Virally delivered CMYA5 enhances the assembly of cardiac dyads.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) lack nanoscale structures essential for efficient excitation-contraction coupling. Such nanostructures, known as dyads, are frequently disrupted in heart failure. Here we show that the reduced expression of cardiomyopathy-associated 5 (CMYA5), a master protein that establishes dyads, contributes to dyad disorganization in heart failure and to impaired dyad assembly in hiPSC-CMs, and that a miniaturized form of CMYA5 suitable for delivery via an adeno-associated virus substantially improved dyad architecture and normalized cardiac function under pressure overload. In hiPSC-CMs, the miniaturized form of CMYA5 increased contractile forces, improved Ca2+ handling and enhanced the alignment of sarcomere Z-lines with ryanodine receptor 2, a protein that mediates the sarcoplasmic release of stored Ca2+. Our findings clarify the mechanisms responsible for impaired dyad structure in diseased cardiomyocytes, and suggest strategies for promoting dyad assembly and stability in heart disease and during the derivation of hiPSC-CMs.

Cardiac NAD+ depletion in mice promotes hypertrophic cardiomyopathy and arrhythmias prior to impaired bioenergetics.

Nicotinamide adenine dinucleotide (NAD+) is an essential co-factor in metabolic reactions and co-substrate for signaling enzymes. Failing human hearts display decreased expression of the major NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (Nampt) and lower NAD+ levels, and supplementation with NAD+ precursors is protective in preclinical models. Here we show that Nampt loss in adult cardiomyocytes caused depletion of NAD+ along with marked metabolic derangements, hypertrophic remodeling and sudden cardiac deaths, despite unchanged ejection fraction, endurance and mitochondrial respiratory capacity. These effects were directly attributable to NAD+ loss as all were ameliorated by restoring cardiac NAD+ levels with the NAD+ precursor nicotinamide riboside (NR). Electrocardiograms revealed that loss of myocardial Nampt caused a shortening of QT intervals with spontaneous lethal arrhythmias causing sudden cardiac death. Thus, changes in NAD+ concentration can have a profound influence on cardiac physiology even at levels sufficient to maintain energetics.

Phosphorylation of CRYAB Induces a Condensatopathy to Worsen Post-Myocardial Infarction Left Ventricular Remodeling.

Protein aggregates are emerging therapeutic targets in rare monogenic causes of cardiomyopathy and amyloid heart disease, but their role in more prevalent heart failure syndromes remains mechanistically unexamined. We observed mis-localization of desmin and sarcomeric proteins to aggregates in human myocardium with ischemic cardiomyopathy and in mouse hearts with post-myocardial infarction ventricular remodeling, mimicking findings of autosomal-dominant cardiomyopathy induced by R120G mutation in the cognate chaperone protein, CRYAB. In both syndromes, we demonstrate increased partitioning of CRYAB phosphorylated on serine-59 to NP40-insoluble aggregate-rich biochemical fraction. While CRYAB undergoes phase separation to form condensates, the phospho-mimetic mutation of serine-59 to aspartate (S59D) in CRYAB mimics R120G-CRYAB mutants with reduced condensate fluidity, formation of protein aggregates and increased cell death. Conversely, changing serine to alanine (phosphorylation-deficient mutation) at position 59 (S59A) restored condensate fluidity, and reduced both R120G-CRYAB aggregates and cell death. In mice, S59D CRYAB knock-in was sufficient to induce desmin mis-localization and myocardial protein aggregates, while S59A CRYAB knock-in rescued left ventricular systolic dysfunction post-myocardial infarction and preserved desmin localization with reduced myocardial protein aggregates. 25-Hydroxycholesterol attenuated CRYAB serine-59 phosphorylation and rescued post-myocardial infarction adverse remodeling. Thus, targeting CRYAB phosphorylation-induced condensatopathy is an attractive strategy to counter ischemic cardiomyopathy.

Endogenous adenine is a potential driver of the cardiovascular-kidney-metabolic syndrome.

Mechanisms underlying the cardiovascular-kidney-metabolic (CKM) syndrome are unknown, although key small molecule metabolites may be involved. Bulk and spatial metabolomics identified adenine to be upregulated and specifically enriched in coronary blood vessels in hearts from patients with diabetes and left ventricular hypertrophy. Single nucleus gene expression studies revealed that endothelial methylthioadenosine phosphorylase (MTAP) was increased in human hearts with hypertrophic cardiomyopathy. The urine adenine/creatinine ratio in patients was predictive of incident heart failure with preserved ejection fraction. Heart adenine and MTAP gene expression was increased in a 2-hit mouse model of hypertrophic heart disease and in a model of diastolic dysfunction with diabetes. Inhibition of MTAP blocked adenine accumulation in the heart, restored heart dysfunction in mice with type 2 diabetes and prevented ischemic heart damage in a rat model of myocardial infarction. Mechanistically, adenine-induced impaired mitophagy was reversed by reduction of mTOR. These studies indicate that endogenous adenine is in a causal pathway for heart failure and ischemic heart disease in the context of CKM syndrome.

