SIRT6 Mitigates Heart Failure With Preserved Ejection Fraction in Diabetes.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a growing health problem without effective therapies. Epidemiological studies indicate that diabetes is a strong risk factor for HFpEF, and about 45% of patients with HFpEF are suffering from diabetes, yet the underlying mechanisms remain elusive. METHODS: Using a combination of echocardiography, hemodynamics, RNA-sequencing, molecular biology, in vitro and in vivo approaches, we investigated the roles of SIRT6 (sirtuin 6) in regulation of endothelial fatty acid (FA) transport and HFpEF in diabetes. RESULTS: We first observed that endothelial SIRT6 expression was markedly diminished in cardiac tissues from heart failure patients with diabetes. We then established an experimental mouse model of HFpEF in diabetes induced by a combination of the long-term high-fat diet feeding and a low-dose streptozocin challenge. We also generated a unique humanized SIRT6 transgenic mouse model, in which a single copy of human SIRT6 transgene was engineered at mouse Rosa26 locus and conditionally induced with the Cre-loxP technology. We found that genetically restoring endothelial SIRT6 expression in the diabetic mice ameliorated diastolic dysfunction concurrently with decreased cardiac lipid accumulation. SIRT6 gain- or loss-of-function studies showed that SIRT6 downregulated endothelial FA uptake. Mechanistically, SIRT6 suppressed endothelial expression of PPARγ through SIRT6-dependent deacetylation of histone H3 lysine 9 around PPARγ promoter region; and PPARγ reduction mediated SIRT6-dependent inhibition of endothelial FA uptake. Importantly, oral administration of small molecule SIRT6 activator MDL-800 to diabetic mice mitigated cardiac lipid accumulation and diastolic dysfunction. CONCLUSIONS: The impairment of endothelial SIRT6 expression links diabetes to HFpEF through the alteration of FA transport across the endothelial barrier. Genetic and pharmacological strategies that restored endothelial SIRT6 function in mice with diabetes alleviated experimental HFpEF by limiting FA uptake and improving cardiac metabolism, thus warranting further clinical evaluation.

GSK-3ß Localizes to the Cardiac Z-Disc to Maintain Length Dependent Activation.

BACKGROUND: Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3ß (glycogen synthase kinase 3ß) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3ß's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance. METHODS: Inducible cardiomyocyte-specific GSK-3ß knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied. RESULTS: Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3ß. Agreeing with the localization of its targets, GSK-3ß that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3ß in neonatal rat ventricular cardiomyocytes. One of GSK-3ß's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3ß. Last, GSK-3ß myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA. CONCLUSIONS: We identified a novel mechanism by which GSK-3ß localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3ß levels were reduced in patients with heart failure, indicating z-disc localized GSK-3ß is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

BAG3 expression and sarcomere localization in the human heart are linked to HSF-1 and are differentially affected by sex and disease.

Mutations to the sarcomere-localized cochaperone protein Bcl2-associated athanogene 3 (BAG3) are associated with dilated cardiomyopathy (DCM) and display greater penetrance in male patients. Decreased protein expression of BAG3 is also associated with nongenetic heart failure; however, the factors regulating cardiac BAG3 expression are unknown. Using left ventricular (LV) tissue from nonfailing and DCM human samples, we found that whole LV BAG3 expression was not significantly impacted by DCM or sex; however, myofilament localized BAG3 was significantly decreased in males with DCM. Females with DCM displayed no changes in BAG3 compared with nonfailing. This sex difference appears to be estrogen independent, as estrogen treatment in ovariectomized female rats had no impact on BAG3 expression. BAG3 gene expression in noncardiac cells is primarily regulated by the heat shock transcription factor-1 (HSF-1). We show whole LV HSF-1 expression and nuclear localized/active HSF-1 each displayed a striking positive correlation with whole LV BAG3 expression. We further found that HSF-1 localizes to the sarcomere Z-disc in cardiomyocytes and that this myofilament-associated HSF-1 pool decreases in heart failure. The decrease of HSF-1 was more pronounced in male patients and tightly correlated with myofilament BAG3 expression. Together our findings indicate that cardiac BAG3 expression and myofilament localization are differentially impacted by sex and disease and are linked to HSF-1.NEW & NOTEWORTHY Myofilament BAG3 expression decreases in male patients with nonischemic DCM but is preserved in female patients with DCM. BAG3 expression in the human heart is tightly linked to HSF-1 expression and nuclear translocation. HSF-1 localizes to the sarcomere Z-disc in the human heart. HSF-1 expression in the myofilament fraction decreases in male patients with DCM and positively correlates with myofilament BAG3.

