, Raza U, Chen J, Raj UJ, Gou D

Pulmonary arterial hypertension (PAH) is a disease with complex pathobiology, significant morbidity and mortality, and remains without a cure. It is characterized by vascular remodelling associated with uncontrolled proliferation of pulmonary artery smooth muscle cells, endothelial cell proliferation and dysfunction, and endothelial-to-mesenchymal transition, leading to narrowing of the vascular lumen, increased vascular resistance and pulmonary arterial pressure, which inevitably results in right heart failure and death. There are multiple molecules and signalling pathways that are involved in the vascular remodelling, including non-coding RNAs, i.e. microRNAs and long non-coding RNAs (lncRNAs). It is only in recent years that the role of lncRNAs in the pathobiology of pulmonary vascular remodelling and right ventricular dysfunction is being vigorously investigated. In this review, we have summarized the current state of knowledge about the role of lncRNAs as key drivers and gatekeepers in regulating major cellular and molecular trafficking involved in the pathogenesis of PAH. In addition, we have discussed the limitations and challenges in translating lncRNA research in vivo and in therapeutic applications of lncRNAs in PAH.

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

Cardiovasc Res: 30 Sep 2020; 116:1937-1947

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. Whilst 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 data sets 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 modeling 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 pro-arrhythmia. 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.
Conclusions
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.
Translational perspective
Here, we show, using meta-analysis of multiple independent human gene expression datasets, that there is co-expression of KCNH2-CACNA1C in human heart tissue which was then confirmed in human cardiac myocytes derived from induced pluripotent stem cells. Both in silico and functional studies show that the co-expression of CACNA1C and KCNH2 increases the robustness of cardiac electrical signalling. Our data also suggest that those patients who express higher levels of CACNA1C and KCNH2 are likely to be more susceptible to arrhythmias when exposed to drugs that block IKr, the major cause of drug-induced cardiac arrhythmias.

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

Cardiovasc Res: 30 Sep 2020; epub ahead of print

Transcriptome Analysis of Non-Human Primate Induced Pluripotent Stem Cell-Derived Cardiomyocytes in 2D Monolayer Culture versus 3D Engineered Heart Tissue.

Yang H, Shao N, Holmström A, Zhao X, ... Abilez OJ, Wu JC
Aims
Stem cell therapy has shown promise for treating myocardial infarction (MI) via re-muscularization and paracrine signaling in both small and large animals. Non-human primates (NHPs), such as rhesus macaques (Macaca mulatta), are primarily utilized in preclinical trials due to their similarity to humans, both genetically and physiologically. Currently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are delivered into the infarcted myocardium by either direct cell injection or an engineered tissue patch. Although both approaches have advantages in terms of sample preparation, cell-host interaction, and engraftment, how the iPSC-CMs respond to ischemic conditions in the infarcted heart under these two different delivery approaches remain unclear. Here we aim to gain a better understanding of the effects of hypoxia on iPSC-CMs at the transcriptome level.
Methods and results
NHP iPSC-CMs in both monolayer culture (2 D) and engineered heart tissue (EHT) (3 D) format were exposed to hypoxic conditions to serve as surrogates of direct cell injection and tissue implantation in vivo, respectively. Outcomes were compared at the transcriptome level. We found the 3 D EHT model was more sensitive to ischemic conditions and similar to the native in vivo myocardium in terms of cell-extracellular matrix/cell-cell interactions, energy metabolism, and paracrine signaling.
Conclusions
By exposing NHP iPSC-CMs to different culture conditions, transcriptome profiling improves our understanding of the mechanism of ischemic injury.
Translational perspective
Stem cell therapy has shown promise for treating ischemic heart tissue. However, how stem cells respond following different delivery method is unclear. Here hypoxic conditioning was applied to non-human primate iPSC-CMs in 2 D monolayer culture and 3 D engineered heart tissue to model cell injection versus patch implantation, respectively, in an ischemic milieu. The differential transcriptome of hypoxic effects on iPSC-CMs show upregulation of ECM-cell/cell-cell interactions (COL9A1, ITGB6, CTSV, and EPHA1), energy metabolism/hypoxia (ALDOC, ENO2, PFKFB4, CA3, and CA9), and paracrine signaling (WNT, PDGF, FGFR, EGFR, PI3K, and VEGF) in the 3 D format, which suggest engineered heart tissue as more suitable model for evaluating cardiac regenerative therapy.

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

Cardiovasc Res: 30 Sep 2020; epub ahead of print

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

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

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

Cardiovasc Res: 09 Aug 2020; epub ahead of print

Stimulating pro-reparative immune responses to prevent adverse cardiac remodelling: consensus document from the joint 2019 meeting of the ESC Working Groups of cellular biology of the heart and myocardial function.

Steffens S, Van Linthout S, Sluijter JPG, Tocchetti CG, Thum T, Madonna R

Cardiac injury may have multiple causes, including ischaemic, non-ischaemic, autoimmune, and infectious triggers. Independent of the underlying pathophysiology, cardiac tissue damage induces an inflammatory response to initiate repair processes. Immune cells are recruited to the heart to remove dead cardiomyocytes, which is essential for cardiac healing. Insufficient clearance of dying cardiomyocytes after myocardial infarction (MI) has been shown to promote unfavourable cardiac remodelling, which may result in heart failure (HF). Although immune cells are integral key players of cardiac healing, an unbalanced or unresolved immune reaction aggravates tissue damage that triggers maladaptive remodelling and HF. Neutrophils and macrophages are involved in both, inflammatory as well as reparative processes. Stimulating the resolution of cardiac inflammation seems to be an attractive therapeutic strategy to prevent adverse remodelling. Along with numerous experimental studies, the promising outcomes from recent clinical trials testing canakinumab or colchicine in patients with MI are boosting the interest in novel therapies targeting inflammation in cardiovascular disease patients. The aim of this review is to discuss recent experimental studies that provide new insights into the signalling pathways and local regulators within the cardiac microenvironment promoting the resolution of inflammation and tissue regeneration. We will cover ischaemia- and non-ischaemic-induced as well as infection-related cardiac remodelling and address potential targets to prevent adverse cardiac remodelling.

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

Cardiovasc Res: 31 Aug 2020; 116:1850-1862

A key role for the novel coronary artery disease gene JCAD in atherosclerosis via shear stress mechanotransduction.

Douglas G, Mehta V, Al Haj Zen A, Akoumianakis I, ... Tzima E, Channon KM
Aims
Genome-wide association studies (GWAS) have consistently identified an association between coronary artery disease (CAD) and a locus on chromosome 10 containing a single gene, JCAD (formerly KIAA1462). However, little is known about the mechanism by which JCAD could influence the development of atherosclerosis.
Methods and results
Vascular function was quantified in subjects with CAD by flow-mediated dilatation (FMD) and vasorelaxation responses in isolated blood vessel segments. The JCAD risk allele identified by GWAS was associated with reduced FMD and reduced endothelial-dependent relaxations. To study the impact of loss of Jcad on atherosclerosis, Jcad-/- mice were crossed to an ApoE-/- background and fed a high-fat diet from 6 to16 weeks of age. Loss of Jcad did not affect blood pressure or heart rate. However, Jcad-/-ApoE-/- mice developed significantly less atherosclerosis in the aortic root and the inner curvature of the aortic arch. En face analysis revealed a striking reduction in pro-inflammatory adhesion molecules at sites of disturbed flow on the endothelial cell layer of Jcad-/- mice. Loss of Jcad lead to a reduced recovery perfusion in response to hind limb ischaemia, a model of altered in vivo flow. Knock down of JCAD using siRNA in primary human aortic endothelial cells significantly reduced the response to acute onset of flow, as evidenced by reduced phosphorylation of NF-КB, eNOS, and Akt.
Conclusion
The novel CAD gene JCAD promotes atherosclerotic plaque formation via a role in the endothelial cell shear stress mechanotransduction pathway.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 31 Aug 2020; 116:1863-1874

Transient receptor potential vanilloid-4 contributes to stretch-induced hypercontractility and time-dependent dysfunction in the aged heart.

Veteto AB, Peana D, Lambert MD, McDonald KS, Domeier TL
Aims
Cardiovascular disease remains the greatest cause of mortality in Americans over 65. The stretch-activated transient receptor potential vanilloid-4 (TRPV4) ion channel is expressed in cardiomyocytes of the aged heart. This investigation tests the hypothesis that TRPV4 alters Ca2+ handling and cardiac function in response to increased ventricular preload and cardiomyocyte stretch.
Methods and results
Left ventricular maximal pressure (PMax) was monitored in isolated working hearts of Aged (24-27 months) mice following preload elevation from 5 to 20mmHg, with and without TRPV4 antagonist HC067047 (HC, 1 µmol/L). In preload responsive hearts, PMax prior to and immediately following preload elevation (i.e. Frank-Starling response) was similar between Aged and Aged+HC. Within 1 min following preload elevation, Aged hearts demonstrated secondary PMax augmentation (Aged>Aged+HC) suggesting a role for stretch-activated TRPV4 in cardiac hypercontractility. However, after 20 min at 20 mmHg Aged exhibited depressed PMax (AgedConclusion
TRPV4 contributes to a stretch-induced increase in cardiomyocyte Ca2+ and cardiac hypercontractility, yet sustained stretch leads to cardiomyocyte Ca2+ overload and contractile dysfunction in the aged heart.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Aug 2020; 116:1887-1896

In peripartum cardiomyopathy plasminogen activator inhibitor-1 is a potential new biomarker with controversial roles.

