PBX/Knotted 1 homeobox-2 (PKNOX2) is a novel regulator of myocardial fibrosis.

Much effort has been made to uncover the cellular heterogeneities of human hearts by single-nucleus RNA sequencing. However, the cardiac transcriptional regulation networks have not been systematically described because of the limitations in detecting transcription factors. In this study, we optimized a pipeline for isolating nuclei and conducting single-nucleus RNA sequencing targeted to detect a higher number of cell signal genes and an optimal number of transcription factors. With this unbiased protocol, we characterized the cellular composition of healthy human hearts and investigated the transcriptional regulation networks involved in determining the cellular identities and functions of the main cardiac cell subtypes. Particularly in fibroblasts, a novel regulator, PKNOX2, was identified as being associated with physiological fibroblast activation in healthy hearts. To validate the roles of these transcription factors in maintaining homeostasis, we used single-nucleus RNA-sequencing analysis of transplanted failing hearts focusing on fibroblast remodelling. The trajectory analysis suggested that PKNOX2 was abnormally decreased from fibroblast activation to pathological myofibroblast formation. Both gain- and loss-of-function in vitro experiments demonstrated the inhibitory role of PKNOX2 in pathological fibrosis remodelling. Moreover, fibroblast-specific overexpression and knockout of PKNOX2 in a heart failure mouse model induced by transverse aortic constriction surgery significantly improved and aggravated myocardial fibrosis, respectively. In summary, this study established a high-quality pipeline for single-nucleus RNA-sequencing analysis of heart muscle. With this optimized protocol, we described the transcriptional regulation networks of the main cardiac cell subtypes and identified PKNOX2 as a novel regulator in suppressing fibrosis and a potential therapeutic target for future translational studies.

BACH1 controls hepatic insulin signaling and glucose homeostasis in mice.

Hepatic insulin resistance is central to the metabolic syndrome. Here we investigate the role of BTB and CNC homology 1 (BACH1) in hepatic insulin signaling. BACH1 is elevated in the hepatocytes of individuals with obesity and patients with non-alcoholic fatty liver disease (NAFLD). Hepatocyte-specific Bach1 deletion in male mice on a high-fat diet (HFD) ameliorates hyperglycemia and insulin resistance, improves glucose homeostasis, and protects against steatosis, whereas hepatic overexpression of Bach1 in male mice leads to the opposite phenotype. BACH1 directly interacts with the protein-tyrosine phosphatase 1B (PTP1B) and the insulin receptor ß (IR-ß), and loss of BACH1 reduces the interaction between PTP1B and IR-ß upon insulin stimulation and enhances insulin signaling in hepatocytes. Inhibition of PTP1B significantly attenuates BACH1-mediated suppression of insulin signaling in HFD-fed male mice. Hepatic BACH1 knockdown ameliorates hyperglycemia and improves insulin sensitivity in diabetic male mice. These results demonstrate a critical function for hepatic BACH1 in the regulation of insulin signaling and glucose homeostasis.

BACH1 regulates the differentiation of vascular smooth muscle cells from human embryonic stem cells via CARM1-mediated methylation of H3R17.

The role of BACH1 in the process of vascular smooth muscle cell (VSMC) differentiation from human embryonic stem cells (hESCs) remains unknown. Here, we find that the loss of BACH1 in hESCs attenuates the expression of VSMC marker genes, whereas overexpression of BACH1 after mesoderm induction increases the expression of VSMC markers during in vitro hESC-VSMC differentiation. Mechanistically, BACH1 binds directly to coactivator-associated arginine methyltransferase 1 (CARM1) during in vitro hESC-VSMC differentiation, and this interaction is mediated by the BACH1 bZIP domain. BACH1 recruits CARM1 to VSMC marker gene promoters and promotes VSMC marker expression by increasing H3R17me2 modification, thus facilitating in vitro VSMC differentiation from hESCs after the mesoderm induction. The increased expression of VSMC marker genes by BACH1 overexpression is partially abolished by inhibition of CARM1 or the H3R17me2 inhibitor TBBD in hESC-derived cells. These findings highlight the critical role of BACH1 in hESC differentiation into VSMCs by CARM1-mediated methylation of H3R17.

Cardiac-specific BACH1 ablation attenuates pathological cardiac hypertrophy by inhibiting the Ang II type 1 receptor expression and the Ca2+/CaMKII pathway.

