Integrating Clinical Phenotype With Multiomics Analyses of Human Cardiac Tissue Unveils Divergent Metabolic Remodeling in Genotype-Positive and Genotype-Negative Patients With Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by sarcomere gene mutations (genotype-positive HCM) in ≈50% of patients and occurs in the absence of mutations (genotype-negative HCM) in the other half of patients. We explored how alterations in the metabolomic and lipidomic landscape are involved in cardiac remodeling in both patient groups. METHODS: We performed proteomics, metabolomics, and lipidomics on myectomy samples (genotype-positive N=19; genotype-negative N=22; and genotype unknown N=6) from clinically well-phenotyped patients with HCM and on cardiac tissue samples from sex- and age-matched and body mass index-matched nonfailing donors (N=20). These data sets were integrated to comprehensively map changes in lipid-handling and energy metabolism pathways. By linking metabolomic and lipidomic data to variability in clinical data, we explored patient group-specific associations between cardiac and metabolic remodelings. RESULTS: HCM myectomy samples exhibited (1) increased glucose and glycogen metabolism, (2) downregulation of fatty acid oxidation, and (3) reduced ceramide formation and lipid storage. In genotype-negative patients, septal hypertrophy and diastolic dysfunction correlated with the lowering of acylcarnitines, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines. In contrast, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines were positively associated with septal hypertrophy and diastolic impairment in genotype-positive patients. CONCLUSIONS: We provide novel insights into both general and genotype-specific metabolic changes in HCM. Distinct metabolic alterations underlie cardiac disease progression in genotype-negative and genotype-positive patients with HCM.

Functional Impact of Alternative Metabolic Substrates in Failing Human Cardiomyocytes.

Recent studies suggest that metabolic dysregulation in patients with heart failure might contribute to myocardial contractile dysfunction. To understand the correlation between function and energy metabolism, we studied the impact of different fuel substrates on human nonfailing or failing cardiomyocytes. Consistent with the concept of metabolic flexibility, nonfailing myocytes exhibited excellent contractility in all fuels provided. However, impaired contractility was observed in failing myocytes when carbohydrates alone were used but was improved when additional substrates were added. This study demonstrates the functional significance of fuel utilization shifts in failing human cardiomyocytes.

Discovery adductomics provides a comprehensive portrait of tissue-, age- and sex-specific DNA modifications in rodents and humans.

DNA damage causes genomic instability underlying many diseases, with traditional analytical approaches providing minimal insight into the spectrum of DNA lesions in vivo. Here we used untargeted chromatography-coupled tandem mass spectrometry-based adductomics (LC-MS/MS) to begin to define the landscape of DNA modifications in rat and human tissues. A basis set of 114 putative DNA adducts was identified in heart, liver, brain, and kidney in 1-26-month-old rats and 111 in human heart and brain by 'stepped MRM' LC-MS/MS. Subsequent targeted analysis of these species revealed species-, tissue-, age- and sex-biases. Structural characterization of 10 selected adductomic signals as known DNA modifications validated the method and established confidence in the DNA origins of the signals. Along with strong tissue biases, we observed significant age-dependence for 36 adducts, including N2-CMdG, 5-HMdC and 8-Oxo-dG in rats and 1,N6-ϵdA in human heart, as well as sex biases for 67 adducts in rat tissues. These results demonstrate the potential of adductomics for discovering the true spectrum of disease-driving DNA adducts. Our dataset of 114 putative adducts serves as a resource for characterizing dozens of new forms of DNA damage, defining mechanisms of their formation and repair, and developing them as biomarkers of aging and disease.

