Single-Cell RNA Sequencing Reveals Cardiac Fibroblast-Specific Transcriptomic Changes in Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is the most common cause of heart failure, with a complex aetiology involving multiple cell types. We aimed to detect cell-specific transcriptomic alterations in DCM through analysis that leveraged recent advancements in single-cell analytical tools. Single-cell RNA sequencing (scRNA-seq) data from human DCM cardiac tissue were subjected to an updated bioinformatic workflow in which unsupervised clustering was paired with reference label transfer to more comprehensively annotate the dataset. Differential gene expression was detected primarily in the cardiac fibroblast population. Bulk RNA sequencing was performed on an independent cohort of human cardiac tissue and compared with scRNA-seq gene alterations to generate a stratified list of higher-confidence, fibroblast-specific expression candidates for further validation. Concordant gene dysregulation was confirmed in TGFß-induced fibroblasts. Functional assessment of gene candidates showed that AEBP1 may play a significant role in fibroblast activation. This unbiased approach enabled improved resolution of cardiac cell-type-specific transcriptomic alterations in DCM.

Circulating biomarkers for management of cancer therapeutics-related cardiac dysfunction.

Cancer therapeutics-related cardiac dysfunction (CTRCD) has emerged as a major cause of morbidity and mortality in cancer survivors. Effective clinical management of CTRCD is impeded by a lack of sensitive diagnostic and prognostic strategies. Circulating molecular markers could potentially address this need as they are often indicative of cardiac stress before cardiac damage can be detected clinically. A growing understanding of the underlying physiological mechanisms for CTRCD has inspired research efforts to identify novel pathophysiologically relevant biomarkers that may also guide development of cardio-protective therapeutic approaches. The purpose of this review is to evaluate current circulating biomarkers of cardiac stress and their potential role in diagnosis and management of CTRCD. We also discuss some emerging avenues for CTRCD-focused biomarker investigations.

Biomarker profiling for risk of future heart failure (HFpEF) development.

BACKGROUND: The purpose of this study was to investigate the utility of BNP, hsTroponin-I, interleukin-6, sST2, and galectin-3 in predicting the future development of new onset heart failure with preserved ejection fraction (HFpEF) in asymptomatic patients at-risk for HF. METHODS: This is a retrospective analysis of the longitudinal STOP-HF study of thirty patients who developed HFpEF matched to a cohort that did not develop HFpEF (n = 60) over a similar time period. Biomarker candidates were quantified at two time points prior to initial HFpEF diagnosis. RESULTS: HsTroponin-I and BNP at baseline and follow-up were statistically significant predictors of future new onset HFpEF, as was galectin-3 at follow-up and concentration change over time. Interleukin-6 and sST2 were not predictive of future development of new onset HFpEF in this study. Unadjusted biomarker combinations of hsTroponin-I, BNP, and galectin-3 could significantly predict future HFpEF using both baseline (AUC 0.82 [0.73,0.92]) and follow-up data (AUC 0.86 [0.79,0.94]). A relative-risk matrix was developed to categorize the relative-risk of new onset of HFpEF based on biomarker threshold levels. CONCLUSION: We provided evidence for the utility of BNP, hsTroponin-I, and Galectin-3 in the prediction of future HFpEF in asymptomatic event-free populations with cardiovascular disease risk factors.

Investigation of association of genetic variant rs3918242 of matrix metalloproteinase-9 with hypertension, myocardial infarction and progression of ventricular dysfunction in Irish Caucasian patients with diabetes: a report from the STOP-HF follow-up programme.

