MOF Acetyl Transferase Regulates Transcription and Respiration in Mitochondria.

A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism.

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.

Integrated multi-omic characterization of congenital heart disease.

The heart, the first organ to develop in the embryo, undergoes complex morphogenesis that when defective results in congenital heart disease (CHD). With current therapies, more than 90% of patients with CHD survive into adulthood, but many suffer premature death from heart failure and non-cardiac causes1. Here, to gain insight into this disease progression, we performed single-nucleus RNA sequencing on 157,273 nuclei from control hearts and hearts from patients with CHD, including those with hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot, two common forms of cyanotic CHD lesions, as well as dilated and hypertrophic cardiomyopathies. We observed CHD-specific cell states in cardiomyocytes, which showed evidence of insulin resistance and increased expression of genes associated with FOXO signalling and CRIM1. Cardiac fibroblasts in HLHS were enriched in a low-Hippo and high-YAP cell state characteristic of activated cardiac fibroblasts. Imaging mass cytometry uncovered a spatially resolved perivascular microenvironment consistent with an immunodeficient state in CHD. Peripheral immune cell profiling suggested deficient monocytic immunity in CHD, in agreement with the predilection in CHD to infection and cancer2. Our comprehensive phenotyping of CHD provides a roadmap towards future personalized treatments for CHD.

Aficamten for Symptomatic Obstructive Hypertrophic Cardiomyopathy.

BACKGROUND: One of the major determinants of exercise intolerance and limiting symptoms among patients with obstructive hypertrophic cardiomyopathy (HCM) is an elevated intracardiac pressure resulting from left ventricular outflow tract obstruction. Aficamten is an oral selective cardiac myosin inhibitor that reduces left ventricular outflow tract gradients by mitigating cardiac hypercontractility. METHODS: In this phase 3, double-blind trial, we randomly assigned adults with symptomatic obstructive HCM to receive aficamten (starting dose, 5 mg; maximum dose, 20 mg) or placebo for 24 weeks, with dose adjustment based on echocardiography results. The primary end point was the change from baseline to week 24 in the peak oxygen uptake as assessed by cardiopulmonary exercise testing. The 10 prespecified secondary end points (tested hierarchically) were change in the Kansas City Cardiomyopathy Questionnaire clinical summary score (KCCQ-CSS), improvement in the New York Heart Association (NYHA) functional class, change in the pressure gradient after the Valsalva maneuver, occurrence of a gradient of less than 30 mm Hg after the Valsalva maneuver, and duration of eligibility for septal reduction therapy (all assessed at week 24); change in the KCCQ-CSS, improvement in the NYHA functional class, change in the pressure gradient after the Valsalva maneuver, and occurrence of a gradient of less than 30 mm Hg after the Valsalva maneuver (all assessed at week 12); and change in the total workload as assessed by cardiopulmonary exercise testing at week 24. RESULTS: A total of 282 patients underwent randomization: 142 to the aficamten group and 140 to the placebo group. The mean age was 59.1 years, 59.2% were men, the baseline mean resting left ventricular outflow tract gradient was 55.1 mm Hg, and the baseline mean left ventricular ejection fraction was 74.8%. At 24 weeks, the mean change in the peak oxygen uptake was 1.8 ml per kilogram per minute (95% confidence interval [CI], 1.2 to 2.3) in the aficamten group and 0.0 ml per kilogram per minute (95% CI, -0.5 to 0.5) in the placebo group (least-squares mean between-group difference, 1.7 ml per kilogram per minute; 95% CI, 1.0 to 2.4; P<0.001). The results for all 10 secondary end points were significantly improved with aficamten as compared with placebo. The incidence of adverse events appeared to be similar in the two groups. CONCLUSIONS: Among patients with symptomatic obstructive HCM, treatment with aficamten resulted in a significantly greater improvement in peak oxygen uptake than placebo. (Funded by Cytokinetics; SEQUOIA-HCM ClinicalTrials.gov number, NCT05186818.).

Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration.

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the ß-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

The penetrance of rare variants in cardiomyopathy-associated genes: A cross-sectional approach to estimating penetrance for secondary findings.

Understanding the penetrance of pathogenic variants identified as secondary findings (SFs) is of paramount importance with the growing availability of genetic testing. We estimated penetrance through large-scale analyses of individuals referred for diagnostic sequencing for hypertrophic cardiomyopathy (HCM; 10,400 affected individuals, 1,332 variants) and dilated cardiomyopathy (DCM; 2,564 affected individuals, 663 variants), using a cross-sectional approach comparing allele frequencies against reference populations (293,226 participants from UK Biobank and gnomAD). We generated updated prevalence estimates for HCM (1:543) and DCM (1:220). In aggregate, the penetrance by late adulthood of rare, pathogenic variants (23% for HCM, 35% for DCM) and likely pathogenic variants (7% for HCM, 10% for DCM) was substantial for dominant cardiomyopathy (CM). Penetrance was significantly higher for variant subgroups annotated as loss of function or ultra-rare and for males compared to females for variants in HCM-associated genes. We estimated variant-specific penetrance for 316 recurrent variants most likely to be identified as SFs (found in 51% of HCM- and 17% of DCM-affected individuals). 49 variants were observed at least ten times (14% of affected individuals) in HCM-associated genes. Median penetrance was 14.6% (±14.4% SD). We explore estimates of penetrance by age, sex, and ancestry and simulate the impact of including future cohorts. This dataset reports penetrance of individual variants at scale and will inform the management of individuals undergoing genetic screening for SFs. While most variants had low penetrance and the costs and harms of screening are unclear, some individuals with highly penetrant variants may benefit from SFs.

Exploring the Connection Between Relaxed Myosin States and the Anrep Effect.

The Anrep effect is an adaptive response that increases left ventricular contractility following an acute rise in afterload. Although the mechanistic origin remains undefined, recent findings suggest a two-phase activation of resting myosin for contraction, involving strain-sensitive and posttranslational phases. We propose that this mobilization represents a transition among the relaxed states of myosin-specifically, from the super-relaxed (SRX) to the disordered-relaxed (DRX)-with DRX myosin ready to participate in force generation. This hypothesis offers a unified explanation that connects myosin's SRX-DRX equilibrium and the Anrep effect as parts of a singular phenomenon. We underscore the significance of this equilibrium in modulating contractility, primarily studied in the context of hypertrophic cardiomyopathy, the most common inherited cardiomyopathy associated with diastolic dysfunction, hypercontractility, and left ventricular hypertrophy. As we posit that the cellular basis of the Anrep effect relies on a two-phased transition of myosin from the SRX to the contraction-ready DRX configuration, any dysregulation in this equilibrium may result in the pathological manifestation of the Anrep phenomenon. For instance, in hypertrophic cardiomyopathy, hypercontractility is linked to a considerable shift of myosin to the DRX state, implying a persistent activation of the Anrep effect. These valuable insights call for additional research to uncover a clinical Anrep fingerprint in pathological states. Here, we demonstrate through noninvasive echocardiographic pressure-volume measurements that this fingerprint is evident in 12 patients with hypertrophic obstructive cardiomyopathy before septal myocardial ablation. This unique signature is characterized by enhanced contractility, indicated by a leftward shift and steepening of the end-systolic pressure-volume relationship, and a prolonged systolic ejection time adjusted for heart rate, which reverses post-procedure. The clinical application of this concept has potential implications beyond hypertrophic cardiomyopathy, extending to other genetic cardiomyopathies and even noncongenital heart diseases with complex etiologies across a broad spectrum of left ventricular ejection fractions.