Adult human cardiomyocyte mechanics in osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is an extracellular matrix disorder characterized by defects in collagen-1 transport or synthesis, resulting in bone abnormalities. Although reduced collagen in OI hearts has been associated with reduced myocardial stiffness and left ventricular remodeling, its impact on cardiomyocyte (CM) function has not been studied. Here, we explore the tissue-level and CM-level properties of a heart from a deceased organ donor with OI type I. Proteomics and histology confirmed strikingly low expression of collagen 1. Trabecular stretch confirmed low stiffness on the tissue level. However, CMs retained normal viscoelastic properties as revealed by nanoindentation. Interestingly, OI CMs were hypercontractile relative to nonfailing controls after 24 h of culture. In response to 48 h of culture on surfaces with physiological (10 kPa) and pathological (50 kPa) stiffness, OI CMs demonstrated a greater reduction in contractility than nonfailing CMs, suggesting that OI CMs may have an impaired stress response. Levels of detyrosinated α-tubulin, known to be responsive to extracellular stiffness, were reduced in OI CMs. Together these data confirm multiple CM-level adaptations to low stiffness that extend our understanding of OI in the heart and how CMs respond to extracellular stiffness.NEW & NOTEWORTHY In a rare donation of a heart from an individual with osteogenesis imperfecta (OI), we explored cardiomyocyte (CM) adaptations to low stiffness. This represents the first assessment of cardiomyocyte mechanics in OI. The data reveal the hypercontractility of OI CMs with rapid rundown when exposed to acute stiffness challenges, extending our understanding of OI. These data demonstrate that the impact of OI on myocardial mechanics includes cardiomyocyte adaptations beyond known direct effects on the extracellular matrix.

Myocardial Metabolomics of Human Heart Failure With Preserved Ejection Fraction.

BACKGROUND: The human heart primarily metabolizes fatty acids, and this decreases as alternative fuel use rises in heart failure with reduced ejection fraction (HFrEF). Patients with severe obesity and diabetes are thought to have increased myocardial fatty acid metabolism, but whether this is found in those who also have heart failure with preserved ejection fraction (HFpEF) is unknown. METHODS: Plasma and endomyocardial biopsies were obtained from HFpEF (n=38), HFrEF (n=30), and nonfailing donor controls (n=20). Quantitative targeted metabolomics measured organic acids, amino acids, and acylcarnitines in myocardium (72 metabolites) and plasma (69 metabolites). The results were integrated with reported RNA sequencing data. Metabolomics were analyzed using agnostic clustering tools, Kruskal-Wallis test with Dunn test, and machine learning. RESULTS: Agnostic clustering of myocardial but not plasma metabolites separated disease groups. Despite more obesity and diabetes in HFpEF versus HFrEF (body mass index, 39.8 kg/m2 versus 26.1 kg/m2; diabetes, 70% versus 30%; both P<0.0001), medium- and long-chain acylcarnitines (mostly metabolites of fatty acid oxidation) were markedly lower in myocardium from both heart failure groups versus control. In contrast, plasma levels were no different or higher than control. Gene expression linked to fatty acid metabolism was generally lower in HFpEF versus control. Myocardial pyruvate was higher in HFpEF whereas the tricarboxylic acid cycle intermediates succinate and fumarate were lower, as were several genes controlling glucose metabolism. Non-branched-chain and branched-chain amino acids (BCAA) were highest in HFpEF myocardium, yet downstream BCAA metabolites and genes controlling BCAA metabolism were lower. Ketone levels were higher in myocardium and plasma of patients with HFrEF but not HFpEF. HFpEF metabolomic-derived subgroups were differentiated by only a few differences in BCAA metabolites. CONCLUSIONS: Despite marked obesity and diabetes, HFpEF myocardium exhibited lower fatty acid metabolites compared with HFrEF. Ketones and metabolites of the tricarboxylic acid cycle and BCAA were also lower in HFpEF, suggesting insufficient use of alternative fuels. These differences were not detectable in plasma and challenge conventional views of myocardial fuel use in HFpEF with marked diabetes and obesity and suggest substantial fuel inflexibility in this syndrome.

Mitochondrial dysfunction in human hypertrophic cardiomyopathy is linked to cardiomyocyte architecture disruption and corrected by improving NADH-driven mitochondrial respiration.