BACKGROUND: Hypertension and/or myocardial infarction are common causes of heart failure in Type 2 diabetes. Progression to heart failure is usually preceded by ventricular dysfunction, linked to matrix metalloproteinase (MMP) mediated extracellular matrix changes. We hypothesise that the minor allele of genetic variant rs3918242 in the promoter region of the MMP-9 gene is associated with hypertension and/or myocardial infarction, with resultant progression of dysfunctional cardiac remodelling in patients with diabetes without symptomatic heart failure. METHODS: We genotyped 498 diabetes patients participating in the St Vincent's Screening TO Prevent Heart Failure (STOP-HF) follow-up programme for the rs3918242 single nucleotide polymorphism and investigated associations with the co-primary endpoints hypertension and/or myocardial infarction using a dominant model. We also evaluated resulting cardiometabolic phenotype and progression of ventricular dysfunction and cardiac structural abnormalities over a median follow-up period of 3.5 years. RESULTS: The CT/TT genotype comprised 28.1% of the cohort and was associated with a twofold higher risk of myocardial infarction (17.9% vs 8.4%), a reduction in ejection fraction and greater left ventricular systolic dysfunction progression [adjusted OR = 2.56 (1.09, 6.01), p = 0.026] over a median follow-up of 3.5 years [IQR 2.6, 4.9 years]. Conversely, rs3918242 was not associated with hypertension, blood pressure, pulse pressure or left ventricular mass index at baseline or over follow up. CONCLUSIONS: Diabetes patients with the minor T allele of rs3918242 in the STOP-HF follow up programme have greater risk of myocardial infarction, lower ejection fraction and greater progression of left ventricular systolic abnormalities, a precursor to heart failure. These data may support further work on MMP-9 as a biomarker of ventricular dysfunction and the investigation of MMP-9 inhibitors for heart failure prevention in diabetes, particularly in the post-infarction setting. ClinicalTrials.gov Identifier: NCT00921960.

Epigenetic Regulation of Endothelial Cell Function by Nucleic Acid Methylation in Cardiac Homeostasis and Disease.

Pathological remodelling of the myocardium, including inflammation, fibrosis and hypertrophy, in response to acute or chronic injury is central in the development and progression of heart failure (HF). While both resident and infiltrating cardiac cells are implicated in these pathophysiological processes, recent evidence has suggested that endothelial cells (ECs) may be the principal cell type responsible for orchestrating pathological changes in the failing heart. Epigenetic modification of nucleic acids, including DNA, and more recently RNA, by methylation is essential for physiological development due to their critical regulation of cellular gene expression. As accumulating evidence has highlighted altered patterns of DNA and RNA methylation in HF at both the global and individual gene levels, much effort has been directed towards defining the precise role of such cell-specific epigenetic changes in the context of HF. Considering the increasingly apparent crucial role that ECs play in cardiac homeostasis and disease, this article will specifically focus on nucleic acid methylation (both DNA and RNA) in the failing heart, emphasising the key influence of these epigenetic mechanisms in governing EC function. This review summarises current understanding of DNA and RNA methylation alterations in HF, along with their specific role in regulating EC function in response to stress (e.g. hyperglycaemia, hypoxia). Improved appreciation of this important research area will aid in further implicating dysfunctional ECs in HF pathogenesis, whilst informing development of EC-targeted strategies and advancing potential translation of epigenetic-based therapies for specific targeting of pathological cardiac remodelling in HF.

Repurposing From Oncology to Cardiology: Low-Dose 5-Azacytidine Attenuates Pathological Cardiac Remodeling in Response to Pressure Overload Injury.