Ricke-Hoch M, Hoes MF, Pfeffer TJ, Schlothauer S, ... van der Meer P, Hilfiker-Kleiner D
Aims
Peripartum cardiomyopathy (PPCM) is a life-threatening heart disease occurring in previously heart-healthy women. A common pathomechanism in PPCM involves the angiostatic 16 kDa-prolactin (16 kDa-PRL) fragment, which via NF-κB-mediated up-regulation of microRNA-(miR)-146a induces vascular damage and heart failure. We analyse whether the plasminogen activator inhibitor-1 (PAI-1) is involved in the pathophysiology of PPCM.
Methods and results
In healthy age-matched postpartum women (PP-Ctrl, n = 53, left ventricular ejection fraction, LVEF > 55%), PAI-1 plasma levels were within the normal range (21 ± 10 ng/mL), but significantly elevated (64 ± 38 ng/mL, P < 0.01) in postpartum PPCM patients at baseline (BL, n = 64, mean LVEF: 23 ± 8%). At 6-month follow-up (n = 23), PAI-1 levels decreased (36 ± 14 ng/mL, P < 0.01 vs. BL) and LVEF (49 ± 11%) improved. Increased N-terminal pro-brain natriuretic peptide and Troponin T did not correlate with PAI-1. C-reactive protein, interleukin (IL)-6 and IL-1β did not differ between PPCM patients and PP-Ctrl. MiR-146a was 3.6-fold (P < 0.001) higher in BL-PPCM plasma compared with PP-Ctrl and correlated positively with PAI-1. In BL-PPCM serum, 16 kDa-PRL coprecipitated with PAI-1, which was associated with higher (P < 0.05) uPAR-mediated NF-κB activation in endothelial cells compared with PP-Ctrl serum. Cardiac biopsies and dermal fibroblasts from PPCM patients displayed higher PAI-1 mRNA levels (P < 0.05) than healthy controls. In PPCM mice (due to a cardiomyocyte-specific-knockout for STAT3, CKO), cardiac PAI-1 expression was higher than in postpartum wild-type controls, whereas a systemic PAI-1-knockout in CKO mice accelerated peripartum cardiac fibrosis, inflammation, heart failure, and mortality.
Conclusion
In PPCM patients, circulating and cardiac PAI-1 expression are up-regulated. While circulating PAI-1 may add 16 kDa-PRL to induce vascular impairment via the uPAR/NF-κB/miR-146a pathway, experimental data suggest that cardiac PAI-1 expression seems to protect the PPCM heart from fibrosis. Thus, measuring circulating PAI-1 and miR-146a, together with an uPAR/NF-κB-activity assay could be developed into a specific diagnostic marker assay for PPCM, but unrestricted reduction of PAI-1 for therapy may not be advised.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Aug 2020; 116:1875-1886

Development of a new mouse model for coxsackievirus-induced myocarditis by attenuating coxsackievirus B3 virulence in the pancreas.

Pinkert S, Pryshliak M, Pappritz K, Knoch K, ... Klingel K, Fechner H
Aims
The coxsackievirus B3 (CVB3) mouse myocarditis model is the standard model for investigation of virus-induced myocarditis but the pancreas, rather than the heart, is the most susceptible organ in mouse. The aim of this study was to develop a CVB3 mouse myocarditis model in which animals develop myocarditis while attenuating viral infection of the pancreas and the development of severe pancreatitis.
Methods and results
We developed the recombinant CVB3 variant H3N-375TS by inserting target sites (TS) of miR-375, which is specifically expressed in the pancreas, into the 3\'UTR of the genome of the pancreo- and cardiotropic CVB3 variant H3. In vitro evaluation showed that H3N-375TS was suppressed in pancreatic miR-375-expressing EndoC-βH1 cells >5 log10, whereas its replication was not suppressed in isolated primary embryonic mouse cardiomyocytes. In vivo, intraperitoneal (i.p.) administration of H3N-375TS to NMRI mice did not result in pancreatic or cardiac infection. In contrast, intravenous (i.v.) administration of H3N-375TS to NMRI and Balb/C mice resulted in myocardial infection and acute and chronic myocarditis, whereas the virus was not detected in the pancreas and the pancreatic tissue was not damaged. Acute myocarditis was characterized by myocardial injury, inflammation with mononuclear cells, induction of proinflammatory cytokines, and detection of replicating H3N-375TS in the heart. Mice with chronic myocarditis showed myocardial fibrosis and persistence of H3N-375TS genomic RNA but no replicating virus in the heart. Moreover, H3N-375TS infected mice showed distinctly less suffering compared with mice that developed pancreatitis and myocarditis after i.p. or i.v application of control virus.
Conclusion
In this study, we demonstrate that by use of the miR-375-sensitive CVB3 variant H3N-375TS, CVB3 myocarditis can be established without the animals developing severe systemic infection and pancreatitis. As the H3N-375TS myocarditis model depends on pancreas-attenuated H3N-375TS, it can easily be used in different mouse strains and for various applications.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Jul 2020; 116:1756-1766

Blood flow patterns regulate PCSK9 secretion via MyD88-mediated pro-inflammatory cytokines.

Liu S, Deng X, Zhang P, Wang X, ... Mehta JL, Ding Z
Aims
Blood flow patterns play an important role in the localization of atherosclerosis in the sense that low-flow state is pro-atherogenic, and helical flow is protective against atherosclerosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol metabolism via low-density lipoprotein receptor (LDLr) degradation and is highly expressed in the atherosclerotic tissues. This study was designed to investigate the role of different blood flow patterns in the regulation of PCSK9 expression.
Methods and results
We designed an experimental model guider to generate stable helical flow. Our data showed that compared with normal flow, low-flow state induces whereas helical flow inhibits PCSK9 expression in the rabbit thoracic aorta in an inflammatory state. Our data also identified that TLR4-MyD88-NF-κB signalling plays an important role in PCSK9 expression. On the other hand, TRIF pathway had almost no effect. Further studies showed that the signals downstream of NF-κB, such as pro-inflammatory cytokines (IL-1β, IL-18, MCP-1, IL-6, TNF-α, IL-12, IFNγ, and GM-CSF) directly influence PCSK9 expression. Interestingly, high fat diet further enhanced PCSK9 expression in an inflammatory milieu.
Conclusions
These observations suggest a link between abnormal flow patterns and PCSK9 expression in inflammatory states, which may qualify helical flow and pro-inflammatory cytokines as potential targets to treat PCSK9-related cardiovascular diseases.

Published by Oxford University Press on behalf of the European Society of Cardiology 2019. This work is written by US Government employees and is in the public domain in the US.

Cardiovasc Res: 31 Jul 2020; 116:1721-1732

Selective ETA vs. dual ETA/B receptor blockade for the prevention of sunitinib-induced hypertension and albuminuria in WKY rats.

Mirabito Colafella KM, Neves KB, Montezano AC, Garrelds IM, ... Danser AHJ, Versmissen J
Aims
Although effective in preventing tumour growth, angiogenesis inhibitors cause off-target effects including cardiovascular toxicity and renal injury, most likely via endothelin (ET)-1 up-regulation. ET-1 via stimulation of the ETA receptor has pro-hypertensive actions whereas stimulation of the ETB receptor can elicit both pro- or anti-hypertensive effects. In this study, our aim was to determine the efficacy of selective ETA vs. dual ETA/B receptor blockade for the prevention of angiogenesis inhibitor-induced hypertension and albuminuria.
Methods and results
Male Wistar Kyoto (WKY) rats were treated with vehicle, sunitinib (angiogenesis inhibitor; 14 mg/kg/day) alone or in combination with macitentan (ETA/B receptor antagonist; 30 mg/kg/day) or sitaxentan (selective ETA receptor antagonist; 30 or 100 mg/kg/day) for 8 days. Compared with vehicle, sunitinib treatment caused a rapid and sustained increase in mean arterial pressure of ∼25 mmHg. Co-treatment with macitentan or sitaxentan abolished the pressor response to sunitinib. Sunitinib did not induce endothelial dysfunction. However, it was associated with increased aortic, mesenteric, and renal oxidative stress, an effect that was absent in mesenteric arteries of the macitentan and sitaxentan co-treated groups. Albuminuria was greater in the sunitinib- than vehicle-treated group. Co-treatment with sitaxentan, but not macitentan, prevented this increase in albuminuria. Sunitinib treatment increased circulating and urinary prostacyclin levels and had no effect on thromboxane levels. These increases in prostacyclin were blunted by co-treatment with sitaxentan.
Conclusions
Our results demonstrate that both selective ETA and dual ETA/B receptor antagonism prevents sunitinib-induced hypertension, whereas sunitinib-induced albuminuria was only prevented by selective ETA receptor antagonism. In addition, our results uncover a role for prostacyclin in the development of these effects. In conclusion, selective ETA receptor antagonism is sufficient for the prevention of sunitinib-induced hypertension and renal injury.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Jul 2020; 116:1779-1790

Colesevelam enhances the beneficial effects of brown fat activation on hyperlipidaemia and atherosclerosis development.