Small proline-rich protein 2B drives stress-dependent p53 degradation and fibroblast proliferation in heart failure.

Heart disease is associated with the accumulation of resident cardiac fibroblasts (CFs) that secrete extracellular matrix (ECM), leading to the development of pathological fibrosis and heart failure. However, the mechanisms underlying resident CF proliferation remain poorly defined. Here, we report that small proline-rich protein 2b (Sprr2b) is among the most up-regulated genes in CFs during heart disease. We demonstrate that SPRR2B is a regulatory subunit of the USP7/MDM2-containing ubiquitination complex. SPRR2B stimulates the accumulation of MDM2 and the degradation of p53, thus facilitating the proliferation of pathological CFs. Furthermore, SPRR2B phosphorylation by nonreceptor tyrosine kinases in response to TGF-ß1 signaling and free-radical production potentiates SPRR2B activity and cell cycle progression. Knockdown of the Sprr2b gene or inhibition of SPRR2B phosphorylation attenuates USP7/MDM2 binding and p53 degradation, leading to CF cell cycle arrest. Importantly, SPRR2B expression is elevated in cardiac tissue from human heart failure patients and correlates with the proliferative state of patient-derived CFs in a process that is reversed by insulin growth factor-1 signaling. These data establish SPRR2B as a unique component of the USP7/MDM2 ubiquitination complex that drives p53 degradation, CF accumulation, and the development of pathological cardiac fibrosis.

Novel and Annotated Long Noncoding RNAs Associated with Ischemia in the Human Heart.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been implicated in the pathogenesis of cardiovascular diseases. We aimed to identify novel lncRNAs associated with the early response to ischemia in the heart. METHODS AND RESULTS: RNA sequencing data gathered from 81 paired left ventricle samples from patients undergoing cardiopulmonary bypass was collected before and after a period of ischemia. Novel lncRNAs were validated with Oxford Nanopore Technologies long-read sequencing. Gene modules associated with an early ischemic response were identified and the subcellular location of selected lncRNAs was determined with RNAscope. A total of 2446 mRNAs, 270 annotated lncRNAs and one novel lncRNA differed in response to ischemia (adjusted p < 0.001, absolute fold change >1.2). The novel lncRNA belonged to a gene module of highly correlated genes that also included 39 annotated lncRNAs. This module associated with ischemia (Pearson correlation coefficient = -0.69, p = 1 × 10-23) and activation of cell death pathways (p < 6 × 10-9). A further nine novel cardiac lncRNAs were identified, of which, one overlapped five cis-eQTL eSNPs for the gene RWD Domain-Containing Sumoylation Enhancer (RWDD3) and was itself correlated with RWDD3 expression (Pearson correlation coefficient -0.2, p = 0.002). CONCLUSION: We have identified 10 novel lncRNAs, one of which was associated with myocardial ischemia and may have potential as a novel therapeutic target or early marker for myocardial dysfunction.

Yes-Associated Protein (Yap) Is Up-Regulated in Heart Failure and Promotes Cardiac Fibroblast Proliferation.

Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFß and hypoxia to human ventricular cardiac fibroblasts in vitro increased Yap protein levels, down-regulated LATS1 activation, increased cell proliferation and collagen I production, and decreased ribosomal protein S6 and S6 kinase phosphorylation, a hallmark of mTOR activation, without any significant effect on mTOR and raptor protein expression or phosphorylation of mTOR or 4E-binding protein 1 (4EBP1), a downstream effector of mTOR pathway. As previously reported in various cell types, TGFß/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFß/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFß/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF.

Global analysis of histone modifications and long-range chromatin interactions revealed the differential cistrome changes and novel transcriptional players in human dilated cardiomyopathy.