Myocardial ultrastructure of human heart failure with preserved ejection fraction.

Over half of patients with heart failure have a preserved ejection fraction (>50%, called HFpEF), a syndrome with substantial morbidity/mortality and few effective therapies1. Its dominant comorbidity is now obesity, which worsens disease and prognosis1-3. Myocardial data from patients with morbid obesity and HFpEF show depressed myocyte calcium-stimulated tension4 and disrupted gene expression of mitochondrial and lipid metabolic pathways5,6, abnormalities shared by human HF with a reduced EF but less so in HFpEF without severe obesity. The impact of severe obesity on human HFpEF myocardial ultrastructure remains unexplored. Here we assessed the myocardial ultrastructure in septal biopsies from patients with HFpEF using transmission electron microscopy. We observed sarcomere disruption and sarcolysis, mitochondrial swelling with cristae separation and dissolution and lipid droplet accumulation that was more prominent in the most obese patients with HFpEF and not dependent on comorbid diabetes. Myocardial proteomics revealed associated reduction in fatty acid uptake, processing and oxidation and mitochondrial respiration proteins, particularly in very obese patients with HFpEF.

Nuclear ATP-citrate lyase regulates chromatin-dependent activation and maintenance of the myofibroblast gene program.

Differentiation of cardiac fibroblasts to myofibroblasts is necessary for matrix remodeling and fibrosis in heart failure. We previously reported that mitochondrial calcium signaling drives α-ketoglutarate-dependent histone demethylation, promoting myofibroblast formation. Here we investigate the role of ATP-citrate lyase (ACLY), a key enzyme for acetyl-CoA biosynthesis, in histone acetylation regulating myofibroblast fate and persistence in cardiac fibrosis. We show that inactivation of ACLY prevents myofibroblast differentiation and reverses myofibroblasts towards quiescence. Genetic deletion of Acly in post-activated myofibroblasts prevents fibrosis and preserves cardiac function in pressure-overload heart failure. TGFß stimulation enhances ACLY nuclear localization and ACLY-SMAD2/3 interaction, and increases H3K27ac at fibrotic gene loci. Pharmacological inhibition of ACLY or forced nuclear expression of a dominant-negative ACLY mutant prevents myofibroblast formation and H3K27ac. Our data indicate that nuclear ACLY activity is necessary for myofibroblast differentiation and persistence by maintaining histone acetylation at TGFß-induced myofibroblast genes. These findings provide targets to prevent and reverse pathological fibrosis.

Pathophysiological insights into HFpEF from studies of human cardiac tissue.

Heart failure with preserved ejection fraction (HFpEF) is a major, worldwide health-care problem. Few therapies for HFpEF exist because the pathophysiology of this condition is poorly defined and, increasingly, postulated to be diverse. Although perturbations in other organs contribute to the clinical profile in HFpEF, altered cardiac structure, function or both are the primary causes of this heart failure syndrome. Therefore, studying myocardial tissue is fundamental to improve pathophysiological insights and therapeutic discovery in HFpEF. Most studies of myocardial changes in HFpEF have relied on cardiac tissue from animal models without (or with limited) confirmatory studies in human cardiac tissue. Animal models of HFpEF have evolved based on theoretical HFpEF aetiologies, but these models might not reflect the complex pathophysiology of human HFpEF. The focus of this Review is the pathophysiological insights gained from studies of human HFpEF myocardium. We outline the rationale for these studies, the challenges and opportunities in obtaining myocardial tissue from patients with HFpEF and relevant comparator groups, the analytical approaches, the pathophysiological insights gained to date and the remaining knowledge gaps. Our objective is to provide a roadmap for future studies of cardiac tissue from diverse cohorts of patients with HFpEF, coupling discovery biology with measures to account for pathophysiological diversity.