Zhou E, Hoeke G, Li Z, Eibergen AC, ... Berbée JFP, Wang Y
Aims
Brown fat activation accelerates the uptake of cholesterol-enriched remnants by the liver and thereby lowers plasma cholesterol, consequently protecting against atherosclerosis development. Hepatic cholesterol is then converted into bile acids (BAs) that are secreted into the intestine and largely maintained within the enterohepatic circulation. We now aimed to evaluate the effects of prolonged brown fat activation combined with inhibition of intestinal BA reabsorption on plasma cholesterol metabolism and atherosclerosis development.
Methods and results
APOE*3-Leiden.CETP mice with humanized lipoprotein metabolism were treated for 9 weeks with the selective β3-adrenergic receptor (AR) agonist CL316,243 to substantially activate brown fat. Prolonged β3-AR agonism reduced faecal BA excretion (-31%), while markedly increasing plasma levels of total BAs (+258%), cholic acid-derived BAs (+295%), and chenodeoxycholic acid-derived BAs (+217%), and decreasing the expression of hepatic genes involved in BA production. In subsequent experiments, mice were additionally treated with the BA sequestrant Colesevelam to inhibit BA reabsorption. Concomitant intestinal BA sequestration increased faecal BA excretion, normalized plasma BA levels, and reduced hepatic cholesterol. Moreover, concomitant BA sequestration further reduced plasma total cholesterol (-49%) and non-high-density lipoprotein cholesterol (-56%), tended to further attenuate atherosclerotic lesion area (-54%). Concomitant BA sequestration further increased the proportion of lesion-free valves (+34%) and decreased the relative macrophage area within the lesion (-26%), thereby further increasing the plaque stability index (+44%).
Conclusion
BA sequestration prevents the marked accumulation of plasma BAs as induced by prolonged brown fat activation, thereby further improving cholesterol metabolism and reducing atherosclerosis development. These data suggest that combining brown fat activation with BA sequestration is a promising new therapeutic strategy to reduce hyperlipidaemia and cardiovascular diseases.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cardiovasc Res: 31 Jul 2020; 116:1710-1720

Thioredoxin-1 maintains mitochondrial function via mechanistic target of rapamycin signalling in the heart.

Oka SI, Chin A, Park JY, Ikeda S, ... Yodoi J, Sadoshima J
Aims
Thioredoxin 1 (Trx1) is an evolutionarily conserved oxidoreductase that cleaves disulphide bonds in oxidized substrate proteins such as mechanistic target of rapamycin (mTOR) and maintains nuclear-encoded mitochondrial gene expression. The cardioprotective effect of Trx1 has been demonstrated via cardiac-specific overexpression of Trx1 and dominant negative Trx1. However, the pathophysiological role of endogenous Trx1 has not been defined with a loss-of-function model. To address this, we have generated cardiac-specific Trx1 knockout (Trx1cKO) mice.
Methods and results
Trx1cKO mice were viable but died with a median survival age of 25.5 days. They developed heart failure, evidenced by contractile dysfunction, hypertrophy, and increased fibrosis and apoptotic cell death. Multiple markers consistently indicated increased oxidative stress and RNA-sequencing revealed downregulation of genes involved in energy production in Trx1cKO mice. Mitochondrial morphological abnormality was evident in these mice. Although heterozygous Trx1cKO mice did not show any significant baseline phenotype, pressure-overload-induced cardiac dysfunction, and downregulation of metabolic genes were exacerbated in these mice. mTOR was more oxidized and phosphorylation of mTOR substrates such as S6K and 4EBP1 was impaired in Trx1cKO mice. In cultured cardiomyocytes, Trx1 knockdown inhibited mitochondrial respiration and metabolic gene promoter activity, suggesting that Trx1 maintains mitochondrial function in a cell autonomous manner. Importantly, mTOR-C1483F, an oxidation-resistant mutation, prevented Trx1 knockdown-induced mTOR oxidation and inhibition and attenuated suppression of metabolic gene promoter activity.
Conclusion
Endogenous Trx1 is essential for maintaining cardiac function and metabolism, partly through mTOR regulation via Cys1483.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Jul 2020; 116:1742-1755

Delineating the molecular and histological events that govern right ventricular recovery using a novel mouse model of pulmonary artery de-banding.

Boehm M, Tian X, Mao Y, Ichimura K, ... Metzger RJ, Spiekerkoetter E
Aims
The temporal sequence of events underlying functional right ventricular (RV) recovery after improvement of pulmonary hypertension-associated pressure overload is unknown. We sought to establish a novel mouse model of gradual RV recovery from pressure overload and use it to delineate RV reverse-remodelling events.
Methods and results
Surgical pulmonary artery banding (PAB) around a 26-G needle induced RV dysfunction with increased RV pressures, reduced exercise capacity and caused liver congestion, hypertrophic, fibrotic, and vascular myocardial remodelling within 5 weeks of chronic RV pressure overload in mice. Gradual reduction of the afterload burden through PA band absorption (de-PAB)-after RV dysfunction and structural remodelling were established-initiated recovery of RV function (cardiac output and exercise capacity) along with rapid normalization in RV hypertrophy (RV/left ventricular + S and cardiomyocyte area) and RV pressures (right ventricular systolic pressure). RV fibrotic (collagen, elastic fibres, and vimentin+ fibroblasts) and vascular (capillary density) remodelling were equally reversible; however, reversal occurred at a later timepoint after de-PAB, when RV function was already completely restored. Microarray gene expression (ClariomS, Thermo Fisher Scientific, Waltham, MA, USA) along with gene ontology analyses in RV tissues revealed growth factors, immune modulators, and apoptosis mediators as major cellular components underlying functional RV recovery.
Conclusion
We established a novel gradual de-PAB mouse model and used it to demonstrate that established pulmonary hypertension-associated RV dysfunction is fully reversible. Mechanistically, we link functional RV improvement to hypertrophic normalization that precedes fibrotic and vascular reverse-remodelling events.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Jul 2020; 116:1700-1709

NADPH oxidase 4 mediates the protective effects of physical activity against obesity-induced vascular dysfunction.

Brendel H, Shahid A, Hofmann A, Mittag J, ... Morawietz H, Brunssen C
Aims
Physical activity is one of the most potent strategies to prevent endothelial dysfunction. Recent evidence suggests vaso-protective properties of hydrogen peroxide (H2O2) produced by main endothelial NADPH oxidase isoform 4 (Nox4) in the vasculature. Therefore, we hypothesized that Nox4 connects physical activity with vaso-protective effects.
Methods and results
Analysis of the endothelial function using Mulvany Myograph showed endothelial dysfunction in wild-type (WT) as well as in C57BL/6J/ Nox4-/- (Nox4-/-) mice after 20 weeks on high-fat diet (HFD). Access to running wheels during the HFD prevented endothelial dysfunction in WT but not in Nox4-/- mice. Mechanistically, exercise led to an increased H2O2 release in the aorta of WT mice with increased phosphorylation of eNOS pathway member AKT serine/threonine kinase 1 (AKT1). Both H2O2 release and phosphorylation of AKT1 were diminished in aortas of Nox4-/- mice. Deletion of Nox4 also resulted in lower intracellular calcium release proven by reduced phenylephrine-mediated contraction, whilst potassium-induced contraction was not affected. H2O2 scavenger catalase reduced phenylephrine-induced contraction in WT mice. Supplementing H2O2 increased phenylephrine-induced contraction in Nox4-/- mice. Exercise-induced peroxisome proliferative-activated receptor gamma, coactivator 1 alpha (Ppargc1a), as key regulator of mitochondria biogenesis in WT but not Nox4-/- mice. Furthermore, exercise-induced citrate synthase activity and mitochondria mass were reduced in the absence of Nox4. Thus, Nox4-/- mice became less active and ran less compared with WT mice.
Conclusions
Nox4 derived H2O2 plays a key role in exercise-induced adaptations of eNOS and Ppargc1a pathway and intracellular calcium release. Hence, loss of Nox4 diminished physical activity performance and vascular protective effects of exercise.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: [email protected]

Cardiovasc Res: 31 Jul 2020; 116:1767-1778

Cardiomyocyte ageing and cardioprotection: consensus document from the ESC working groups cell biology of the heart and myocardial function.

Ruiz-Meana M, Bou-Teen D, Ferdinandy P, Gyongyosi M, ... Thum T, Madonna R

Advanced age is a major predisposing risk factor for the incidence of coronary syndromes and comorbid conditions which impact the heart response to cardioprotective interventions. Advanced age also significantly increases the risk of developing post-ischaemic adverse remodelling and heart failure after ischaemia/reperfusion (IR) injury. Some of the signalling pathways become defective or attenuated during ageing, whereas others with well-known detrimental consequences, such as glycoxidation or proinflammatory pathways, are exacerbated. The causative mechanisms responsible for all these changes are yet to be elucidated and are a matter of active research. Here, we review the current knowledge about the pathophysiology of cardiac ageing that eventually impacts on the increased susceptibility of cells to IR injury and can affect the efficiency of cardioprotective strategies.