AIMS: BACH1 is up-regulated in hypertrophic hearts, but its function in cardiac hypertrophy remains largely unknown. This research investigates the function and mechanisms of BACH1 in the regulation of cardiac hypertrophy. METHODS AND RESULTS: Male cardiac-specific BACH1 knockout mice or cardiac-specific BACH1 transgenic (BACH1-Tg) mice and their respective wild-type littermates developed cardiac hypertrophy induced by angiotensin II (Ang II) or transverse aortic constriction (TAC). Cardiac-specific BACH1 knockout in mice protected the hearts against Ang II- and TAC-induced cardiac hypertrophy and fibrosis, and preserved cardiac function. Conversely, cardiac-specific BACH1 overexpression markedly exaggerated cardiac hypertrophy and fibrosis and reduced cardiac function in mice with Ang II- and TAC-induced hypertrophy. Mechanistically, BACH1 silencing attenuated Ang II- and norepinephrine-stimulated calcium/calmodulin-dependent protein kinase II (CaMKII) signalling, the expression of hypertrophic genes, and hypertrophic growth of cardiomyocytes. Ang II stimulation promoted the nuclear localization of BACH1, facilitated the recruitment of BACH1 to the Ang II type 1 receptor (AT1R) gene promoter, and then increased the expression of AT1R. Inhibition of BACH1 attenuated Ang II-stimulated AT1R expression, cytosolic Ca2+ levels, and CaMKII activation in cardiomyocytes, whereas overexpression of BACH1 led to the opposite effects. The increased expression of hypertrophic genes induced by BACH1 overexpression upon Ang II stimulation was suppressed by CaMKII inhibitor KN93. The AT1R antagonist, losartan, significantly attenuated BACH1-mediated CaMKII activation and cardiomyocyte hypertrophy under Ang II stimulation in vitro. Similarly, Ang II-induced myocardial pathological hypertrophy, cardiac fibrosis, and dysfunction in BACH1-Tg mice were blunted by treatment with losartan. CONCLUSION: This study elucidates a novel important role of BACH1 in pathological cardiac hypertrophy by regulating the AT1R expression and the Ca2+/CaMKII pathway, and highlights potential therapeutic target in pathological cardiac hypertrophy.

Coronary Collateral Circulation: A New Predictor of Mortality in Heart Transplant Recipients With Allograft Vasculopathy.

Coronary collateral arteries (CCAs) are anastomotic channels between vessels; although beneficial in atherosclerosis, their role in heart transplantation (HT) recipients is underinvestigated. CCAs initially develop as microcirculation and cardiac allograft vasculopathy (CAV), promoting immune-dependent proliferative angiogenic response, and play a role in their development. In our hypothesis, ischemia induced by coronary microvascular dysfunction (CMD) triggers the development of CCAs, which are, in turn, less functional as affected by CAV themselves. Methods: One hundred twenty-one patients receiving HT at our institution were retrospectively evaluated and were included if transthoracic echocardiography with coronary flow velocity reserve (CFVR) assessment and coronary angiography were performed. CMD was defined as CFVR of ≤2.5. Patients with CAV were enrolled, and their angiograms were reviewed to evaluate the presence of CCAs. Cardiovascular mortality was assessed as the main clinical outcome. Results: Forty patients were found to have CCAs. Patients with CCAs have lower CFVR than those without CCAs (2.22 ± 0.72 versus 2.69 ± 0.92;P = 0.003), reflecting in different rates of CMD in the 2 groups (72.5% versus 37%; P < 0.001). CMD is associated with higher CAV grades (P < 0.001), which are also associated with CCAs (P < 0.001). Patients with poorly developed CCAs have lower CFVR (P < 0.001). At multivariable analysis, CMD (P = 0.008) and higher CAV grades (P = 0.005) are independent predictors of CCAs. During the median follow-up time of 10.2 (6.6-13.3) y, patients with CCAs have been found to have higher mortality than those without CCAs (57.5% versus 32.1%; P = 0.007). CCAs are associated with a lower probability of survival also in patients with CMD (P < 0.001) and are independent predictors of mortality (P < 0.001). Conclusions: Our results demonstrate an interplay between CAV, CMD, and CCAs. We confirm that CAV is associated with CMD, and we show, for the first time, that CMD is associated with CCAs. CCAs are pathophysiologically associated with more severe graft vasculopathy and independently predict mortality after HT.

BACH1 deficiency prevents neointima formation and maintains the differentiated phenotype of vascular smooth muscle cells by regulating chromatin accessibility.