AIMS: Genetic hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere protein-encoding genes (i.e. genotype-positive HCM). In an increasing number of patients, HCM occurs in the absence of a mutation (i.e. genotype-negative HCM). Mitochondrial dysfunction is thought to be a key driver of pathological remodelling in HCM. Reports of mitochondrial respiratory function and specific disease-modifying treatment options in patients with HCM are scarce. METHODS AND RESULTS: Respirometry was performed on septal myectomy tissue from patients with HCM (n = 59) to evaluate oxidative phosphorylation and fatty acid oxidation. Mitochondrial dysfunction was most notably reflected by impaired NADH-linked respiration. In genotype-negative patients, but not genotype-positive patients, NADH-linked respiration was markedly depressed in patients with an indexed septal thickness ≥10 compared with <10. Mitochondrial dysfunction was not explained by reduced abundance or fragmentation of mitochondria, as evaluated by transmission electron microscopy. Rather, improper organization of mitochondria relative to myofibrils (expressed as a percentage of disorganized mitochondria) was strongly associated with mitochondrial dysfunction. Pre-incubation with the cardiolipin-stabilizing drug elamipretide and raising mitochondrial NAD+ levels both boosted NADH-linked respiration. CONCLUSION: Mitochondrial dysfunction is explained by cardiomyocyte architecture disruption and is linked to septal hypertrophy in genotype-negative HCM. Despite severe myocardial remodelling mitochondria were responsive to treatments aimed at restoring respiratory function, eliciting the mitochondria as a drug target to prevent and ameliorate cardiac disease in HCM. Mitochondria-targeting therapy may particularly benefit genotype-negative patients with HCM, given the tight link between mitochondrial impairment and septal thickening in this subpopulation.

Single-nucleus RNA sequencing in ischemic cardiomyopathy reveals common transcriptional profile underlying end-stage heart failure.

Ischemic cardiomyopathy (ICM) is the leading cause of heart failure worldwide, yet the cellular and molecular signature of this disease is largely unclear. Using single-nucleus RNA sequencing (snRNA-seq) and integrated computational analyses, we profile the transcriptomes of over 99,000 human cardiac nuclei from the non-infarct region of the left ventricle of 7 ICM transplant recipients and 8 non-failing (NF) controls. We find the cellular composition of the ischemic heart is significantly altered, with decreased cardiomyocytes and increased proportions of lymphatic, angiogenic, and arterial endothelial cells in patients with ICM. We show that there is increased LAMININ signaling from endothelial cells to other cell types in ICM compared with NF. Finally, we find that the transcriptional changes that occur in ICM are similar to those in hypertrophic and dilated cardiomyopathies and that the mining of these combined datasets can identify druggable genes that could be used to target end-stage heart failure.

Noncanonical Form of ERAD Regulates Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy increases demands on protein folding, which causes an accumulation of misfolded proteins in the endoplasmic reticulum (ER). These misfolded proteins can be removed by the adaptive retrotranslocation, polyubiquitylation, and a proteasome-mediated degradation process, ER-associated degradation (ERAD), which, as a biological process and rate, has not been studied in vivo. To investigate a role for ERAD in a pathophysiological model, we examined the function of the functional initiator of ERAD, valosin-containing protein-interacting membrane protein (VIMP), positing that VIMP would be adaptive in pathological cardiac hypertrophy in mice. METHODS: We developed a new method involving cardiac myocyte-specific adeno-associated virus serovar 9-mediated expression of the canonical ERAD substrate, TCRα, to measure the rate of ERAD, ie, ERAD flux, in the heart in vivo. Adeno-associated virus serovar 9 was also used to either knock down or overexpress VIMP in the heart. Then mice were subjected to transverse aortic constriction to induce pressure overload-induced cardiac hypertrophy. RESULTS: ERAD flux was slowed in both human heart failure and mice after transverse aortic constriction. Surprisingly, although VIMP adaptively contributes to ERAD in model cell lines, in the heart, VIMP knockdown increased ERAD and ameliorated transverse aortic constriction-induced cardiac hypertrophy. Coordinately, VIMP overexpression exacerbated cardiac hypertrophy, which was dependent on VIMP engaging in ERAD. Mechanistically, we found that the cytosolic protein kinase SGK1 (serum/glucocorticoid regulated kinase 1) is a major driver of pathological cardiac hypertrophy in mice subjected to transverse aortic constriction, and that VIMP knockdown decreased the levels of SGK1, which subsequently decreased cardiac pathology. We went on to show that although it is not an ER protein, and resides outside of the ER, SGK1 is degraded by ERAD in a noncanonical process we call ERAD-Out. Despite never having been in the ER, SGK1 is recognized as an ERAD substrate by the ERAD component DERLIN1, and uniquely in cardiac myocytes, VIMP displaces DERLIN1 from initiating ERAD, which decreased SGK1 degradation and promoted cardiac hypertrophy. CONCLUSIONS: ERAD-Out is a new preferentially favored noncanonical form of ERAD that mediates the degradation of SGK1 in cardiac myocytes, and in so doing is therefore an important determinant of how the heart responds to pathological stimuli, such as pressure overload.