Deficiency of Transcription Factor Sp1 Contributes to Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disorder. However, the pathogenesis of HCM, especially its nongenetic mechanisms, remains largely unclear. Transcription factors are known to be involved in various biological processes including cell growth. We hypothesized that SP1 (specificity protein 1), the first purified TF in mammals, plays a role in the cardiomyocyte growth and cardiac hypertrophy of HCM. METHODS: Cardiac-specific conditional knockout of Sp1 mice were constructed to investigate the role of SP1 in the heart. The echocardiography, histochemical experiment, and transmission electron microscope were performed to analyze the cardiac phenotypes of cardiac-specific conditional knockout of Sp1 mice. RNA sequencing, chromatin immunoprecipitation sequencing, and adeno-associated virus experiments in vivo were performed to explore the downstream molecules of SP1. To examine the therapeutic effect of SP1 on HCM, an SP1 overexpression vector was constructed and injected into the mutant allele of Myh6 R404Q/+ (Myh6 c. 1211C>T) HCM mice. The human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with HCM were used to detect the potential therapeutic effects of SP1 in human HCM. RESULTS: The cardiac-specific conditional knockout of Sp1 mice developed a typical HCM phenotype, displaying overt myocardial hypertrophy, interstitial fibrosis, and disordered myofilament. In addition, Sp1 knockdown dramatically increased the cell area of hiPSC-CMs and caused intracellular myofibrillar disorganization, which was similar to the hypertrophic cardiomyocytes of HCM. Mechanistically, Tuft1 was identified as the key target gene of SP1. The hypertrophic phenotypes induced by Sp1 knockdown in both hiPSC-CMs and mice could be rescued by TUFT1 (tuftelin 1) overexpression. Furthermore, SP1 overexpression suppressed the development of HCM in the mutant allele of Myh6 R404Q/+ mice and also reversed the hypertrophic phenotype of HCM hiPSC-CMs. CONCLUSIONS: Our study demonstrates that SP1 deficiency leads to HCM. SP1 overexpression exhibits significant therapeutic effects on both HCM mice and HCM hiPSC-CMs, suggesting that SP1 could be a potential intervention target for HCM.

Filamin C is Essential for mammalian myocardial integrity.

FLNC, encoding filamin C, is one of the most mutated genes in dilated and hypertrophic cardiomyopathy. However, the precise role of filamin C in mammalian heart remains unclear. In this study, we demonstrated Flnc global (FlncgKO) and cardiomyocyte-specific knockout (FlnccKO) mice died in utero from severely ruptured ventricular myocardium, indicating filamin C is required to maintain the structural integrity of myocardium in the mammalian heart. Contrary to the common belief that filamin C acts as an integrin inactivator, we observed attenuated activation of ß1 integrin specifically in the myocardium of FlncgKO mice. Although deleting ß1 integrin from cardiomyocytes did not recapitulate the heart rupture phenotype in Flnc knockout mice, deleting both ß1 integrin and filamin C from cardiomyocytes resulted in much more severe heart ruptures than deleting filamin C alone. Our results demonstrated that filamin C works in concert with ß1 integrin to maintain the structural integrity of myocardium during mammalian heart development.

Missense mutations in the central domains of cardiac myosin binding protein-C and their potential contribution to hypertrophic cardiomyopathy.

Myosin binding protein-C (MyBP-C) is a multidomain protein that regulates muscle contraction. Mutations in MYBPC3, the gene encoding for the cardiac variant (henceforth called cMyBP-C), are amongst the most frequent causes of hypertrophic cardiomyopathy. Most mutations lead to a truncated version of cMyBP-C, which is most likely unstable. However, missense mutations have also been reported, which tend to cluster in the central domains of the cMyBP-C molecule. This suggests that these central domains are more than just a passive spacer between the better characterized N- and C-terminal domains. Here, we investigated the potential impact of four different missense mutations, E542Q, G596R, N755K, and R820Q, which are spread over the domains C3 to C6, on the function of MyBP-C on both the isolated protein level and in cardiomyocytes in vitro. Effect on domain stability, interaction with thin filaments, binding to myosin, and subcellular localization behavior were assessed. Our studies show that these missense mutations result in slightly different phenotypes at the molecular level, which are mutation specific. The expected functional readout of each mutation provides a valid explanation for why cMyBP-C fails to work as a brake in the regulation of muscle contraction, which eventually results in a hypertrophic cardiomyopathy phenotype. We conclude that missense mutations in cMyBP-C must be evaluated in context of their domain localization, their effect on interaction with thin filaments and myosin, and their effect on protein stability to explain how they lead to disease.