The transcription factor BTB and CNC homology 1(BACH1) has been linked to coronary artery disease risk by human genome-wide association studies, but little is known about the role of BACH1 in vascular smooth muscle cell (VSMC) phenotype switching and neointima formation following vascular injury. Therefore, this study aims to explore the role of BACH1 in vascular remodeling and its underlying mechanisms. BACH1 was highly expressed in human atherosclerotic plaques and has high transcriptional factor activity in VSMCs of human atherosclerotic arteries. VSMC-specific loss of Bach1 in mice inhibited the transformation of VSMC from contractile to synthetic phenotype and VSMC proliferation and attenuated the neointimal hyperplasia induced by wire injury. Mechanistically, BACH1 suppressed chromatin accessibility at the promoters of VSMC marker genes via recruiting histone methyltransferase G9a and cofactor YAP and maintaining the H3K9me2 state, thereby repressing VSMC marker genes expression in human aortic smooth muscle cells (HASMCs). BACH1-induced repression of VSMC marker genes was abolished by the silencing of G9a or YAP. Thus, these findings demonstrate a crucial regulatory role of BACH1 in VSMC phenotypic transition and vascular homeostasis and shed light on potential future protective vascular disease intervention via manipulation of BACH1.

The AP-1 transcription factor Fosl-2 drives cardiac fibrosis and arrhythmias under immunofibrotic conditions.

Fibrotic changes in the myocardium and cardiac arrhythmias represent fatal complications in systemic sclerosis (SSc), however the underlying mechanisms remain elusive. Mice overexpressing transcription factor Fosl-2 (Fosl-2tg) represent animal model of SSc. Fosl-2tg mice showed interstitial cardiac fibrosis, disorganized connexin-43/40 in intercalated discs and deregulated expression of genes controlling conduction system, and developed higher heart rate (HR), prolonged QT intervals, arrhythmias with prevalence of premature ventricular contractions, ventricular tachycardias, II-degree atrio-ventricular blocks and reduced HR variability. Following stimulation with isoproterenol Fosl-2tg mice showed impaired HR response. In contrast to Fosl-2tg, immunodeficient Rag2-/-Fosl-2tg mice were protected from enhanced myocardial fibrosis and ECG abnormalities. Transcriptomics analysis demonstrated that Fosl-2-overexpression was responsible for profibrotic signature of cardiac fibroblasts, whereas inflammatory component in Fosl-2tg mice activated their fibrotic and arrhythmogenic phenotype. In human cardiac fibroblasts FOSL-2-overexpression enhanced myofibroblast signature under proinflammatory or profibrotic stimuli. These results demonstrate that under immunofibrotic conditions transcription factor Fosl-2 exaggerates myocardial fibrosis, arrhythmias and aberrant response to stress.

Neutral effect of SGLT2 inhibitors on lipoprotein metabolism: From clinical evidence to molecular mechanisms.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are effective, well-tolerated, and safe glucose-lowering compounds for patients with type 2 diabetes mellitus (T2DM). SGLT2i benefit encompasses protection from heart and kidney failure, independently of the presence of diabetes. In addition, SGLT2i consistently reduce the risk of hospitalization for heart failure and, although with some heterogeneity between specific members of the class, favourably affect the risk of cardiovascular outcomes. The molecular mechanisms underlying the cardiovascular favourable effect are not fully clarified. Studies testing the efficacy of SGLT2i in human cohorts and experimental models of atherosclerotic cardiovascular disease (ASCVD) have reported significant differences in circulating levels and composition of lipoprotein classes. In randomized clinical trials, small but significant increases in low-density lipoprotein cholesterol (LDL-C) levels have been observed, with a still undefined clinical significance; on the other hand, favourable (although modest) effects on high-density lipoprotein cholesterol (HDL-C) and triglycerides have been reported. At the molecular level, glycosuria may promote a starving-like state that ultimately leads to a metabolic improvement through the mobilization of fatty acids from the adipose tissue and their oxidation for the production of ketone bodies. This, however, may also fuel hepatic cholesterol synthesis, thus inhibiting atherogenic lipoprotein uptake from the liver. Long-term studies collecting detailed information on lipid-lowering therapies at baseline and during the trials with SGLT2i, as well as regularly monitoring lipid profiles are warranted to disentangle the potential implications of SGLT2i in modulating lipoprotein-mediated atherosclerotic cardiovascular risk.

Translational opportunities of single-cell biology in atherosclerosis.

The advent of single-cell biology opens a new chapter for understanding human biological processes and for diagnosing, monitoring, and treating disease. This revolution now reaches the field of cardiovascular disease (CVD). New technologies to interrogate CVD samples at single-cell resolution are allowing the identification of novel cell communities that are important in shaping disease development and direct towards new therapeutic strategies. These approaches have begun to revolutionize atherosclerosis pathology and redraw our understanding of disease development. This review discusses the state-of-the-art of single-cell analysis of atherosclerotic plaques, with a particular focus on human lesions, and presents the current resolution of cellular subpopulations and their heterogeneity and plasticity in relation to clinically relevant features. Opportunities and pitfalls of current technologies as well as the clinical impact of single-cell technologies in CVD patient care are highlighted, advocating for multidisciplinary and international collaborative efforts to join the cellular dots of CVD.