BACKGROUND: Acetylation and methylation of histones alter the chromatin structure and accessibility that affect transcriptional regulators binding to enhancers and promoters. The binding of transcriptional regulators enables the interaction between enhancers and promoters, thus affecting gene expression. However, our knowledge of these epigenetic alternations in patients with heart failure remains limited. METHODS AND RESULTS: From the comprehensive analysis of major histone modifications, 3-dimensional chromatin interactions, and transcriptome in left ventricular (LV) tissues from dilated cardiomyopathy (DCM) patients and non-heart failure (NF) donors, differential active enhancer and promoter regions were identified between NF and DCM. Moreover, the genome-wide average promoter signal is significantly lower in DCM than in NF. Super-enhancer (SE) analysis revealed that fewer SEs were found in DCM LVs than in NF ones, and three unique SE-associated genes between NF and DCM were identified. Moreover, SEs are enriched within the genomic region associated with long-range chromatin interactions. The differential enhancer-promoter interactions were observed in the known heart failure gene loci and are correlated with the gene expression levels. Motif analysis identified known cardiac factors and possible novel players for DCM. CONCLUSIONS: We have established the cistrome of four histone modifications and chromatin interactome for enhancers and promoters in NF and DCM tissues. Differential histone modifications and enhancer-promoter interactions were found in DCM, which were associated with gene expression levels of a subset of disease-associated genes in human heart failure.

Mechanosensitive Gene Regulation by Myocardin-Related Transcription Factors Is Required for Cardiomyocyte Integrity in Load-Induced Ventricular Hypertrophy.

BACKGROUND: Hypertrophic cardiomyocyte growth and dysfunction accompany various forms of heart disease. The mechanisms responsible for transcriptional changes that affect cardiac physiology and the transition to heart failure are not well understood. The intercalated disc (ID) is a specialized intercellular junction coupling cardiomyocyte force transmission and propagation of electrical activity. The ID is gaining attention as a mechanosensitive signaling hub and hotspot for causative mutations in cardiomyopathy. METHODS: Transmission electron microscopy, confocal microscopy, and single-molecule localization microscopy were used to examine changes in ID structure and protein localization in the murine and human heart. We conducted detailed cardiac functional assessment and transcriptional profiling of mice lacking myocardin-related transcription factor (MRTF)-A and MRTF-B specifically in adult cardiomyocytes to evaluate the role of mechanosensitive regulation of gene expression in load-induced ventricular remodeling. RESULTS: We found that MRTFs localize to IDs in the healthy human heart and accumulate in the nucleus in heart failure. Although mice lacking MRTFs in adult cardiomyocytes display normal cardiac physiology at baseline, pressure overload leads to rapid heart failure characterized by sarcomere disarray, ID disintegration, chamber dilation and wall thinning, cardiac functional decline, and partially penetrant acute lethality. Transcriptional profiling reveals a program of actin cytoskeleton and cardiomyocyte adhesion genes driven by MRTFs during pressure overload. Indeed, conspicuous remodeling of gap junctions at IDs identified by single-molecule localization microscopy may partially stem from a reduction in Mapre1 expression, which we show is a direct mechanosensitive MRTF target. CONCLUSIONS: Our study describes a novel paradigm in which MRTFs control an acute mechanosensitive signaling circuit that coordinates cross-talk between the actin and microtubule cytoskeleton and maintains ID integrity and cardiomyocyte homeostasis in heart disease.

Discovery of Genetic Variation on Chromosome 5q22 Associated with Mortality in Heart Failure.

Failure of the human heart to maintain sufficient output of blood for the demands of the body, heart failure, is a common condition with high mortality even with modern therapeutic alternatives. To identify molecular determinants of mortality in patients with new-onset heart failure, we performed a meta-analysis of genome-wide association studies and follow-up genotyping in independent populations. We identified and replicated an association for a genetic variant on chromosome 5q22 with 36% increased risk of death in subjects with heart failure (rs9885413, P = 2.7x10-9). We provide evidence from reporter gene assays, computational predictions and epigenomic marks that this polymorphism increases activity of an enhancer region active in multiple human tissues. The polymorphism was further reproducibly associated with a DNA methylation signature in whole blood (P = 4.5x10-40) that also associated with allergic sensitization and expression in blood of the cytokine TSLP (P = 1.1x10-4). Knockdown of the transcription factor predicted to bind the enhancer region (NHLH1) in a human cell line (HEK293) expressing NHLH1 resulted in lower TSLP expression. In addition, we observed evidence of recent positive selection acting on the risk allele in populations of African descent. Our findings provide novel genetic leads to factors that influence mortality in patients with heart failure.