2024 AHA/ACC/AMSSM/HRS/PACES/SCMR Guideline for the Management of Hypertrophic Cardiomyopathy: A Report of the American Heart Association/American College of Cardiology Joint Committee on Clinical Practice Guidelines.

AIM: The "2024 AHA/ACC/AMSSM/HRS/PACES/SCMR Guideline for the Management of Hypertrophic Cardiomyopathy" provides recommendations to guide clinicians in the management of patients with hypertrophic cardiomyopathy. METHODS: A comprehensive literature search was conducted from September 14, 2022, to November 22, 2022, encompassing studies, reviews, and other evidence on human subjects that were published in English from PubMed, EMBASE, the Cochrane Library, the Agency for Healthcare Research and Quality, and other selected databases relevant to this guideline. Additional relevant studies, published through May 23, 2023, during the guideline writing process, were also considered by the writing committee and added to the evidence tables, where appropriate. STRUCTURE: Hypertrophic cardiomyopathy remains a common genetic heart disease reported in populations globally. Recommendations from the "2020 AHA/ACC Guideline for the Diagnosis and Treatment of Patients With Hypertrophic Cardiomyopathy" have been updated with new evidence to guide clinicians.

Meta-Analysis of Penetrance and Systematic Review on Transition to Disease in Genetic Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is characterized by unexplained left ventricular hypertrophy and is classically caused by pathogenic or likely pathogenic variants (P/LP) in genes encoding sarcomere proteins. Not all subclinical variant carriers will manifest clinically overt disease because penetrance (proportion of sarcomere or sarcomere-related P/LP variant carriers who develop disease) is variable, age dependent, and not reliably predicted. METHODS: A systematic search of the literature was performed. We used random-effects generalized linear mixed model meta-analyses to contrast the cross-sectional prevalence and penetrance of sarcomere or sarcomere-related genes in 2 different contexts: clinically-based studies on patients and families with HCM versus population or community-based studies. Longitudinal family/clinical studies were additionally analyzed to investigate the rate of phenotypic conversion from subclinical to overt HCM during follow-up. RESULTS: In total, 455 full-text manuscripts and articles were assessed. In family/clinical studies, the prevalence of sarcomere variants in patients diagnosed with HCM was 34%. The penetrance across all genes in nonproband relatives carrying P/LP variants identified during cascade screening was 57% (95% CI, 52%-63%), and the mean age at HCM diagnosis was 38 years (95% CI, 36%-40%). Penetrance varied from ≈32% for MYL3 (myosin light chain 3) to ≈55% for MYBPC3 (myosin-binding protein C3), ≈60% for TNNT2 (troponin T2) and TNNI3 (troponin I3), and ≈65% for MYH7 (myosin heavy chain 7). Population-based genetic studies demonstrate that P/LP sarcomere variants are present in the background population but at a low prevalence of <1%. The penetrance of HCM in incidentally identified P/LP variant carriers was also substantially lower at ≈11%, ranging from 0% in Atherosclerosis Risk in Communities to 18% in UK Biobank. In longitudinal family studies, the pooled phenotypic conversion across all genes was 15% over an average of ≈8 years of follow-up, starting from a mean of ≈16 years of age. However, short-term gene-specific phenotypic conversion varied between ≈12% for MYBPC3 and ≈23% for MYH7. CONCLUSIONS: The penetrance of P/LP variants is highly variable and influenced by currently undefined and context-dependent genetic and environmental factors. Additional longitudinal studies are needed to improve our understanding of true lifetime penetrance in families and in the community and to identify drivers of the transition from subclinical to overt HCM.