How to optimize the adherence to a guideline-directed medical therapy in the secondary prevention of cardiovascular diseases: a clinical consensus statement from the European Association of Preventive Cardiology.

A key factor to successful secondary prevention of cardiovascular disease (CVD) is optimal patient adherence to treatment. However, unsatisfactory rates of adherence to treatment for CVD risk factors and CVD have been observed consistently over the last few decades. Hence, achieving optimal adherence to lifestyle measures and guideline-directed medical therapy in secondary prevention and rehabilitation is a great challenge to many healthcare professionals. Therefore, in this European Association of Preventive Cardiology clinical consensus document, a modern reappraisal of the adherence to optimal treatment is provided, together with simple, practical, and feasible suggestions to achieve this goal in the clinical setting, focusing on evidence-based concepts.

Crosstalk between high-density lipoproteins and endothelial cells in health and disease: Insights into sex-dependent modulation.

Atherosclerotic cardiovascular disease is the leading cause of death worldwide. Intense research in vascular biology has advanced our knowledge of molecular mechanisms of its onset and progression until complications; however, several aspects of the patho-physiology of atherosclerosis remain to be further elucidated. Endothelial cell homeostasis is fundamental to prevent atherosclerosis as the appearance of endothelial cell dysfunction is considered the first pro-atherosclerotic vascular modification. Physiologically, high density lipoproteins (HDLs) exert protective actions for vessels and in particular for ECs. Indeed, HDLs promote endothelial-dependent vasorelaxation, contribute to the regulation of vascular lipid metabolism, and have immune-modulatory, anti-inflammatory and anti-oxidative properties. Sex- and gender-dependent differences are increasingly recognized as important, although not fully elucidated, factors in cardiovascular health and disease patho-physiology. In this review, we highlight the importance of sex hormones and sex-specific gene expression in the regulation of HDL and EC cross-talk and their contribution to cardiovascular disease.

Epigenetics in the primary and secondary prevention of cardiovascular disease: influence of exercise and nutrition.

Increasing evidence links changes in epigenetic systems, such as DNA methylation, histone modification, and non-coding RNA expression, to the occurrence of cardiovascular disease (CVD). These epigenetic modifications can change genetic function under influence of exogenous stimuli and can be transferred to next generations, providing a potential mechanism for inheritance of behavioural intervention effects. The benefits of exercise and nutritional interventions in the primary and secondary prevention of CVD are well established, but the mechanisms are not completely understood. In this review, we describe the acute and chronic epigenetic effects of physical activity and dietary changes. We propose exercise and nutrition as potential triggers of epigenetic signals, promoting the reshaping of transcriptional programmes with effects on CVD phenotypes. Finally, we highlight recent developments in epigenetic therapeutics with implications for primary and secondary CVD prevention.

Coronary Flow Evaluation in Heart Transplant Patients Compared to Healthy Controls Documents the Superiority of Coronary Flow Velocity Reserve Companion as Diagnostic and Prognostic Tool.