The "How and Why" of Group Biofeedback for Chronic Disease Management.

Biofeedback has been shown to have some level of efficacy for the treatment of a number of chronic medical conditions; however, individualized biofeedback treatment is not always feasible. While group- based interventions are growing in practice due to numerous advantages, the dearth of research examining the efficacy of Group Biofeedback (GBF) suggests that this treatment modality may not be commonly utilized. Thus, the current paper highlights some advantages and constructively addresses potential challenges of utilizing GBF. Obstacles specific to GBF include equipment for participants, need for support staffing, and billing. However, the potential benefits are numerous, and pertain to cost-effectiveness, improved patient access, and additive benefits specific to group-based treatment. We offer a six-session GBF protocol to be used to guide future clinical work in this area. We hope that through the ideas and protocol presented in this paper, biofeedback practitioners will be more inclined to implement GBF.

An enhancer polymorphism at the cardiomyocyte intercalated disc protein NOS1AP locus is a major regulator of the QT interval.

QT interval variation is assumed to arise from variation in repolarization as evidenced from rare Na- and K-channel mutations in Mendelian QT prolongation syndromes. However, in the general population, common noncoding variants at a chromosome 1q locus are the most common genetic regulators of QT interval variation. In this study, we use multiple human genetic, molecular genetic, and cellular assays to identify a functional variant underlying trait association: a noncoding polymorphism (rs7539120) that maps within an enhancer of NOS1AP and affects cardiac function by increasing NOS1AP transcript expression. We further localized NOS1AP to cardiomyocyte intercalated discs (IDs) and demonstrate that overexpression of NOS1AP in cardiomyocytes leads to altered cellular electrophysiology. We advance the hypothesis that NOS1AP affects cardiac electrical conductance and coupling and thereby regulates the QT interval through propagation defects. As further evidence of an important role for propagation variation affecting QT interval in humans, we show that common polymorphisms mapping near a specific set of 170 genes encoding ID proteins are significantly enriched for association with the QT interval, as compared to genome-wide markers. These results suggest that focused studies of proteins within the cardiomyocyte ID are likely to provide insights into QT prolongation and its associated disorders.

Contribution of two-pore K+ channels to cardiac ventricular action potential revealed using human iPSC-derived cardiomyocytes.

Two-pore K+ (K2p) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K2p channels in the heart. Comparing quantitative PCR expression of K2p channels between human heart tissue and iPSC-CMs revealed K2p1.1, K2p2.1, K2p5.1, and K2p17.1 to be higher expressed in cHVT, whereas K2p3.1 and K2p13.1 were higher in iPSC-CMs. Notably, K2p17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K2p2.1, K2p3.1, K2p6.1, and K2p17.1. Here, we report the expression level of 10 human K2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K2p17.1 as significantly reduced in niHF tissues and K2p4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias.NEW & NOTEWORTHY Two-pore K+ (K2p) channels are traditionally regarded as merely background leak channels in myriad physiological systems. Here, we describe the expression profile of K2p channels in human-induced pluripotent stem cell-derived cardiomyocytes and outline a salient role in cardiac repolarization and pathology for multiple K2p channels.

RNA-Seq identifies novel myocardial gene expression signatures of heart failure.

Heart failure is a complex clinical syndrome and has become the most common reason for adult hospitalization in developed countries. Two subtypes of heart failure, ischemic heart disease (ISCH) and dilated cardiomyopathy (DCM), have been studied using microarray platforms. However, microarray has limited resolution. Here we applied RNA sequencing (RNA-Seq) to identify gene signatures for heart failure from six individuals, including three controls, one ISCH and two DCM patients. Using genes identified from this small RNA-Seq dataset, we were able to accurately classify heart failure status in a much larger set of 313 individuals. The identified genes significantly overlapped with genes identified via genome-wide association studies for cardiometabolic traits and the promoters of those genes were enriched for binding sites for transcriptions factors. Our results indicate that it is possible to use RNA-Seq to classify disease status for complex diseases such as heart failure using an extremely small training dataset.