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

Cardiovasc Res: 31 Aug 2020; 116:1835-1849

Non-coding RNAs: update on mechanisms and therapeutic targets from the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart.

Bär C, Chatterjee S, Falcão Pires I, Rodrigues P, ... Madonna R, Thum T

Vast parts of mammalian genomes are actively transcribed, predominantly giving rise to non-coding RNA (ncRNA) transcripts including microRNAs, long ncRNAs, and circular RNAs among others. Contrary to previous opinions that most of these RNAs are non-functional molecules, they are now recognized as critical regulators of many physiological and pathological processes including those of the cardiovascular system. The discovery of functional ncRNAs has opened up new research avenues aiming at understanding ncRNA-related disease mechanisms as well as exploiting them as novel therapeutics in cardiovascular therapy. In this review, we give an update on the current progress in ncRNA research, particularly focusing on cardiovascular physiological and disease processes, which are under current investigation at the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart. This includes a range of topics such as extracellular vesicle-mediated communication, neurohormonal regulation, inflammation, cardiac remodelling, cardio-oncology as well as cardiac development and regeneration, collectively highlighting the wide-spread involvement and importance of ncRNAs in the cardiovascular system.

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

Cardiovasc Res: 31 Aug 2020; 116:1805-1819

Cardiac dysfunction in cancer patients: beyond direct cardiomyocyte damage of anticancer drugs: novel cardio-oncology insights from the joint 2019 meeting of the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart.

Tocchetti CG, Ameri P, de Boer RA, D\'Alessandra Y, ... Heymans S, Thum T

In western countries, cardiovascular (CV) disease and cancer are the leading causes of death in the ageing population. Recent epidemiological data suggest that cancer is more frequent in patients with prevalent or incident CV disease, in particular, heart failure (HF). Indeed, there is a tight link in terms of shared risk factors and mechanisms between HF and cancer. HF induced by anticancer therapies has been extensively studied, primarily focusing on the toxic effects that anti-tumour treatments exert on cardiomyocytes. In this Cardio-Oncology update, members of the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart discuss novel evidence interconnecting cardiac dysfunction and cancer via pathways in which cardiomyocytes may be involved but are not central. In particular, the multiple roles of cardiac stromal cells (endothelial cells and fibroblasts) and inflammatory cells are highlighted. Also, the gut microbiota is depicted as a new player at the crossroads between HF and cancer. Finally, the role of non-coding RNAs in Cardio-Oncology is also addressed. All these insights are expected to fuel additional research efforts in the field of Cardio-Oncology.

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

Cardiovasc Res: 31 Aug 2020; 116:1820-1834

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

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

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

Cardiovasc Res: 26 Jul 2020; epub ahead of print

Heart Failure with Preserved Ejection Fraction: Insights into Diagnosis and Pathophysiology.

Nagueh SF

Heart failure with preserved ejection fraction (HFpEF) accounts for at least half the cases of heart failure, currently diagnosed. There are several cardiac and non-cardiac manifestations of the syndrome. Structure and function abnormalities can include all four cardiac chambers. The left ventricle (LV) has abnormal systolic and diastolic functions which can be examined by invasive and noninvasive measurements. In addition, the left atrium (LA) enlarges with abnormal LA function, pulmonary hypertension occurs, and the right ventricle can develop hypertrophy, enlargement, and systolic dysfunction. There are a paucity of data on calcium handling in HFpEF patients. Growing literature supports the presence of abnormalities in titin and its phosphorylation, and increased interstitial fibrosis contributing to increased chamber stiffness. A systemic inflammatory state causing reduced myocardial c-GMP along with defects in the unfolded protein response have been recently reported. Diagnosis relies on signs and symptoms of heart failure, preserved EF, and detection of diastolic function abnormalities based on echocardiographic findings and abnormally elevated natriuretic peptide levels or invasive measurements of wedge pressure at rest or with exercise. There are currently two diagnostic algorithms: H2FPEF, and HFA-PEFF with limited data comparing their performance head to head in the same patient population. Despite the growing understanding of the syndrome\'s pathophysiology, there have been little success in developing specific treatment for patients with HFpEF.

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

Cardiovasc Res: 26 Jul 2020; epub ahead of print

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

Roy-Chowdhury E, Brauns N, Helmke A, Nordlohne J, ... Haller H, von Vietinghoff S
Aims
Monocytes are central for atherosclerotic vascular inflammation. The human non-classical, patrolling subtype, which expresses high levels of CD16 and fractalkine receptor CX3CR1, strongly associates with cardiovascular events. This is most marked in renal failure, a condition with excess atherosclerosis morbidity. The underlying mechanism is not understood. This study investigated how human CD16+ monocytes modulate endothelial cell function.
Methods and results
In patients with kidney failure, CD16+ monocyte counts were elevated and dynamically decreased within a year after transplantation, chiefly due to a drop in CD14+CD16+ cells. The CX3CR1 ligand CX3CL1 was similarly elevated in the circulation of humans and mice with renal impairment. CX3CL1 upregulation was also observed close to macrophage rich human coronary artery plaques. To investigate a mechanistic basis of this association, CD16+CX3CR1HIGH monocytes were co-incubated with primary human endothelium in vitro. Compared to classical CD14+ monocytes or transwell cocultures, CD16+ monocytes enhanced endothelial STAT1 and NFκB p65 phosphorylation, upregulated expression of CX3CL1 and IL-1β, numerous CCL and CXCL chemokines and molecules promoting leukocyte patrolling and adhesion such as ICAM1 and VCAM1. Genes required for vasodilatation including eNOS decreased while endothelial collagen production increased. Uremic patients\' monocytes enhanced endothelial CX3CL1 even more markedly. Their receptor CX3CR1 was required for enhanced aortic endothelial stiffness in murine atherosclerosis with renal impairment. CX3CR1 dose-dependently modulated monocyte-contact-dependent gene expression in human endothelium.
Conclusions
By demonstrating endothelial proatherosclerotic gene regulation in direct contact with CD16+ monocytes, in part via cellular CX3CR1-CX3CL1 interaction, our data delineate a mechanism how this celltype can increase cardiovascular risk.
Translational perspective
Human CD16+ monocytes strongly associate with cardiovascular disease. Our data show that they induce primary human arterial and venous endothelial chemokine and adhesion molecule expression and increase mediators of vascular stiffness in direct interaction of the endothelium with CX3CR1. CD16+ monocytes reversibly increase in human kidney failure. Our results propose CD16+ monocytes as mediators of cardiovascular events including the reversible vascular phenotype of patients with kidney failure. Interference with their direct contact to the endothelium may represent a causal therapeutic approach in this high-risk patient group.

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

Cardiovasc Res: 26 Jul 2020; epub ahead of print

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

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

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

Cardiovasc Res: 30 Jul 2020; epub ahead of print

Impaired Retinal Microvascular Function Predicts Long-Term Adverse Events in Patients with Cardiovascular Disease.

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

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

Cardiovasc Res: 03 Aug 2020; epub ahead of print

Endothelial dysfunction in COVID-19: a position paper of the ESC Working Group for Atherosclerosis and Vascular Biology, and the ESC Council of Basic Cardiovascular Science.

Evans PC, Ed Rainger G, Mason JC, Guzik TJ, ... Bochaton-Piallat ML, Bäck M

The COVID-19 pandemic is an unprecedented healthcare emergency causing mortality and illness across the world. Although primarily affecting the lungs, the SARS-CoV-2 virus also affects the cardiovascular system. In addition to cardiac effects, e.g. myocarditis, arrhythmias, and myocardial damage, the vasculature is affected in COVID-19, both directly by the SARS-CoV-2 virus, and indirectly as a result of a systemic inflammatory cytokine storm. This includes the role of the vascular endothelium in the recruitment of inflammatory leucocytes where they contribute to tissue damage and cytokine release, which are key drivers of acute respiratory distress syndrome (ARDS), in disseminated intravascular coagulation, and cardiovascular complications in COVID-19. There is also evidence linking endothelial cells (ECs) to SARS-CoV-2 infection including: (i) the expression and function of its receptor angiotensin-converting enzyme 2 (ACE2) in the vasculature; (ii) the prevalence of a Kawasaki disease-like syndrome (vasculitis) in COVID-19; and (iii) evidence of EC infection with SARS-CoV-2 in patients with fatal COVID-19. Here, the Working Group on Atherosclerosis and Vascular Biology together with the Council of Basic Cardiovascular Science of the European Society of Cardiology provide a Position Statement on the importance of the endothelium in the underlying pathophysiology behind the clinical presentation in COVID-19 and identify key questions for future research to address. We propose that endothelial biomarkers and tests of function (e.g. flow-mediated dilatation) should be evaluated for their usefulness in the risk stratification of COVID-19 patients. A better understanding of the effects of SARS-CoV-2 on endothelial biology in both the micro- and macrovasculature is required, and endothelial function testing should be considered in the follow-up of convalescent COVID-19 patients for early detection of long-term cardiovascular complications.