Background: Distinct contributions by functional or structural alterations of coronary microcirculation in heart transplantation (HT) and their prognostic role have not been fully elucidated. We aimed to identify the mechanisms of coronary microvascular dysfunction (CMD) in HT and their prognostic implications. Methods: 134 patients, surviving at least 5 years after HT, without evidence of angiographic vasculopathy or symptoms/signs of rejection were included. 50 healthy volunteers served as controls. All underwent the assessment of rest and hyperemic coronary diastolic peak flow velocity (DPVr and DPVh) and coronary flow velocity reserve (CFVR) and its inherent companion that is based on the adjusted quadratic mean: CCFVR = √{(DPVr)2 + (DPVh)2}. Additionally, basal and hyperemic coronary microvascular resistance (BMR and HMR) were estimated. Results: Based on CFVR and DPVh, HT patients can be assigned to four endotypes: endotype 1, discordant with preserved CFVR (3.1 ± 0.4); endotype 2, concordant with preserved CFVR (3.4 ± 0.5); endotype 3, concordant with impaired CFVR (1.8 ± 0.3) and endotype 4, discordant with impaired CFVR (2.0 ± 0.2). Intriguingly, endotype 1 showed lower DPVr (p < 0.0001) and lower DPVh (p < 0.0001) than controls with lower CFVR (p < 0.0001) and lower CCFVR (p < 0.0001) than controls. Moreover, both BMR and HMR were higher in endotype 1 than in controls (p = 0.001 and p < 0.0001, respectively), suggesting structural microvascular remodeling. Conversely, endotype 2 was comparable to controls. A 13/32 (41%) patients in endotype 1 died in a follow up of 28 years and mortality rate was comparable to endotype 3 (14/31, 45%). However, CCFVR was < 80 cm/s in all 13 deaths of endotype 1 (characterized by preserved CFVR). At multivariable analysis, CMD, DPVh < 75 cm/s and CCFVR < 80 cm/s were independent predictors of mortality. The inclusion of CCFVR < 80 cm/s to models with clinical indicators of mortality better predicted survival, compared to only adding CMD or DPVh < 75 cm/s (p < 0.0001 and p = 0.03, respectively). Conclusion: A normal CFVR could hide detection of microvasculopathy with high flow resistance and low flow velocities at rest. This microvasculopathy seems to be secondary to factors unrelated to HT (less rejections and more often diabetes). The combined use of CFVR and CCFVR provides more complete clinical and prognostic information on coronary microvasculopathy in HT.

From novel discovery tools and biomarkers to precision medicine-basic cardiovascular science highlights of 2021/22.

Here, we review the highlights of cardiovascular basic science published in 2021 and early 2022 on behalf of the European Society of Cardiology Council for Basic Cardiovascular Science. We begin with non-coding RNAs which have emerged as central regulators cardiovascular biology, and then discuss how technological developments in single-cell 'omics are providing new insights into cardiovascular development, inflammation, and disease. We also review recent discoveries on the biology of extracellular vesicles in driving either protective or pathogenic responses. The Nobel Prize in Physiology or Medicine 2021 recognized the importance of the molecular basis of mechanosensing and here we review breakthroughs in cardiovascular sensing of mechanical force. We also summarize discoveries in the field of atherosclerosis including the role of clonal haematopoiesis of indeterminate potential, and new mechanisms of crosstalk between hyperglycaemia, lipid mediators, and inflammation. The past 12 months also witnessed major advances in the field of cardiac arrhythmia including new mechanisms of fibrillation. We also focus on inducible pluripotent stem cell technology which has demonstrated disease causality for several genetic polymorphisms in long-QT syndrome and aortic valve disease, paving the way for personalized medicine approaches. Finally, the cardiovascular community has continued to better understand COVID-19 with significant advancement in our knowledge of cardiovascular tropism, molecular markers, the mechanism of vaccine-induced thrombotic complications and new anti-viral therapies that protect the cardiovascular system.

Effects of acute administration of trimethylamine N-oxide on endothelial function: a translational study.

Elevated circulating levels of nutrient-derived trimethylamine N-oxide (TMAO) have been associated with the onset and progression of cardiovascular disease by promoting athero-thrombosis. However, in conditions like bariatric surgery (Roux-en-Y gastric bypass, RYGB), stable increases of plasma TMAO are associated with improved endothelial function and reduced cardiovascular morbidity and mortality, thus questioning whether a mechanistic relationship between TMAO and endothelial dysfunction exists. Herein, we translationally assessed the effects of acute TMAO exposure on endothelial dysfunction, thrombosis and stroke. After RYGB, fasting circulating levels of TMAO increased in patients and obese rats, in parallel with an improved gluco-lipid profile and higher circulating bile acids. The latter enhanced FXR-dependent signalling in rat livers, which may lead to higher TMAO synthesis post RYGB. In lean rats, acute TMAO injection (7 mg kg-1) 1.5-h before sacrifice and ex-vivo 30-min incubation of thoracic aortas with 10-6 M TMAO did not impair vasodilation in response to acetylcholine (Ach), glucagon-like peptide 1, or insulin. Similarly, in lean WT mice (n = 5-6), TMAO injection prior to subjecting mice to ischemic stroke or arterial thrombosis did not increase its severity compared to vehicle treated mice. Endothelial nitric oxide synthase (eNOS) activity and intracellular stress-activated pathways remained unaltered in aorta of TMAO-injected rats, as assessed by Western Blot. Pre-incubation of human aortic endothelial cells with TMAO (10-6 M) did not alter NO release in response to Ach. Our results indicate that increased plasmatic TMAO in the near-physiological range seems to be a neutral bystander to vascular function as translationally seen in patients after bariatric surgery or in healthy lean rodent models and in endothelial cells exposed acutely to TMAO.