MetaDiff: differential isoform expression analysis using random-effects meta-regression.

BACKGROUND: RNA sequencing (RNA-Seq) allows an unbiased survey of the entire transcriptome in a high-throughput manner. A major application of RNA-Seq is to detect differential isoform expression across experimental conditions, which is of great biological interest due to its direct relevance to protein function and disease pathogenesis. Detection of differential isoform expression is challenging because of uncertainty in isoform expression estimation owing to ambiguous reads and variability in precision of the estimates across samples. It is desirable to have a method that can account for these issues and is flexible enough to allow adjustment for covariates. RESULTS: In this paper, we present MetaDiff, a random-effects meta-regression model that naturally fits for the above purposes. Through extensive simulations and analysis of an RNA-Seq dataset on human heart failure, we show that the random-effects meta-regression approach is computationally fast, reliable, and can improve the power of differential expression analysis while controlling for false positives due to the effect of covariates or confounding variables. In contrast, several existing methods either fail to control false discovery rate or have reduced power in the presence of covariates or confounding variables. The source code, compiled JAR package and documentation of MetaDiff are freely available at https://github.com/jiach/MetaDiff. CONCLUSION: Our results indicate that random-effects meta-regression offers a flexible framework for differential expression analysis of isoforms, particularly when gene expression is influenced by other variables.

Biofeedback Assisted Stress Management in Patients with Lung Cancer: A Feasibility Study.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death for men and women in the United States. NSCLC causes a variety of symptoms which result in significant distress and reduced quality of life for patients. Behavioral and other non-pharmacologic treatment interventions for NSCLC have resulted in improved quality of life, reduced emotional distress, and improved longevity. This study investigates the feasibility and effectiveness of biofeedback assisted stress management (BFSM) to reduce stress in patients with NSCLC. Because of patient dropout, this study was terminated prematurely. Despite this, evaluation of data revealed positive trends, with patients learning to reduce their stress, improve their respiration and heart rate variability, and improve coping. These trends suggest that patients with NSCLC can learn to self-regulate physiology and BFSM may be useful for them, although a less ill patient population may be desirable for future investigations.

Comparative gene expression analysis between coronary arteries and internal mammary arteries identifies a role for the TES gene in endothelial cell functions relevant to coronary artery disease.

Coronary artery disease (CAD) is the leading cause of death worldwide. It has been established that internal mammary arteries (IMA) are resistant to the development of atherosclerosis, whereas left anterior descending (LAD) coronary arteries are athero-prone. The contrasting properties of these two arteries provide an innovative strategy to identify the genes that play important roles in the development of atherosclerosis. We carried out microarray analysis to identify genes differentially expressed between IMA and LAD. Twenty-nine genes showed significant differences in their expression levels between IMA and LAD, which included the TES gene encoding Testin. The role of TES in the cardiovascular system is unknown. Here we show that TES is involved in endothelial cell (EC) functions relevant to atherosclerosis. Western blot analysis showed higher TES expression in IMA than in LAD. Reverse transcription polymerase chain reaction and western blot analyses showed that TES was consistently and markedly down-regulated by more than 6-fold at both mRNA and protein levels in patients with CAD compared with controls without CAD (P= 0.000049). The data suggest that reduced TES expression is associated with the development of CAD. Knockdown of TES expression by small-interfering RNA promoted oxidized-LDL-mediated monocyte adhesion to ECs, EC migration and the transendothelial migration of monocytes, while the over-expression of TES in ECs blunted these processes. These results demonstrate association between reduced TES expression and CAD, establish a novel role for TES in EC functions and raise the possibility that reduced TES expression increases susceptibility to the development of CAD.

Sex differences in myosin heavy chain isoforms of human failing and nonfailing atria.