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

Cardiovasc Res: 03 Aug 2020; epub ahead of print

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

Singla B, Lin HP, Chen A, Ahn W, ... Stansfield BK, Csányi G
Background
Impaired lymphatic drainage of the arterial wall results in intimal lipid accumulation and atherosclerosis. However, the mechanisms regulating lymphangiogenesis in atherosclerotic arteries are not well understood. Our studies identified elevated levels of matrix protein R-Spondin 2 (RSPO2) in atherosclerotic arteries. In this study, we investigated the role of RSPO2 in lymphangiogenesis, arterial cholesterol efflux into lesion-draining lymph nodes and development of atherosclerosis.
Methods and results
The effect of RSPO2 on lymphangiogenesis was investigated using human lymphatic endothelial cells in vitro and implanted Matrigel plugs in vivo. Cellular and molecular approaches, pharmacological agents, and siRNA silencing of RSPO2 receptor LGR4 were used to investigate RSPO2-mediated signaling in lymphatic endothelial cells. In vivo LDL tracking and perivascular blockade of RSPO2-LGR4 signaling using LGR4-ECD pluronic gel in hypercholesterolemic mice were utilized to investigate the role of RSPO2 in arterial reverse cholesterol transport and atherosclerosis. Immunoblotting and imaging experiments demonstrated increased RSPO2 expression in human and mouse atherosclerotic arteries compared to non-atherosclerotic controls. RSPO2 treatment inhibited lymphangiogenesis both in vitro and in vivo. LGR4 silencing and inhibition of RSPO2-LGR4 signaling abrogated RSPO2-induced inhibition of lymphangiogenesis. Mechanistically, we found that RSPO2 inhibits PI3K-AKT-eNOS signaling via LGR4 and inhibits activation of the canonical Wnt-β-catenin pathway. ApoE-/- mice treated with LGR4-ECD developed significantly less atherosclerosis compared with control treatment. Finally, increased arterial lymphatic vessel density and improved lymphatic drainage of fluorescently-labeled LDL to deep cervical lymph nodes were observed in LGR4-ECD-treated mice.
Conclusions
These findings demonstrate that RSPO2 inhibits lymphangiogenesis via LGR4 and downstream impairment of AKT-eNOS-NO signaling. These results may also inform new therapeutic strategies to promote lymphangiogenesis and improve cholesterol efflux from atherosclerotic arteries.
Translational perspective
Atherosclerotic cardiovascular disease is the leading cause of death worldwide. Thus, attenuation of atherosclerotic lesion formation and prevention of its cardiovascular complications is an urgent medical need. The findings of the present study that inhibition of LGR4-mediated signaling increases arterial lymphangiogenesis, improves lymphatic drainage from the vessel wall and attenuates atherosclerosis, provide a framework from which novel therapeutic strategies to augment lymphatic vessel density and reduce atherosclerotic lesion formation can be developed and used for the treatment of patients with atherosclerosis. This pathway may also have important implications in other pathological conditions associated with lymphatic dysfunction, such as lymphedema, obesity, hypertension, and impaired wound healing.

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

Cardiovasc Res: 03 Aug 2020; epub ahead of print

Mechanism of succinate efflux upon reperfusion of the ischemic heart.

Prag HA, Gruszczyk AV, Huang MM, Beach TE, ... Murphy MP, Aksentijević D
Aims
Succinate accumulates several-fold in the ischemic heart and is then rapidly oxidised upon reperfusion, contributing to reactive oxygen species (ROS) production by mitochondria. In addition, a significant amount of the accumulated succinate is released from the heart into the circulation at reperfusion, potentially activating the G-protein coupled succinate receptor (SUCNR1). However, the factors that determine the proportion of succinate oxidation or release, and the mechanism of this release, are not known.
Methods and results
To address these questions, we assessed the fate of accumulated succinate upon reperfusion of anoxic cardiomyocytes, and of the ischemic heart both ex vivo and in vivo. The release of accumulated succinate was selective and was enhanced by acidification of the intracellular milieu. Furthermore, pharmacological inhibition, or haploinsufficiency of the monocarboxylate transporter 1 (MCT1) significantly decreased succinate efflux from the reperfused heart.
Conclusion
Succinate release upon reperfusion of the ischemic heart is mediated by MCT1 and is facilitated by the acidification of the myocardium during ischemia. These findings will allow the signalling interaction between succinate released from reperfused ischemic myocardium and SUCNR1 to be explored.
Translational perspectives
In this study we demonstrate that succinate efflux upon reperfusion of the ischemic myocardium is mediated by the monocarboxylate transporter 1 (MCT1) and is enhanced by the ischemic acidification of the heart. These findings are an important advance in understanding how succinate is released upon reperfusion of ischemic organs. While this pathway is therapeutically tractable, greater understanding of the effects of succinate release is required before exploring this possibility.

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

Cardiovasc Res: 06 Aug 2020; epub ahead of print

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

Rathjens FS, Blenkle A, Iyer LM, Renger A, ... Zelarayan LC, Zafeiriou MP
Aims
Arrhythmias and sudden cardiac death (SCD) occur commonly in patients with heart failure. We found T-box 5 (TBX5) dysregulated in ventricular myocardium from heart failure patients and thus we hypothesized that TBX5 reduction contributes to arrhythmia development in these patients. To understand the underlying mechanisms, we aimed to reveal the ventricular TBX5-dependent transcriptional network and further test the therapeutic potential of TBX5 level normalization in mice with documented arrhythmias.
Methods and results
We used a mouse model of TBX5 conditional deletion in ventricular cardiomyocytes. Ventricular (v) TBX5 loss in mice resulted in mild cardiac dysfunction and arrhythmias and was associated with a high mortality rate (60%) due to SCD. Upon angiotensin stimulation, vTbx5KO mice showed exacerbated cardiac remodelling and dysfunction suggesting a cardioprotective role of TBX5. RNA sequencing of a ventricular specific TBX5KO mouse and TBX5 chromatin immunoprecipitation were used to dissect TBX5 transcriptional network in cardiac ventricular tissue. Overall, we identified 47 transcripts expressed under the control of TBX5, which may have contributed to the fatal arrhythmias in vTbx5KO mice. These included transcripts encoding for proteins implicated in cardiac conduction and contraction (Gja1, Kcnj5, Kcng2, Cacna1g, Chrm2), in cytoskeleton organization (Fstl4, Pdlim4, Emilin2, Cmya5), and cardiac protection upon stress (Fhl2, Gpr22, Fgf16). Interestingly, after TBX5 loss and arrhythmia development in vTbx5KO mice, TBX5 protein level normalization by systemic adeno-associated-virus (AAV) 9 application, re-established TBX5-dependent transcriptome. Consequently, cardiac dysfunction was ameliorated and the propensity of arrhythmia occurrence was reduced.
Conclusions
This study uncovers a novel cardioprotective role of TBX5 in the adult heart and provides preclinical evidence for the therapeutic value of TBX5 protein normalization in the control of arrhythmia.
Translational perspective
Cardiovascular disease (CVD) is the number one cause of death worldwide (WHO factsheets 09/2016). Although more than 60% of CVD-related deaths are due to out-of-hospital sudden cardiac death (SCD), we have little insight in the mechanisms underlying SCD pathophysiology. Our data show a link between TBX5 dysregulation and arrhythmia occurrence in patients. To test the therapeutic potential of TBX5, we normalized TBX5 levels in a mouse model with TBX5 dysregulation, which developed arrhythmias and SCD. TBX5 normalization re-established TBX5 target gene expression and more importantly, rescued the arrhythmia phenotype. Altogether, we provide proof-of-concept for the therapeutic potential of TBX5 expression restoration against arrhythmia and SCD.

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

Cardiovasc Res: 09 Aug 2020; epub ahead of print

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

Déri S, Borbás J, Hartai T, Hategan L, ... Sepp R, Ördög B
Aims
Subunit interactions at the cytoplasmic domain interface (CD-I) have recently been shown to control gating in inward rectifier potassium channels. Here we report the novel KCNJ2 variant p.Glu293Lys that has been found in a patient with Andersen-Tawil Syndrome type 1 (ATS1), causing amino acid substitution at the CD-I of the inward rectifier potassium channel subunit Kir2.1. Neither has the role of Glu293 in gating control been investigated, nor has a pathogenic variant been described at this position. This study aimed to assess the involvement of Glu293 in CD-I subunit interactions and to establish the pathogenic role of the p.Glu293Lys variant in ATS1.
Methods and results
The p.Glu293Lys variant produced no current in homomeric form and showed dominant negative effect over wild type (WT) subunits. Immunocytochemical labelling showed the p.Glu293Lys subunits to distribute in the subsarcolemmal space. Salt bridge prediction indicated the presence of an intersubunit salt bridge network at the CD-I of Kir2.1, with the involvement of Glu293. Subunit interactions were studied by the NanoBiT split reporter assay. Reporter constructs carrying NanoBiT tags on the intracellular termini produced no bioluminescent signal above background with the p.Glu293Lys variant in homomeric configuration and significantly reduced signals in cells co-expressing WT and p.Glu293Lys subunits simultaneously. Extracellularly presented reporter tags, however, generated comparable bioluminescent signals with heteromeric WT and p.Glu293Lys subunits and with homomeric WT channels.
Conclusions
Loss of function and dominant negative effect confirm the causative role of p.Glu293Lys in ATS1. Co-assembly of Kir2.1 subunits is impaired in homomeric channels consisting of p.Glu293Lys subunits and is partially rescued in heteromeric complexes of WT and p.Glu293Lys Kir2.1 variants. These data point to an important role of Glu293 in mediating subunit assembly, as well as in gating of Kir2.1 channels.
Translational perspective
Andersen-Tawil Syndrome (ATS) is a rare genetic disorder characterized by the triad of periodic paralysis, dysmorphic features and ventricular arrhythmias. Symptoms can be mild and atypical, therefore, genetic screening of affected families is pivotal. This study describes the p.Glu293Lys variant of KCNJ2 encoding the Kir2.1 ion channel subunit as pathogenic, thereby aiding genetic testing of ATS. The study also identifies disturbed interactions between the cytoplasmic domains of Kir2.1 subunits as the molecular mechanism of loss-of-function in the p.Glu293Lys variant. Targeting cytoplasmic domain interactions may represent a promising strategy for the development of Kir2.1 agonists.