Extra-cardiac BCAA catabolism lowers blood pressure and protects from heart failure.

Pharmacologic activation of branched-chain amino acid (BCAA) catabolism is protective in models of heart failure (HF). How protection occurs remains unclear, although a causative block in cardiac BCAA oxidation is widely assumed. Here, we use in vivo isotope infusions to show that cardiac BCAA oxidation in fact increases, rather than decreases, in HF. Moreover, cardiac-specific activation of BCAA oxidation does not protect from HF even though systemic activation does. Lowering plasma and cardiac BCAAs also fails to confer significant protection, suggesting alternative mechanisms of protection. Surprisingly, activation of BCAA catabolism lowers blood pressure (BP), a known cardioprotective mechanism. BP lowering occurred independently of nitric oxide and reflected vascular resistance to adrenergic constriction. Mendelian randomization studies revealed that elevated plasma BCAAs portend higher BP in humans. Together, these data indicate that BCAA oxidation lowers vascular resistance, perhaps in part explaining cardioprotection in HF that is not mediated directly in cardiomyocytes.

Aortic Cellular Diversity and Quantitative Genome-Wide Association Study Trait Prioritization Through Single-Nuclear RNA Sequencing of the Aneurysmal Human Aorta.

BACKGROUND: Mural cells in ascending aortic aneurysms undergo phenotypic changes that promote extracellular matrix destruction and structural weakening. To explore this biology, we analyzed the transcriptional features of thoracic aortic tissue. METHODS: Single-nuclear RNA sequencing was performed on 13 samples from human donors, 6 with thoracic aortic aneurysm, and 7 without aneurysm. Individual transcriptomes were then clustered based on transcriptional profiles. Clusters were used for between-disease differential gene expression analyses, subcluster analysis, and analyzed for intersection with genetic aortic trait data. RESULTS: We sequenced 71 689 nuclei from human thoracic aortas and identified 14 clusters, aligning with 11 cell types, predominantly vascular smooth muscle cells (VSMCs) consistent with aortic histology. With unbiased methodology, we found 7 vascular smooth muscle cell and 6 fibroblast subclusters. Differentially expressed genes analysis revealed a vascular smooth muscle cell group accounting for the majority of differential gene expression. Fibroblast populations in aneurysm exhibit distinct behavior with almost complete disappearance of quiescent fibroblasts. Differentially expressed genes were used to prioritize genes at aortic diameter and distensibility genome-wide association study loci highlighting the genes JUN, LTBP4 (latent transforming growth factor beta-binding protein 1), and IL34 (interleukin 34) in fibroblasts, ENTPD1, PDLIM5 (PDZ and LIM domain 5), ACTN4 (alpha-actinin-4), and GLRX in vascular smooth muscle cells, as well as LRP1 in macrophage populations. CONCLUSIONS: Using nuclear RNA sequencing, we describe the cellular diversity of healthy and aneurysmal human ascending aorta. Sporadic aortic aneurysm is characterized by differential gene expression within known cellular classes rather than by the appearance of novel cellular forms. Single-nuclear RNA sequencing of aortic tissue can be used to prioritize genes at aortic trait loci.

Single-nucleus profiling of human dilated and hypertrophic cardiomyopathy.