Mammalian hearts express two myosin heavy chain (MHC) isoforms, which drive contractions with different kinetics and power-generating ability. The expression of the isoform that is associated with more rapid contraction kinetics and greater power output, MHC-α, is downregulated, with a concurrent increase in the relative amount of the slower isoform, MHC-ß, during the progression to experimentally induced or disease-related heart failure. This change in protein expression has been well studied in right and left ventricles in heart failure models and in humans with failure. Relatively little quantitative data exists regarding MHC isoform expression shifts in human failing atria. We previously reported significant increases in the relative amount of MHC-ß in the human failing left atrium. The results of that study suggested that there might be a sex-related difference in the level of MHC-ß in the left atrium, but the number of female subjects was insufficient for statistical analysis. The objective of this study was to test whether there is, in fact, a sex-related difference in the level of MHC-ß in the right and left atria of humans with cardiomyopathy. The results indicate that significant differences exist in atrial MHC isoform expression between men and women who are in failure. The results also revealed an unexpected twofold greater amount of MHC-ß in the nonfailing left atrium of women, compared with men. The observed sex-related differences in MHC isoform expression could impact ventricular diastolic filling during normal daily activities, as well as during physiologically stressful events.

Novel proteins associated with human dilated cardiomyopathy: selective reduction in α(1A)-adrenergic receptors and increased desensitization proteins.

Abstract Therapeutics to treat human heart failure (HF) and the identification of proteins associated with HF are still limited. We analyzed α(1)-adrenergic receptor (AR) subtypes in human HF and performed proteomic analysis on more uniform samples to identify novel proteins associated with human HF. Six failing hearts with end-stage dilated cardiomyopathy (DCM) and four non-failing heart controls were subjected to proteomic analysis. Out of 48 identified proteins, 26 proteins were redundant between samples. Ten of these 26 proteins were previously reported to be associated with HF. Of the newly identified proteins, we found several muscle proteins and mitochondrial/electron transport proteins, while novel were functionally similar to previous reports. However, we also found novel proteins involved in functional classes such as ß-oxidation and G-protein coupled receptor signaling and desensitization not previously associated with HF. We also performed radioligand-binding studies on the heart samples and not only confirmed a large loss of ß(1)-ARs in end-stage DCM, but also found a selective decrease in the α(1A)-AR subtype not previously reported. We have identified new proteins and functional categories associated with end-stage DCM. We also report that similar to the previously characterized loss of ß(1)-AR in HF, there is also a concomitant loss of α(1A)-ARs, which are considered cardioprotective proteins.

Decreased FAM13B Expression Increases Atrial Fibrillation Susceptibility by Regulating Sodium Current and Calcium Handling.

A specific genetic variant associated with atrial fibrillation risk, rs17171731, was identified as a regulatory variant responsible for controlling FAM13B expression. The atrial fibrillation risk allele decreases FAM13B expression, whose knockdown alters the expression of many genes in stem cell-derived cardiomyocytes, including SCN2B, and led to pro-arrhythmogenic changes in the late sodium current and Ca2+ cycling. Fam13b knockout mice had increased P-wave and QT interval duration and were more susceptible to pacing-induced arrhythmias vs control mice. FAM13B expression, its regulation, and downstream effects are potential targets for investigation of patient-specific therapeutics.

Epistasis regulates genetic control of cardiac hypertrophy.

The combinatorial effect of genetic variants is often assumed to be additive. Although genetic variation can clearly interact non-additively, methods to uncover epistatic relationships remain in their infancy. We develop low-signal signed iterative random forests to elucidate the complex genetic architecture of cardiac hypertrophy. We derive deep learning-based estimates of left ventricular mass from the cardiac MRI scans of 29,661 individuals enrolled in the UK Biobank. We report epistatic genetic variation including variants close to CCDC141, IGF1R, TTN, and TNKS. Several loci not prioritized by univariate genome-wide association analysis are identified. Functional genomic and integrative enrichment analyses reveal a complex gene regulatory network in which genes mapped from these loci share biological processes and myogenic regulatory factors. Through a network analysis of transcriptomic data from 313 explanted human hearts, we show that these interactions are preserved at the level of the cardiac transcriptome. We assess causality of epistatic effects via RNA silencing of gene-gene interactions in human induced pluripotent stem cell-derived cardiomyocytes. Finally, single-cell morphology analysis using a novel high-throughput microfluidic system shows that cardiomyocyte hypertrophy is non-additively modifiable by specific pairwise interactions between CCDC141 and both TTN and IGF1R. Our results expand the scope of genetic regulation of cardiac structure to epistasis.