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

Cardiovasc Res: 17 Aug 2020; epub ahead of print

High Spatial Endothelial Shear Stress Gradient Independently Predicts Site of Acute Coronary Plaque Rupture and Erosion.

Thondapu V, Mamon C, Poon EKW, Kurihara O, ... Jang IK,
Aims
To investigate local haemodynamics in the setting of acute coronary plaque rupture and erosion.
Methods and results
Intracoronary optical coherence tomography performed in 37 patients with acute coronary syndromes caused by plaque rupture (n = 19) or plaque erosion (n = 18) was used for 3D reconstruction and computational fluid dynamic simulation. Endothelial shear stress (ESS), spatial ESS gradient (ESSG), and oscillatory shear index (OSI) were compared between plaque rupture and erosion through mixed-effects logistic regression. Lipid, calcium, macrophages, layered plaque, and cholesterol crystals were also analysed. By multivariable analysis, only high ESSG (odds ratio [OR] 5.29, 95% confidence interval [CI] 2.57-10.89, p < 0.001), lipid (OR 12.98, 95% CI 6.57-25.67 p < 0.001), and layered plaque (OR 3.17, 95% CI 1.82-5.50, p < 0.001) were independently associated with plaque rupture. High ESSG (OR 13.28, 95% CI 6.88-25.64, p < 0.001), ESS (OR 2.70, 95% CI 1.34-5.42, p = 0.005) and OSI (OR 2.18, 95% CI 1.33-3.54, p = 0.002) independently associated with plaque erosion. ESSG was higher at rupture sites than erosion sites (median (interquartile range): 5.78 (2.47, 21.15) versus 2.62 (1.44, 6.18) Pa/mm, p = 0.009), OSI was higher at erosion sites than rupture sites (1.04x10-2 (2.3x10-3, 4.74x10-2) versus 1.29x10-3 (9.39x10-5, 3.0x10-2), p < 0.001), but ESS was similar (p = 0.29).
Conclusions
High ESSG is independently associated with plaque rupture while high ESSG, ESS, and OSI associate with plaque erosion. While ESSG is higher at rupture sites than erosion sites, OSI is higher at erosion sites and ESS was similar. These results suggest that ESSG and OSI may play critical roles in acute plaque rupture and erosion, respectively.
Translational perspective
Plaque rupture and erosion are distinct pathological and clinical entities with possibly different optimal treatments. This study demonstrates that high endothelial shear stress gradient is independently associated with site of both rupture and erosion, and is significantly higher in rupture. High oscillatory shear index is independently associated with the site of erosion only, and is higher in erosion than rupture. Larger studies are necessary to determine whether these indices may detect and distinguish plaque rupture and erosion in a clinical setting or to assess overall risk for acute coronary syndromes.

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

Cardiovasc Res: 23 Aug 2020; epub ahead of print

Autonomic Innervation of the Carotid Body as a Determinant of its Sensitivity - Implications for Cardiovascular Physiology and Pathology.

Brognara F, Felippe ISA, Salgado HC, Paton JFR

The motivation for this review comes from the emerging complexity of the autonomic innervation of the carotid body and its putative role in regulating chemoreceptor sensitivity. With the carotid bodies as a potential therapeutic target for numerous cardiorespiratory and metabolic diseases, an understanding of the neural control of its circulation is most relevant. Since nerve fibres track blood vessels and receive autonomic innervation, we initiate our review by describing the origins of arterial feed to the carotid body and its unique vascular architecture and blood flow. Arterial feed(s) vary amongst species and, unequivocally, the arterial blood supply is relatively high to this organ. The vasculature appears to form separate circuits inside the carotid body with one having an arterial venous anastomoses. Both sympathetic and parasympathetic nerves are present with postganglionic neurons located within the carotid body or close to it in the form of paraganglia. Their role in arterial vascular resistance control is described as is how carotid blood flow relates to carotid sinus afferent activity. We discuss non-vascular targets of autonomic nerves, their possible role in controlling glomus cell activity, and how certain transmitters may relate to function. We propose that the autonomic nerves sub-serving the carotid body provide a rapid mechanism to tune the gain of peripheral chemoreflex sensitivity based on alterations in blood flow and oxygen delivery, and might provide future therapeutic targets. However, there remain a number of unknowns regarding these mechanisms that require further research that is discussed.

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

Cardiovasc Res: 23 Aug 2020; epub ahead of print

Mechanism of Ventricular Premature Beats Elicited by Left Stellate Ganglion Stimulation During Acute Ischemia of the Anterior Left Ventricle.

Boukens BJD, Dacey M, Meijborg VMF, Janse MJ, ... Shivkumar K, Coronel R
Aims
Enhanced sympathetic activity during acute ischemia is arrhythmogenic, but the underlying mechanism is unknown. During ischemia, a diastolic current flows from the ischemic to the non-ischemic myocardium. This \"injury\" current can cause ventricular premature beats originating in the non-ischemic myocardium, especially during a deeply negative T wave in the ischemic zone. We reasoned that shortening of repolarization in myocardium adjacent to ischemic myocardium increases the \"injury\" current and causes earlier deeply negative T waves in the ischemic zone, and re-excitation of the normal myocardium. We tested this hypothesis by activation and repolarization mapping during stimulation of the left stellate ganglion (LSGS) during left anterior descending coronary artery (LAD) occlusion.
Methods and results
In 9 pigs, five subsequent episodes of acute ischemia, separated by 20 min of reperfusion, were produced by occlusion of the LAD and 121 epicardial local unipolar electrograms were recorded. During the third occlusion, LSGS was initiated after 3 min for a 30-sec period, causing a shortening of repolarization in the normal myocardium by about 100 msec. This resulted in more negative T waves in the ischemic zone and more ventricular premature beats (VPBs) than during the second, control, occlusion. Following decentralization of the LSG (including removal of the right stellate ganglion and bilateral cervical vagotomy), fewer VPBs occurred during ischemia without LSGS. During LSGS, the number of VPBs was similar to that recorded before decentralization.
Conclusion
LSGS, by virtue of shortening of repolarization in the non-ischemic myocardium by about 100 msec, causes deeply negative T waves in the ischemic tissue and VPBs originating from the normal tissue adjacent to the ischemic border. In this setting, decentralization of the LSG is antiarrhythmic.
Translational perspective
Cardiac sympathetic denervation is a promising therapy for reducing arrhythmias during acute ischemia. Currently it is not clear which patients with ischemic heart disease would benefit from cardiac sympathetic denervation and for which patients it is unlikely to have an effect. This is important because cardiac sympathetic denervation by removing the stellate ganglia often results in severe side effects and morbidity. Our results indicate that left stellate ganglion activity is pro-arrhythmic and that left stellectomy is beneficial for the prevention of arrhythmias during anterior wall ischemia. When the ischemic zone is in the lateral and posterior wall, the effects of LSGS will be different because it will directly affect the ischemic myocardium, and might even be antiarrhythmic. Thus, the effect of left stellate stimulation can be pro or antiarrhythmic, depending on the location of myocardial ischemia.