INTRODUCTION: Recent evidence suggests that transcriptional reprogramming is involved in the pathogenesis of cardiac remodeling (cardiomyocyte hypertrophy and fibrosis) and the development of heart failure. 5-Azacytidine (5aza), an inhibitor of DNA methylation approved for hematological malignancies, has previously demonstrated beneficial effects on cardiac remodeling in hypertension. The aim of our work was to investigate whether pressure overload is associated with alterations in DNA methylation and if intervention with low-dose 5aza can attenuate the associated pathological changes. METHODS AND RESULTS: C57Bl6/J mice underwent surgical constriction of the aortic arch for 8 weeks. Mice began treatment 4 weeks post-surgery with either vehicle or 5aza (5 mg/kg). Cardiac structure and function was examined in vivo using echocardiography followed by post mortem histological assessment of hypertrophy and fibrosis. Global DNA methylation was examined by immunostaining for 5-methylcytosine (5MeC) and assessment of DNA methyltransferase expression. The results highlighted that pressure overload-induced pathological cardiac remodeling is associated with increased DNA methylation (elevated cardiac 5MeC positivity and Dnmt1 expression). Administration of 5aza attenuated pathological remodeling and diastolic dysfunction. These beneficial changes were mirrored by a treatment-related reduction in global 5MeC levels and expression of Dnmt1 and Dnmt3B in the heart. CONCLUSION: DNA methylation plays an important role in the pathogenesis of pressure overload-induced cardiac remodeling. Therapeutic intervention with 5aza, at a dose 5 times lower than clinically given for oncology treatment, attenuated myocardial hypertrophy and fibrosis. Our work supports the rationale for its potential use in cardiac pathologies associated with aberrant cardiac wound healing.

Tetranectin, a potential novel diagnostic biomarker of heart failure, is expressed within the myocardium and associates with cardiac fibrosis.

Heart failure (HF) screening strategies require biomarkers to predict disease manifestation to aid HF surveillance and management programmes. The aim of this study was to validate a previous proteomics discovery programme that identified Tetranectin as a potential HF biomarker candidate based on expression level changes in asymptomatic patients at future risk for HF development. The initial study consisted of 132 patients, comprising of HF (n = 40), no-HF controls (n = 60), and cardiac surgery patients (n = 32). Serum samples were quantified for circulating levels of Tetranectin and a panel of circulating fibro-inflammatory markers. Cardiac tissue served as a resource to investigate the relationship between cardiac Tetranectin levels and fibrosis and inflammation within the myocardium. An independent cohort of 224 patients with or without HF was used to validate serum Tetranectin levels. Results show that circulating Tetranectin levels are significantly reduced in HF patients (p < 0.0001), and are associated with HF more closely than B-type natriuretic peptide (AUC = 0.97 versus 0.84, p = 0.011). Serum Tetranectin negatively correlated with circulating fibrosis markers, whereas cardiac tissue Tetranectin correlated positively with fibrotic genes and protein within the myocardium. In conclusion, we report for the first time that Tetranectin is a promising HF biomarker candidate linked with fibrotic processes within the myocardium.

The effects of genetic deletion of Macrophage migration inhibitory factor on the chronically hypoxic pulmonary circulation.

While it is well established that the haemodynamic cause of hypoxic pulmonary hypertension is increased pulmonary vascular resistance, the molecular pathogenesis of the increased resistance remains incompletely understood. Macrophage migration inhibitory factor is a pleiotropic cytokine with endogenous tautomerase enzymatic activity as well as both intracellular and extracellular signalling functions. In several diseases, macrophage migration inhibitory factor has pro-inflammatory roles that are dependent upon signalling through the cell surface receptors CD74, CXCR2 and CXCR4. Macrophage migration inhibitory factor expression is increased in animal models of hypoxic pulmonary hypertension and macrophage migration inhibitory factor tautomerase inhibitors, which block some of the functions of macrophage migration inhibitory factor, and have been shown to attenuate hypoxic pulmonary hypertension in mice and monocrotaline-induced pulmonary hypertension in rats. However, because of the multiple pathways through which it acts, the integrated actions of macrophage migration inhibitory factor during the development of hypoxic pulmonary hypertension were unclear. We report here that isolated lungs from adult macrophage migration inhibitory factor knockout (MIF-/- ) mice maintained in normoxic conditions showed greater acute hypoxic vasoconstriction than the lungs of wild type mice (MIF+/+ ). Following exposure to hypoxia for three weeks, isolated lungs from MIF-/- mice had significantly higher pulmonary vascular resistance than those from MIF+/+ mice. The major mechanism underlying the greater increase in pulmonary vascular resistance in the hypoxic MIF-/- mice was reduction of the pulmonary vascular bed due to an impairment of the normal hypoxia-induced expansion of the alveolar capillary network. Taken together, these results demonstrate that macrophage migration inhibitory factor plays a central role in the development of the pulmonary vascular responses to chronic alveolar hypoxia.