Heart failure encompasses a heterogeneous set of clinical features that converge on impaired cardiac contractile function1,2 and presents a growing public health concern. Previous work has highlighted changes in both transcription and protein expression in failing hearts3,4, but may overlook molecular changes in less prevalent cell types. Here we identify extensive molecular alterations in failing hearts at single-cell resolution by performing single-nucleus RNA sequencing of nearly 600,000 nuclei in left ventricle samples from 11 hearts with dilated cardiomyopathy and 15 hearts with hypertrophic cardiomyopathy as well as 16 non-failing hearts. The transcriptional profiles of dilated or hypertrophic cardiomyopathy hearts broadly converged at the tissue and cell-type level. Further, a subset of hearts from patients with cardiomyopathy harbour a unique population of activated fibroblasts that is almost entirely absent from non-failing samples. We performed a CRISPR-knockout screen in primary human cardiac fibroblasts to evaluate this fibrotic cell state transition; knockout of genes associated with fibroblast transition resulted in a reduction of myofibroblast cell-state transition upon TGFß1 stimulation for a subset of genes. Our results provide insights into the transcriptional diversity of the human heart in health and disease as well as new potential therapeutic targets and biomarkers for heart failure.

Direct anabolic metabolism of three-carbon propionate to a six-carbon metabolite occurs in vivo across tissues and species.

Anabolic metabolism of carbon in mammals is mediated via the one- and two-carbon carriers S-adenosyl methionine and acetyl-coenzyme A. In contrast, anabolic metabolism of three-carbon units via propionate has not been shown to extensively occur. Mammals are primarily thought to oxidize the three-carbon short chain fatty acid propionate by shunting propionyl-CoA to succinyl-CoA for entry into the TCA cycle. Here, we found that this may not be absolute as, in mammals, one nonoxidative fate of propionyl-CoA is to condense to two three-carbon units into a six-carbon trans-2-methyl-2-pentenoyl-CoA (2M2PE-CoA). We confirmed this reaction pathway using purified protein extracts provided limited substrates and verified the product via LC-MS using a synthetic standard. In whole-body in vivo stable isotope tracing following infusion of 13C-labeled valine at steady state, 2M2PE-CoA was found to form via propionyl-CoA in multiple murine tissues, including heart, kidney, and to a lesser degree, in brown adipose tissue, liver, and tibialis anterior muscle. Using ex vivo isotope tracing, we found that 2M2PE-CoA also formed in human myocardial tissue incubated with propionate to a limited extent. While the complete enzymology of this pathway remains to be elucidated, these results confirm the in vivo existence of at least one anabolic three- to six-carbon reaction conserved in humans and mice that utilizes propionate.

Transcriptional, Post-Transcriptional, and Post-Translational Mechanisms Rewrite the Tubulin Code During Cardiac Hypertrophy and Failure.

A proliferated and post-translationally modified microtubule network underlies cellular growth in cardiac hypertrophy and contributes to contractile dysfunction in heart failure. Yet how the heart achieves this modified network is poorly understood. Determining how the "tubulin code"-the permutations of tubulin isoforms and post-translational modifications-is rewritten upon cardiac stress may provide new targets to modulate cardiac remodeling. Further, while tubulin can autoregulate its own expression, it is unknown if autoregulation is operant in the heart or tuned in response to stress. Here we use heart failure patient samples and murine models of cardiac remodeling to interrogate transcriptional, autoregulatory, and post-translational mechanisms that contribute to microtubule network remodeling at different stages of heart disease. We find that autoregulation is operant across tubulin isoforms in the heart and leads to an apparent disconnect in tubulin mRNA and protein levels in heart failure. We also find that within 4 h of a hypertrophic stimulus and prior to cardiac growth, microtubule detyrosination is rapidly induced to help stabilize the network. This occurs concomitant with rapid transcriptional and autoregulatory activation of specific tubulin isoforms and microtubule motors. Upon continued hypertrophic stimulation, there is an increase in post-translationally modified microtubule tracks and anterograde motors to support cardiac growth, while total tubulin content increases through progressive transcriptional and autoregulatory induction of tubulin isoforms. Our work provides a new model for how the tubulin code is rapidly rewritten to establish a proliferated, stable microtubule network that drives cardiac remodeling, and provides the first evidence of tunable tubulin autoregulation during pathological progression.