Effect of Mavacamten in Women Compared With Men With Obstructive Hypertrophic Cardiomyopathy: Insights From EXPLORER-HCM.

BACKGROUND: Compared with men, women with hypertrophic cardiomyopathy (HCM) have a higher incidence of heart failure and worse outcomes. We investigated baseline clinical and echocardiographic characteristics and response to mavacamten among women compared with men in the EXPLORER-HCM study (Clinical Study to Evaluate Mavacamten [MYK-461] in Adults With Symptomatic Obstructive Hypertrophic Cardiomyopathy). METHODS: A prespecified post hoc analysis of sex from the blinded, randomized EXPLORER-HCM trial of mavacamten versus placebo in symptomatic patients with obstructive HCM was performed. Baseline characteristics were compared with t tests for continuous variables (expressed as mean values) and χ2 tests for categorical variables. Prespecified primary, secondary, and exploratory end points and echocardiographic measurements from baseline to end of treatment (week 30) were analyzed with ANCOVA for continuous end points and a generalized linear model with binomial distribution for binary end points, with adjustment for each outcome's baseline value, New York Heart Association class, ß-blocker use, and ergometer type. RESULTS: At baseline, women (n=102) were older (62 years versus 56 years; P<0.0001), had lower peak oxygen consumption (16.7 mL·kg-1·min-1 versus 21.3 mL·kg-1·min-1; P<0.0001), were more likely to be assigned New York Heart Association class III (42% versus 17%; P<0.0001), had worse health status (Kansas City Cardiomyopathy Questionnaire-Clinical Summary Score 64 versus 75; P<0.0001), and had higher baseline plasma NT-proBNP (N-terminal pro-B-type natriuretic peptide) levels (1704 ng/L versus 990 ng/L; P=0.004) than men (n=149). After 30 weeks of mavacamten treatment, similar improvements were observed in women and men in the primary composite end point (percentage difference on mavacamten versus placebo, 22% versus 19%, respectively; P=0.759) and in the secondary end points of change in postexercise left ventricular outflow tract gradient (-42.4 mm Hg versus -33.6 mm Hg; P=0.348), change in peak oxygen consumption (1.2 mL·kg-1·min-1 versus 1.6 mL·kg-1·min-1; P=0.633), and percentage achieving ≥1 New York Heart Association class improvement (41% versus 28%; P=0.254). However, women had greater improvement in health status (Kansas City Cardiomyopathy Questionnaire-Clinical Summary Score 14.8 versus 6.1; P=0.026) and in the exploratory end point of NT-proBNP levels (-1322 ng/L versus -649 ng/L; P=0.0008). CONCLUSIONS: Although at baseline women with symptomatic obstructive HCM enrolled in EXPLORER-HCM were older and had worse heart failure and health status than men, treatment with mavacamten resulted in similar improvements in the primary and most secondary EXPLORER-HCM end points and greater improvements in health status and NT-proBNP. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03470545.