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

Cardiovasc Res: 26 Aug 2020; epub ahead of print

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

Chao CT, Yeh HY, Tsai YT, Chiang CK, Chen HW
Aims
Vascular calcification (VC) increases the future risk of cardiovascular events in uremic patients, but effective therapies are still unavailable. Accurate identification of those at risk of developing VC using pathogenesis-based biomarkers is of particular interest and may facilitate individualized risk stratification. We aimed to uncover miRNA-target protein-based biomarker panels for evaluating uremic VC probability and severity.
Methods and results
We created a 3-tiered in vitro VC model and an in vivo uremic rat model receiving high phosphate diet to mimic uremic VC. RNAs from the 3-tiered in vitro and in vivo uremic VC models underwent miRNA and mRNA microarray, with results screened for differentially expressed miRNAs and their target genes as biomarkers. Findings were validated in original models and additionally in an ex vivo VC model and human cells, followed by functional assays of identified miRNAs and target proteins, and tests of sera from end-stage renal disease (ESRD) and non-dialysis dependent chronic kidney disease (CKD) patients without and with VC. Totally 122 down-regulated and 119 up-regulated miRNAs during calcification progression were identified initially; further list-narrowing based on miRNA-mRNA pairing, anti-correlation, and functional enrichment left 16 and 14 differentially expressed miRNAs and mRNAs. Levels of 4 miRNAs (miR-10b-5p, miR-195, miR-125b-2-3p, and miR-378a-3p) were shown to decrease throughout all models tested, while 1 mRNA (SULF1, a potential target of miR-378a-3p) exhibited the opposite trend concurrently. Among 96 ESRD (70.8% with VC) and 59 CKD patients (61% with VC), serum miR-125b2-3p and miR-378a-3p decreased with greater VC severity, while serum SULF1 levels increased. Adding serum miR-125b-2-3p, miR-378a-3p, and SULF1 into regression models for VC substantially improved performance compared to using clinical variables alone.
Conclusion
Using a translational approach, we discovered a novel panel of biomarkers for gauging the probability/severity of uremic VC based on miRNAs/target proteins, which improved the diagnostic accuracy.
Translational perspective
Through miRNA and transcriptomic profiling with serial result validation in different models, we discover that miR-378a-3p and its target gene, SULF1, are an important pair of circulating biomarkers for the diagnosis and severity classification of uremic VC among patients with non-dialysis CKD and ESRD. This approach improves the predictive performance compared to that based on traditional clinical features alone. These findings suggest that a combined miRNA/target protein panel may represent a potentially useful approach for detecting uremic VC.

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

Cardiovasc Res: 30 Aug 2020; epub ahead of print

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 aging in HFrEF potentially leading to targeted therapies in this vulnerable population.
Methods and results
From a panel of 363 cardiovascular biomarkers available in 1,611 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 > 75yr versus <65yr. Secondly, pathway overrepresentation analyses were performed to identify biological pathways linked to higher plasma concentrations of biomarkers in elderly versus younger patients. After adjustment, multiple test correction (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 tumor 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 tumorigenesis. Among the 27 biomarkers, WFDC2 (WAP Four-Disulfide-Core-Domain-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 tumor cell regulation were activated, while pathways associated with tumor proliferation functions were down-regulated. These findings may help in a better understanding of the aging processes in HFrEF and identify potential therapeutic targets.
Translational perspective
Elderly patients with heart failure with reduced ejection fraction (HFrEF) have worse prognosis and less often receive guideline-recommended therapies. Using a large set of circulating proteins, elderly patients had higher concentrations of proteins associated with tumor cell regulation, extra-cellular matrix organization, and inflammatory processes, whereas pathways that may point to cell proliferation and tumorigenesis were down-regulated. WAP Four-Disulfide-Core-Domain-2 was associated with older age and had the strongest association with an increased risk of all outcomes. Understanding the underlying pathophysiological processes associated with aging in HFrEF may potentially lead to targeted therapies in this vulnerable population.

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

Cardiovasc Res: 30 Sep 2020; epub ahead of print

Angiogenesis After Acute Myocardial Infarction.

Wu X, Reboll MR, Korf-Klingebiel M, Wollert KC

Acute myocardial infarction (MI) inflicts massive injury to the coronary microcirculation leading to vascular disintegration and capillary rarefication in the infarct region. Tissue repair after MI involves a robust angiogenic response that commences in the infarct border zone and extends into the necrotic infarct core. Technological advances in several areas have provided novel mechanistic understanding of postinfarction angiogenesis and how it may be targeted to improve heart function after MI. Cell lineage tracing studies indicate that new capillary structures arise by sprouting angiogenesis from preexisting endothelial cells (ECs) in the infarct border zone with no meaningful contribution from non-endothelial cell sources. Single cell RNA sequencing (scRNAseq) shows that ECs in infarcted hearts may be grouped into clusters with distinct gene expression signatures, likely reflecting functionally distinct cell populations. EC-specific multicolor lineage tracing reveals that EC subsets clonally expand after MI. Expanding EC clones may arise from tissue-resident ECs with stem cell characteristics that have been identified in multiple organs including the heart. Tissue repair after MI involves interactions among multiple cell types which occur, to a large extent, through secreted proteins and their cognate receptors. While we are only beginning to understand the full complexity of this intercellular communication, macrophage and fibroblast populations have emerged as major drivers of the angiogenic response after MI. Animal data support the view that the endogenous angiogenic response after MI can be boosted to reduce scarring and adverse left ventricular remodeling. The improved mechanistic understanding of infarct angiogenesis therefore creates multiple therapeutic opportunities. During preclinical development, all proangiogenic strategies should be tested in animal models that replicate both cardiovascular risk factor(s) and the pharmacotherapy typically prescribed to patients with acute MI. Considering that the majority of patients nowadays do well after MI, clinical translation will require careful selection of patients in need of proangiogenic therapies.

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

Cardiovasc Res: 15 Oct 2020; epub ahead of print

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 a LC diet impacts on cardiac pathology during hemodynamic stress remains elusive. This study evaluated the effects of a LC diet high in either fat (Fat-LC) or protein (Pro-LC) in a mouse model of chronic hypertensive cardiac remodeling.
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, hemodynamic, 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 signaling.
Conclusions
Strict carbohydrate restriction suppresses pathological cardiac growth and heart failure after pressure overload through distinct anti-hypertrophic mechanisms elicited by supplemented macronutrients.
Translational perspective
Hemodynamic stress, such as hypertension, induces pathological cardiac hypertrophy, leading to heart failure. There is growing evidence that modulating components of diet affects cardiac function in humans, although the causality and underlying mechanisms are poorly understood. Our study demonstrates that strict restriction of dietary carbohydrates supplemented with either fat or proteins during acute hemodynamic stress attenuates the development and progression of cardiac hypertrophy and heart failure by activating distinct anti-hypertrophic and cardioprotective signaling mechanisms. The study suggests that it would be useful to investigate the therapeutic benefit of carbohydrate restriction in patients with hypertension and cardiac hypertrophy in clinical studies.

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

Cardiovasc Res: 17 Oct 2020; epub ahead of print

Omega-3 fatty acids improve flow-induced vasodilation by enhancing TRPV4 in arteries from diet-induced obese mice.

Zhu Y, Wen L, Wang S, Zhang K, ... Wang R, Ma X
Aims
Previous studies have shown the intake of omega-3 polyunsaturated fatty acids are associated with low rates of obesity and ischemic pathologies. Omega-3 also have anti-inflammatory and plaque-stabilization effects and regulate vasodilation and constriction. However, there are few studies of the role of omega-3 in flow-induced vasodilation involving Ca2+-permeable ion channel TRPV4 in high-fat diet-induced obese (DIO) mouse. Here, we determined whether omega-3 protect against vascular dysfunction induced by high-fat diet by enhancing TRPV4 activity and subsequently improving flow-mediated vasodilation.
Methods and results
Flow-mediated vasodilation in 2nd-order mesenteric arteries from mice was measured using a pressure myograph. The intracellular Ca2+ concentration in response to flow and GSK1016790A (a TRPV4 agonist) was measured by Fluo-4 fluorescence. Whole-cell current was measured by patch clamp. Cell membrane tether force was measured by atomic force microscopy. Impairment of flow-mediated vasodilation in arteries and Ca2+ influx in endothelial cells from DIO mice was restored by omega-3 treatment. The improved flow-induced vasodilation was inhibited by the TRPV4 antagonist HC067047 and in TRPV4-/- mice. Omega-3 treatment enhanced endothelial TRPV4 activity and altered cell membrane mechanic property, as indicated by enhanced GSK1016790A-induced Ca2+ influx and whole-cell current and altered membrane mean tether force in endothelial cells from DIO mice.
Conclusion
Omega-3 improve vascular function by improving flow-induced vasodilation via enhancing TRPV4 activity in the endothelium of obese mice which may be related to improved cell membrane physical property. Activation of TRPV4 in endothelium plays an important role in the protective mechanisms of omega-3 against vascular dysfunction in obesity by improving flow-mediated vasodilation.
Translational perspective
Omega-3 improve the endothelial function via enhancing TRPV4 activity and augmenting the endothelial-dependent flow-induced vasodilation in DIO mice resistance arteries. This study provides a strategy of improving vascular function under obesity, namely, by targeting TRPV4 via the usage of omega-3.

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

Cardiovasc Res: 17 Oct 2020; epub ahead of print

Haplo-insufficiency 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 (EMT) and age-dependent expression of myocardial fibrosis and apoptosis in the Myh6-Cre: Tmem43W/F mice.
Conclusions
TMEM43 haplo-insufficiency 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.
Translational perspective
The data indicate that the DNA damage response (DDR) to double stranded DNA breaks (DSBs) is activated in a mouse model of cardiomyopathy caused by haplo-insufficiency of the Tmem43 gene. The TMEM43 gene is a known cause of arrhythmogenic cardiomyopathy in humans. The DDR activates the TP53 pathway and leads to expression of senescence associated secretary phenotype (SASP), such as TGFβ1, which induce a senescence-associated pro-fibrotic cardiomyopathy.These findings along with our previous data identify the DDR as a putative common mechanism in the pathogenesis of cardiomyopathies, and likely in the pathogenesis of over two dozen diseases, caused by mutations in the nuclear envelope proteins (envelopathies).