Quantitative subcellular acyl-CoA analysis reveals distinct nuclear metabolism and isoleucine-dependent histone propionylation.

Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.

Integrated landscape of cardiac metabolism in end-stage human nonischemic dilated cardiomyopathy.

Heart failure (HF) is a leading cause of mortality. Failing hearts undergo profound metabolic changes, but a comprehensive evaluation in humans is lacking. We integrate plasma and cardiac tissue metabolomics of 678 metabolites, genome-wide RNA-sequencing, and proteomic studies to examine metabolic status in 87 explanted human hearts from 39 patients with end-stage HF compared with 48 nonfailing donors. We confirm bioenergetic defects in human HF and reveal selective depletion of adenylate purines required for maintaining ATP levels. We observe substantial reductions in fatty acids and acylcarnitines in failing tissue, despite plasma elevations, suggesting defective import of fatty acids into cardiomyocytes. Glucose levels, in contrast, are elevated. Pyruvate dehydrogenase, which gates carbohydrate oxidation, is de-repressed, allowing increased lactate and pyruvate burning. Tricarboxylic acid cycle intermediates are significantly reduced. Finally, bioactive lipids are profoundly reprogrammed, with marked reductions in ceramides and elevations in lysoglycerophospholipids. These data unveil profound metabolic abnormalities in human failing hearts.

Nuclear deformation guides chromatin reorganization in cardiac development and disease.

In cardiovascular tissues, changes in the mechanical properties of the extracellular matrix are associated with cellular de-differentiation and with subsequent functional declines. However, the underlying mechanoreceptive mechanisms are largely unclear. Here, by generating high-resolution, full-field strain maps of cardiomyocyte nuclei during contraction in vitro, complemented with evidence from tissues from patients with cardiomyopathy and from mice with reduced cardiac performance, we show that cardiomyocytes establish a distinct nuclear organization during maturation, characterized by the reorganization of H3K9me3-marked chromatin towards the nuclear border. Specifically, we show that intranuclear tension is spatially correlated with H3K9me3-marked chromatin, that reductions in nuclear deformation (through environmental stiffening or through the disruption of complexes of the linker of nucleoskeleton and cytoskeleton) abrogate chromatin reorganization and lead to the dissociation of H3K9me3-marked chromatin from the nuclear periphery, and that the suppression of H3K9 methylation induces chromatin reorganization and reduces the expression of cardiac developmental genes. Overall, our findings indicate that, by integrating environmental mechanical cues, the nuclei of cardiomyocytes guide and stabilize the fate of cells through the reorganization of epigenetically marked chromatin.

Truncated titin proteins in dilated cardiomyopathy.

Truncating variants in TTN (TTNtvs) are the most common known cause of nonischemic dilated cardiomyopathy (DCM), but how TTNtvs cause disease has remained controversial. Efforts to detect truncated titin proteins in affected human DCM hearts have failed, suggesting that disease is caused by haploinsufficiency, but reduced amounts of titin protein have not yet been demonstrated. Here, we leveraged a collection of 184 explanted posttransplant DCM hearts to show, using specialized electrophoretic gels, Western blotting, allelic phasing, and unbiased proteomics, that truncated titin proteins can quantitatively be detected in human DCM hearts. The sizes of truncated proteins corresponded to that predicted by their respective TTNtvs; the truncated proteins were encoded by the TTNtv-bearing allele; and no degradation fragments from protein encoded by either allele were detectable. In parallel, full-length titin was less abundant in TTNtv+ than in TTNtv− DCM hearts. Disease severity or need for transplantation did not correlate with TTNtv location. Transcriptomic profiling revealed few differences in splicing or allelic imbalance of the TTN transcript between TTNtv+ and TTNtv− DCM hearts. Studies with isolated human adult cardiomyocytes revealed no defects in contractility in cells from TTNtv+ compared to TTNtv− DCM hearts. Together, these data demonstrate the presence of truncated titin protein in human TTNtv+ DCM, show reduced amounts of full-length titin protein in TTNtv+ DCM hearts, and support combined dominant-negative and haploinsufficiency contributions to disease.