FARS2 Deficiency Causes Cardiomyopathy by Disrupting Mitochondrial Homeostasis and the Mitochondrial Quality Control System.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a common heritable heart disease. Although HCM has been reported to be associated with many variants of genes involved in sarcomeric protein biomechanics, pathogenic genes have not been identified in patients with partial HCM. FARS2 (the mitochondrial phenylalanyl-tRNA synthetase), a type of mitochondrial aminoacyl-tRNA synthetase, plays a role in the mitochondrial translation machinery. Several variants of FARS2 have been suggested to cause neurological disorders; however, FARS2-associated diseases involving other organs have not been reported. We identified FARS2 as a potential novel pathogenic gene in cardiomyopathy and investigated its effects on mitochondrial homeostasis and the cardiomyopathy phenotype. METHODS: FARS2 variants in patients with HCM were identified using whole-exome sequencing, Sanger sequencing, molecular docking analyses, and cell model investigation. Fars2 conditional mutant (p.R415L) or knockout mice, fars2-knockdown zebrafish, and Fars2-knockdown neonatal rat ventricular myocytes were engineered to construct FARS2 deficiency models both in vivo and in vitro. The effects of FARS2 and its role in mitochondrial homeostasis were subsequently evaluated using RNA sequencing and mitochondrial functional analyses. Myocardial tissues from patients were used for further verification. RESULTS: We identified 7 unreported FARS2 variants in patients with HCM. Heart-specific Fars2-deficient mice presented cardiac hypertrophy, left ventricular dilation, progressive heart failure accompanied by myocardial and mitochondrial dysfunction, and a short life span. Heterozygous cardiac-specific Fars2R415L mice displayed a tendency to cardiac hypertrophy at age 4 weeks, accompanied by myocardial dysfunction. In addition, fars2-knockdown zebrafish presented pericardial edema and heart failure. FARS2 deficiency impaired mitochondrial homeostasis by directly blocking the aminoacylation of mt-tRNAPhe and inhibiting the synthesis of mitochondrial proteins, ultimately contributing to an imbalanced mitochondrial quality control system by accelerating mitochondrial hyperfragmentation and disrupting mitochondrion-related autophagy. Interfering with the mitochondrial quality control system using adeno-associated virus 9 or specific inhibitors mitigated the cardiac and mitochondrial dysfunction triggered by FARS2 deficiency by restoring mitochondrial homeostasis. CONCLUSIONS: Our findings unveil the previously unrecognized role of FARS2 in heart and mitochondrial homeostasis. This study may provide new insights into the molecular diagnosis and prevention of heritable cardiomyopathy as well as therapeutic options for FARS2-associated cardiomyopathy.

Progressive liver, kidney, and heart degeneration in children and adults affected by TULP3 mutations.

Organ fibrosis is a shared endpoint of many diseases, yet underlying mechanisms are not well understood. Several pathways governed by the primary cilium, a sensory antenna present on most vertebrate cells, have been linked with fibrosis. Ciliopathies usually start early in life and represent a considerable disease burden. We performed massively parallel sequencing by using cohorts of genetically unsolved individuals with unexplained liver and kidney failure and correlated this with clinical, imaging, and histopathological analyses. Mechanistic studies were conducted with a vertebrate model and primary cells. We detected bi-allelic deleterious variants in TULP3, encoding a critical adaptor protein for ciliary trafficking, in a total of 15 mostly adult individuals, originating from eight unrelated families, with progressive degenerative liver fibrosis, fibrocystic kidney disease, and hypertrophic cardiomyopathy with atypical fibrotic patterns on histopathology. We recapitulated the human phenotype in adult zebrafish and confirmed disruption of critical ciliary cargo composition in several primary cell lines derived from affected individuals. Further, we show interaction between TULP3 and the nuclear deacetylase SIRT1, with roles in DNA damage repair and fibrosis, and report increased DNA damage ex vivo. Transcriptomic studies demonstrated upregulation of profibrotic pathways with gene clusters for hypertrophic cardiomyopathy and WNT and TGF-ß signaling. These findings identify variants in TULP3 as a monogenic cause for progressive degenerative disease of major organs in which affected individuals benefit from early detection and improved clinical management. Elucidation of mechanisms crucial for DNA damage repair and tissue maintenance will guide novel therapeutic avenues for this and similar genetic and non-genomic diseases.

Metabolic profiling of aortic stenosis and hypertrophic cardiomyopathy identifies mechanistic contrasts in substrate utilization.