Targeted DNA Methylation Profiling of Human Cardiac Tissue Reveals Novel Epigenetic Traits and Gene Deregulation Across Different Heart Failure Patient Subtypes.

BACKGROUND: Limited knowledge exists of the extent of epigenetic alterations, such as DNA methylation, in heart failure (HF). We conducted targeted DNA methylation sequencing to identify DNA methylation alterations in coding and noncoding RNA (ncRNA) across different etiological subtypes of HF. METHODS AND RESULTS: A targeted bisulfite sequence capture sequencing platform was applied to DNA extracted from cardiac interventricular septal tissue of 30 male HF patients encompassing causes including hypertrophic obstructive cardiomyopathy, ischemic cardiomyopathy, dilated cardiomyopathy, and 9 control patients with nonfailing hearts. We detected 62 678 differentially methylated regions in the studied HF cohort. By comparing each HF subgroup to the nonfailing control group, we identified 195 unique differentially methylated regions: 5 in hypertrophic obstructive cardiomyopathy, 151 in dilated cardiomyopathy, and 55 in ischemic cardiomyopathy. These translated to 4 genes/1 ncRNA in hypertrophic obstructive cardiomyopathy, 131 genes/17 ncRNA in dilated cardiomyopathy, and 51 genes/5 ncRNA in ischemic cardiomyopathy. Subsequent gene/ncRNA expression analysis was assessed using quantitative reverse transcription polymerase chain reaction and revealed 6 genes: 4 hypermethylated ( HEY2, MSR1, MYOM3, and COX17), 2 hypomethylated ( CTGF and MMP2); and 2 microRNA: 1 hypermethylated (miR-24-1), 1 hypomethylated (miR-155) with significantly upregulated or downregulated expression levels consistent with the direction of methylation in the particular HF subgroup. CONCLUSIONS: For the first time DNA methylation alterations and associated gene expression changes were identified in etiologically variant pathological HF tissue. The methylation-sensitive and disease-associated genes/ncRNA identified from this study represent a unique cohort of loci that demonstrate a plausible potential as a novel diagnostic and therapeutic target in HF and warrant further investigation.

Epigenetics of Aberrant Cardiac Wound Healing.

Remodeling of cardiac tissue architecture is essential for normal organ development and maintaining homeostasis after injury. Injurious insults to the heart, such as hypertension and myocardial infarction, promote cellular responses including stimulation of resident inflammatory cells, activation of endothelial cells and recruitment of immune cells, hypertrophy of cardiomyocytes, and activation of fibroblasts. The physiological goal of this coordinated cellular response is to repair damaged tissue while maintaining or restoring cardiac contractile function. Persistent uncontrolled inflammation, hypertrophy, and fibrosis in the heart due to hyperactive wound healing are detrimental and impair cardiac performance, facilitating the progression to heart failure. Abnormal changes in gene expression promote acquisition of aberrant cellular phenotypes that drive cardiac remodeling. DNA methylation and histone modifications are epigenetic mechanisms that critically regulate chromatin structure and gene expression, and are essential for normal physiology and development. Increasing clinical and experimental evidence suggests that these epigenetic mechanisms are involved in driving aberrant wound healing and the development of heart failure. While most of our knowledge to date is on the heart as a whole, the precise contribution of DNA methylation and histone modifications in regulating aberrant cardiac remodeling at the cellular level is less defined. Therefore, this overview aims to summarize the role of DNA methylation and histone modifications (acetylation and methylation) in heart failure and to comprehensively dissect the role these mechanisms play in regulating the function of cardiomyocytes, fibroblasts, and immune cells in response to injury. © 2018 American Physiological Society. Compr Physiol 8:451-491, 2018.