S-Nitrosylation of Histone Deacetylase 2 by Neuronal Nitric Oxide Synthase as a Mechanism of Diastolic Dysfunction.

BACKGROUND: Although the clinical importance of heart failure with preserved ejection fraction has been extensively explored, most therapeutic regimens, including nitric oxide (NO) donors, lack therapeutic benefit. Although the clinical characteristics of heart failure with preserved ejection fraction are somewhat heterogeneous, diastolic dysfunction (DD) is one of the most important features. Here we report that neuronal NO synthase (nNOS) induces DD by S-nitrosylation of HDAC2 (histone deacetylase 2). METHODS: Two animal models of DD-SAUNA (SAlty drinking water/Unilateral Nephrectomy/Aldosterone) and mild transverse aortic constriction mice-as well as human heart samples from patients with left ventricular hypertrophy were used. Genetically modified mice that were either nNOS-ablated or HDAC2 S-nitrosylation-resistant were also challenged. N(ω)-propyl-L-arginine, an nNOS selective inhibitor, and dimethyl fumarate, an NRF2 (nuclear factor erythroid 2-related factor 2) inducer, were used. Molecular events were further checked in human left ventricle specimens. RESULTS: SAUNA or mild transverse aortic constriction stress impaired diastolic function and exercise tolerance without overt systolic failure. Among the posttranslational modifications tested, S-nitrosylation was most dramatically increased in both models. Utilizing heart samples from both mice and humans, we observed increases in nNOS expression and NO production. N(ω)-propyl-L-arginine alleviated the development of DD in vivo. Similarly, nNOS knockout mice were resistant to SAUNA stress. nNOS-induced S-nitrosylation of HDAC2 was relayed by transnitrosylation of GAPDH. HDAC2 S-nitrosylation was confirmed in both DD mouse and human left ventricular hypertrophy. S-nitrosylation of HDAC2 took place at C262 and C274. When DD was induced, HDAC2 S-nitrosylation was detected in wild-type mouse, but not in HDAC2 knock-in mouse heart that expressed HDAC2 C262A/C274A. In addition, HDAC2 C262A/C274A mice maintained normal diastolic function under DD stimuli. Gene delivery with adenovirus-associated virus 9 (AAV9)-NRF2, a putative denitrosylase of HDAC2, or pharmacological intervention by dimethyl fumarate successfully induced HDAC2 denitrosylation and mitigated DD in vivo. CONCLUSIONS: Our observations are the first to demonstrate a new mechanism underlying DD pathophysiology. Our results provide theoretical and experimental evidence to explain the ineffectiveness of conventional NO enhancement trials for improving DD with heart failure symptoms. More important, our results suggest that reduction of NO or denitrosylation of HDAC2 may provide a new therapeutic platform for the treatment of refractory heart failure with preserved ejection fraction.

Pathogenic LMNA variants disrupt cardiac lamina-chromatin interactions and de-repress alternative fate genes.

Pathogenic mutations in LAMIN A/C (LMNA) cause abnormal nuclear structure and laminopathies. These diseases have myriad tissue-specific phenotypes, including dilated cardiomyopathy (DCM), but how LMNA mutations result in tissue-restricted disease phenotypes remains unclear. We introduced LMNA mutations from individuals with DCM into human induced pluripotent stem cells (hiPSCs) and found that hiPSC-derived cardiomyocytes, in contrast to hepatocytes or adipocytes, exhibit aberrant nuclear morphology and specific disruptions in peripheral chromatin. Disrupted regions were enriched for transcriptionally active genes and regions with lower LAMIN B1 contact frequency. The lamina-chromatin interactions disrupted in mutant cardiomyocytes were enriched for genes associated with non-myocyte lineages and correlated with higher expression of those genes. Myocardium from individuals with LMNA variants similarly showed aberrant expression of non-myocyte pathways. We propose that the lamina network safeguards cellular identity and that pathogenic LMNA variants disrupt peripheral chromatin with specific epigenetic and molecular characteristics, causing misexpression of genes normally expressed in other cell types.