Vasohibin inhibition improves myocardial relaxation in a rat model of heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome associated with increased myocardial stiffness and cardiac filling abnormalities. Prior studies implicated increased α-tubulin detyrosination, which is catalyzed by the vasohibin enzymes, as a contributor to increased stabilization of the cardiomyocyte microtubule network (MTN) and stiffness in failing human hearts. We explored whether increased MTN detyrosination contributed to impaired diastolic function in the ZSF1 obese rat model of HFpEF and designed a small-molecule vasohibin inhibitor to ablate MTN detyrosination in vivo. Compared with ZSF1 lean and Wistar Kyoto rats, obese rats exhibited increased tubulin detyrosination concomitant with diastolic dysfunction, left atrial enlargement, and cardiac hypertrophy with a preserved left ventricle ejection fraction, consistent with an HFpEF phenotype. Ex vivo myocardial phenotyping assessed cardiomyocyte mechanics and contractility. Vasohibin inhibitor treatment of isolated cardiomyocytes from obese rats resulted in reduced stiffness and faster relaxation. Acute in vivo treatment with vasohibin inhibitor improved diastolic relaxation in ZSF1 obese rats compared with ZSF1 lean and Wistar Kyoto rats. Vasohibin inhibition also improved relaxation in isolated human cardiomyocytes from both failing and nonfailing hearts. Our data suggest the therapeutic potential for vasohibin inhibition to reduce myocardial stiffness and improve relaxation in HFpEF.

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 remodeling. 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 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.

Perinuclear damage from nuclear envelope deterioration elicits stress responses that contribute to LMNA cardiomyopathy.

Mutations in the LMNA gene encoding lamins A/C cause an array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the perturbations emanating from LMNA mutations, an integrative understanding of the pathogenesis underlying cardiac dysfunction remains elusive. Using a novel conditional deletion model capable of translatome profiling, we observed that cardiomyocyte-specific Lmna deletion in adult mice led to rapid cardiomyopathy with pathological remodeling. Before cardiac dysfunction, Lmna-deleted cardiomyocytes displayed nuclear abnormalities, Golgi dilation/fragmentation, and CREB3-mediated stress activation. Translatome profiling identified MED25 activation, a transcriptional cofactor that regulates Golgi stress. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the Golgi. Systemic administration of modulators of autophagy or ER stress significantly delayed cardiac dysfunction and prolonged survival. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the LMNA cardiomyopathy development.

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.

Failing to Make the Grade: Conventional Cardiac Allograft Rejection Grading Criteria Are Inadequate for Predicting Rejection Severity.

BACKGROUND: Cardiac allograft rejection is the leading cause of early graft failure and is a major focus of postheart transplant patient care. While histological grading of endomyocardial biopsy samples remains the diagnostic standard for acute rejection, this standard has limited diagnostic accuracy. Discordance between biopsy rejection grade and patient clinical trajectory frequently leads to both overtreatment of indolent processes and delayed treatment of aggressive ones, spurring the need to investigate the adequacy of the current histological criteria for assessing clinically important rejection outcomes. METHODS: N=2900 endomyocardial biopsy images were assigned a rejection grade label (high versus low grade) and a clinical trajectory label (evident versus silent rejection). Using an image analysis approach, n=370 quantitative morphology features describing the lymphocytes and stroma were extracted from each slide. Two models were constructed to compare the subset of features associated with rejection grades versus those associated with clinical trajectories. A proof-of-principle machine learning pipeline-the cardiac allograft rejection evaluator-was then developed to test the feasibility of identifying the clinical severity of a rejection event. RESULTS: The histopathologic findings associated with conventional rejection grades differ substantially from those associated with clinically evident allograft injury. Quantitative assessment of a small set of well-defined morphological features can be leveraged to more accurately reflect the severity of rejection compared with that achieved by the International Society of Heart and Lung Transplantation grades. CONCLUSIONS: Conventional endomyocardial samples contain morphological information that enables accurate identification of clinically evident rejection events, and this information is incompletely captured by the current, guideline-endorsed, rejection grading criteria.

Distinct cytoskeletal regulators of mechanical memory in cardiac fibroblasts and cardiomyocytes.