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

Cardiovasc Res: 17 Oct 2020; epub ahead of print

TNAP as a therapeutic target for cardiovascular calcification - a discussion of its pleiotropic functions in the body.

Claudia G, Agnieszka SK, Laurence B, Thibaut Q, ... Caroline F, David M

Cardiovascular calcification (CVC) is associated with increased morbidity and mortality. It develops in several diseases and locations, such as in the tunica intima in atherosclerosis plaques, in the tunica media in type 2 diabetes and chronic kidney disease, and in aortic valves. In spite of the wide occurrence of CVC and its detrimental effects on cardiovascular diseases (CVD), no treatment is yet available. Most of CVC involve mechanisms similar to those occurring during endochondral and/or intramembranous ossification. Logically, since tissue-nonspecific alkaline phosphatase (TNAP) is the key-enzyme responsible for skeletal/dental mineralization, it is a promising target to limit CVC. Tools have recently been developed to inhibit its activity and preclinical studies conducted in animal models of vascular calcification already provided promising results. Nevertheless, as its name indicates, TNAP is ubiquitous and recent data indicate that it dephosphorylates different substrates in vivo to participate in other important physiological functions besides mineralization. For instance, TNAP is involved in the metabolism of pyridoxal phosphate and the production of neurotransmitters. TNAP has also been described as an anti-inflammatory enzyme able to dephosphorylate adenosine nucleotides and lipopolysaccharide. A better understanding of the full spectrum of TNAP\'s functions is needed to better characterize the effects of TNAP inhibition in diseases associated with CVC. In this review, after a brief description of the different types of CVC, we describe the newly uncovered additional functions of TNAP and discuss the expected consequences of its systemic inhibition in vivo.

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

Cardiovasc Res: 17 Oct 2020; epub ahead of print

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

Monaghan RM, Page DJ, Ostergaard P, Keavney BD

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

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

Cardiovasc Res: 16 Oct 2020; epub ahead of print

SPEG: a key regulator of cardiac calcium homeostasis.

Campbell H, Aguilar-Sanchez Y, Quick AP, Dobrev D, Wehrens X

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 five 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: 16 Oct 2020; epub ahead of print

Full-length dhh and N-terminal shh act as competitive antagonists to regulate angiogenesis and vascular permeability.

Hollier PL, Chapouly C, Diop A, Guimbal S, ... Gadeau AP, Renault MA

The therapeutic potential of Hedgehog (Hh) signalling agonists for vascular diseases is of growing interest. However, molecular and cellular mechanisms underlying the role of the Hh signalling in vascular biology remain poorly understood.
Aims
The purpose of the present paper is to clarify some conflicting literature data.
Methods and results
: With this goal we have demonstrated that, unexpectedly, ectopically administered N-terminal Sonic Hedgehog (N-Shh) and endogenous endothelial-derived Desert Hedgehog (Dhh) induce opposite effects in endothelial cells (ECs). Notably, endothelial Dhh acts under its full-length soluble form (FL-Dhh) and activates Smoothened in ECs, while N-Shh inhibits it. At molecular level, N-Shh prevents FL-Dhh binding to Patched-1 demonstrating that N-Shh acts as competitive antagonist to FL-Dhh. Besides, we found that even though FL-Hh ligands and N-Hh ligands all bind Patched-1, they induce distinct Patched-1 localization. Finally, we confirmed that in a pathophysiological setting i.e. brain inflammation, astrocyte-derived N-Shh act as a FL-Dhh antagonist.
Conclusion
The present study highlights for the first time that FL-Dhh and N-Hh ligands have antagonistic properties especially in ECs.
Translational perspective
As a consequence, Hh ligands or forms of Hh ligands cannot be used instead of another for therapeutic purposes.

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

Cardiovasc Res: 15 Oct 2020; epub ahead of print

Tc17 CD8+ T-cells accumulate in murine atherosclerotic lesions, but do not contribute to early atherosclerosis development.

van Duijn J, de Jong MJM, Benne N, Leboux RJT, ... Kuiper J, Slütter B
Aims
CD8+ T-cells can differentiate into subpopulations that are characterized by a specific cytokine profile, such as the Tc17 population that produces IL-17. The role of this CD8+ T-cell subset in atherosclerosis remains elusive. In this study, we therefore investigated the contribution of Tc17 cells to the development of atherosclerosis.
Methods and results
Flow cytometry analysis of atherosclerotic lesions from apoE-/- mice revealed a pronounced increase in RORγt+CD8+ T-cells compared to the spleen, indicating a lesion-specific increase in Tc17 cells. To study whether and how the Tc17 subset affects atherosclerosis, we performed an adoptive transfer of Tc17 cells or undifferentiated Tc0 cells into CD8-/-LDLr-/- mice fed a Western-type diet. Using flow cytometry, we showed that Tc17 cells retained a high level of IL-17A production in vivo. Moreover, Tc17 cells produced lower levels of IFN-γ than their Tc0 counterparts. Analysis of the aortic root revealed that the transfer of Tc17 cells did not increase atherosclerotic lesion size, in contrast to Tc0-treated mice.
Conclusion
These findings demonstrate a lesion-localized increase in Tc17 cells in an atherosclerotic mouse model. Tc17 cells appeared to be non-atherogenic, in contrast to their Tc0 counterpart.
Translational perspective
CD8+ T-cells are present in high numbers in human atherosclerotic plaques, however their role in inflammation and the pathogenesis of atherosclerosis remains elusive. Our results indicate that the majority of CD8+ T-cells in atherosclerotic plaques of mice have lost their ability to produce the pro-inflammatory cytokine IFN-γ and gain traits of IL-17-producing CD8+ T-cells (Tc17 cells). We show that this subset of CD8+ T-cells is less atherogenic then IFN-γ producing Tc1 cells.

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

Cardiovasc Res: 15 Oct 2020; epub ahead of print

Higher mortality of COVID-19 in males: Sex differences in immune response and cardiovascular comorbidities.

Bienvenu LA, Noonan J, Wang X, Peter K

The high mortality rate of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is a critical concern of the Coronavirus Disease 2019 (COVID-19) pandemic. Strikingly, men account for the majority of COVID-19 deaths, with current figures ranging from 59-75% of total mortality. However, despite clear implications in relation to COVID-19 mortality, most research has not considered sex as a critical factor in data analysis. Here, we highlight fundamental biological differences that exist between males and females, and how these may make significant contributions to the male-biased COVID-19 mortality. We present preclinical evidence identifying the influence of biological sex on the expression and regulation of angiotensin-converting enzyme 2 (ACE2), which is the main receptor used by SARS-CoV-2 to enter cells. However, we note that there is a lack of reports showing that sexual dimorphism of ACE2 expression exists and is of functional relevance in humans. In contrast, there is strong evidence, especially in the context of viral infections, that sexual dimorphism plays a central role in the genetic and hormonal regulation of immune responses, both of the innate as well as the adaptive immune system. We review evidence supporting that ineffective anti-SARS-CoV-2 responses, coupled with a predisposition for inappropriate hyperinflammatory responses, could provide a biological explanation for the male bias in COVID-19 mortality. A prominent finding in COVID-19 is the increased risk of death with pre-existing cardiovascular comorbidities such as hypertension, obesity and age. We contextualise how important features of sexual dimorphism and inflammation in COVID-19 may exhibit a reciprocal relationship with comorbidities, and explain their increased mortality risk. Ultimately, we demonstrate that biological sex is a fundamental variable of critical relevance to our mechanistic understanding of SARS-CoV-2 infection and the pursuit of effective COVID-19 preventative and therapeutic strategies.

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

Cardiovasc Res: 15 Oct 2020; epub ahead of print

Double Trouble: Combined Cardiovascular Effects of Particulate Matter Exposure and COVID-19.

Tanwar V, Adelstein JM, Wold LE

The coronavirus disease-2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly grown into a pandemic. According to initial reports, the lungs were thought to be the primary target, but recent case studies have shown its reach can extend to other organs including the heart and blood vessels. The severity of cardiac complications of COVID-19 depends on multiple underlying factors, with air pollutant exposure being one of them as reported by several recent studies. Airborne particulate matter attracts heightened attention due to its implication in various diseases, especially respiratory and cardiovascular diseases. Inhaled particulate matter not only carries microorganisms inside the body but also elicits local and systemic inflammatory responses resulting in altering host\'s immunity and increasing susceptibility to infection. Previous as well as recent studies have documented that particulate matter acts as a \"carrier\" for the virus and aids in spreading viral infections. This review presents the mechanisms and effects of viral entry and how pollution can potentially modulate pathophysiological processes in the heart. We aimed to concisely summarize studies examining cardiovascular (CV) outcomes in COVID-19 patients and postulate on how particulate matter can influence these outcomes. We have also reviewed evidence on the use of rennin-angiotensin system (RAS) inhibitors, namely, ACE inhibitors and angiotensin receptor blockers, in patients with COVID-19. The interplay of pollution and SARS-CoV-2 is essential to understanding the effects of accentuated cardiovascular effects of COVID-19 and deserves in depth experimental investigations.

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

Cardiovasc Res: 20 Oct 2020; epub ahead of print