Recognizing that cells "feel" and respond to their mechanical environment, recent studies demonstrate that many cells exhibit a phenomenon of "mechanical memory" in which features induced by prior mechanical cues persist after the mechanical stimulus has ceased. While there is a general recognition that different cell types exhibit different responses to changes in extracellular matrix stiffening, the phenomenon of mechanical memory within myocardial cell types has received little attention to date. To probe the dynamics of mechanical memory in cardiac fibroblasts (CFs) and cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs), we employed a magnetorheological elastomer (MRE) cell culture substrate with tunable and reversible stiffness spanning the range from normal to diseased myocardium. In CFs, using increased cell area and increases in α-smooth muscle actin as markers of cellular responses to matrix stiffening, we found that induction of mechanical memory required seven days of stiff priming. Both induction and maintenance of persistent CF activation were blocked with the F-actin inhibitor cytochalasin D, while inhibitors of microtubule detyrosination had no impact on CFs. In iPSC-CMs, mechanical memory was invoked after only 24 h of stiff priming. Moreover, mechanical memory induction and maintenance were microtubule-dependent in CMs with no dependence on F-actin. Overall, these results identify the distinct temporal dynamics of mechanical memory in CFs and iPSC-CMs with different cytoskeletal mediators responsible for inducing and maintaining the stiffness-activated phenotype. Due to its flexibility, this model is broadly applicable to future studies interrogating mechanotransduction and mechanical memory in the heart and might inform strategies for attenuating the impact of load-induced pathology and excess myocardial stiffness.

The HFSA Advanced Heart Failure and Transplant Cardiology Fellowship Consensus Conference.

There is waning interest among cardiology trainees in pursuing an Advanced Heart Failure/Transplant Cardiology (AHFTC) fellowship as evidenced by fewer applicants in the National Resident Matching Program match to this specialty. This trend has generated considerable attention across the heart failure community. In response, the Heart Failure Society of America convened the AHFTC Fellowship Task Force with a charge to develop strategies to increase the value proposition of an AHFTC fellowship. Subsequently, the HFSA sponsored the AHFTC Fellowship Consensus Conference April 26-27, 2023. Before the conference, interviews of 44 expert stakeholders diverse across geography, site of practice (traditional academic medical center or other centers), specialty/area of expertise, sex, and stage of career were conducted virtually. Based on these interviews, potential solutions to address the declining interest in AHFTC fellowship were categorized into five themes: (1) alternative training pathways, (2) regulatory and compensation, (3) educational improvements, (4) exposure and marketing for pipeline development, and (5) quality of life and mental health. These themes provided structure to the deliberations of the AHFTC Fellowship Consensus Conference. The recommendations from the Consensus Conference were subsequently presented to the HFSA Board of Directors to inform strategic plans and interventions. The HFSA Board of Directors later reviewed and approved submission of this document. The purpose of this communication is to provide the HF community with an update summarizing the processes used and concepts that emerged from the work of the HFSA AHFTC Fellowship Task Force and Consensus Conference.

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.

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.

Early Increases in Blood Pressure and Major Adverse Cardiovascular Events in Patients With Renal Cell Carcinoma and Thyroid Cancer Treated With VEGFR TKIs.

BACKGROUND: Although VEGFR tyrosine kinase inhibitors (TKIs) are a preferred systemic treatment approach for patients with advanced renal cell carcinoma (RCC) and thyroid carcinoma (TC), treatment-related cardiovascular (CV) toxicity is an important contributor to morbidity. However, the clinical risk assessment and impact of CV toxicities, including early significant hypertension, among real-world advanced cancer populations receiving VEGFR TKI therapies remain understudied. METHODS: In a multicenter, retrospective cohort study across 3 large and diverse US health systems, we characterized baseline hypertension and CV comorbidity in patients with RCC and those with TC who are newly initiating VEGFR TKI therapy. We also evaluated baseline patient-, treatment-, and disease-related factors associated with the risk for treatment-related early hypertension (within 6 weeks of TKI initiation) and major adverse CV events (MACE), accounting for the competing risk of death in an advanced cancer population, after VEGFR TKI initiation. RESULTS: Between 2008 and 2020, 987 patients (80.3% with RCC, 19.7% with TC) initiated VEGFR TKI therapy. The baseline prevalence of hypertension was high (61.5% and 53.6% in patients with RCC and TC, respectively). Adverse CV events, including heart failure and cerebrovascular accident, were common (occurring in 14.9% of patients) and frequently occurred early (46.3% occurred within 1 year of VEGFR TKI initiation). Baseline hypertension and Black race were the primary clinical factors associated with increased acute hypertensive risk within 6 weeks of VEGFR TKI initiation. However, early significant "on-treatment" hypertension was not associated with MACE. CONCLUSIONS: These multicenter, real-world findings indicate that hypertensive and CV morbidities are highly prevalent among patients initiating VEGFR TKI therapies, and baseline hypertension and Black race represent the primary clinical factors associated with VEGFR TKI-related early significant hypertension. However, early on-treatment hypertension was not associated with MACE, and cancer-specific CV risk algorithms may be warranted for patients initiating VEGFR TKIs.

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.