Aortic stenosis (AS) and hypertrophic cardiomyopathy (HCM) are distinct disorders leading to left ventricular hypertrophy (LVH), but whether cardiac metabolism substantially differs between these in humans remains to be elucidated. We undertook an invasive (aortic root, coronary sinus) metabolic profiling in patients with severe AS and HCM in comparison with non-LVH controls to investigate cardiac fuel selection and metabolic remodeling. These patients were assessed under different physiological states (at rest, during stress induced by pacing). The identified changes in the metabolome were further validated by metabolomic and orthogonal transcriptomic analysis, in separately recruited patient cohorts. We identified a highly discriminant metabolomic signature in severe AS in all samples, regardless of sampling site, characterized by striking accumulation of long-chain acylcarnitines, intermediates of fatty acid transport across the inner mitochondrial membrane, and validated this in a separate cohort. Mechanistically, we identify a downregulation in the PPAR-α transcriptional network, including expression of genes regulating fatty acid oxidation (FAO). In silico modeling of ß-oxidation demonstrated that flux could be inhibited by both the accumulation of fatty acids as a substrate for mitochondria and the accumulation of medium-chain carnitines which induce competitive inhibition of the acyl-CoA dehydrogenases. We present a comprehensive analysis of changes in the metabolic pathways (transcriptome to metabolome) in severe AS, and its comparison to HCM. Our results demonstrate a progressive impairment of ß-oxidation from HCM to AS, particularly for FAO of long-chain fatty acids, and that the PPAR-α signaling network may be a specific metabolic therapeutic target in AS.

iPSC-Based Modeling of Variable Clinical Presentation in Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. Our understanding of the genetic bases and pathogenic mechanisms underlying HCM has improved significantly in the recent past, but the combined effect of various pathogenic gene variants and the influence of genetic modifiers in disease manifestation are very poorly understood. Here, we set out to investigate genotype-phenotype relationships in 2 siblings with an extensive family history of HCM, both carrying a pathogenic truncating variant in the MYBPC3 gene (p.Lys600Asnfs*2), but who exhibited highly divergent clinical manifestations. METHODS: We used a combination of induced pluripotent stem cell (iPSC)-based disease modeling and CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing to generate patient-specific cardiomyocytes (iPSC-CMs) and isogenic controls lacking the pathogenic MYBPC3 variant. RESULTS: Mutant iPSC-CMs developed impaired mitochondrial bioenergetics, which was dependent on the presence of the mutation. Moreover, we could detect altered excitation-contraction coupling in iPSC-CMs from the severely affected individual. The pathogenic MYBPC3 variant was found to be necessary, but not sufficient, to induce iPSC-CM hyperexcitability, suggesting the presence of additional genetic modifiers. Whole-exome sequencing of the mutant carriers identified a variant of unknown significance in the MYH7 gene (p.Ile1927Phe) uniquely present in the individual with severe HCM. We finally assessed the pathogenicity of this variant of unknown significance by functionally evaluating iPSC-CMs after editing the variant. CONCLUSIONS: Our results indicate that the p.Ile1927Phe variant of unknown significance in MYH7 can be considered as a modifier of HCM expressivity when found in combination with truncating variants in MYBPC3. Overall, our studies show that iPSC-based modeling of clinically discordant subjects provides a unique platform to functionally assess the effect of genetic modifiers.

Slower Calcium Handling Balances Faster Cross-Bridge Cycling in Human MYBPC3 HCM.

BACKGROUND: The pathogenesis of MYBPC3-associated hypertrophic cardiomyopathy (HCM) is still unresolved. In our HCM patient cohort, a large and well-characterized population carrying the MYBPC3:c772G>A variant (p.Glu258Lys, E258K) provides the unique opportunity to study the basic mechanisms of MYBPC3-HCM with a comprehensive translational approach. METHODS: We collected clinical and genetic data from 93 HCM patients carrying the MYBPC3:c772G>A variant. Functional perturbations were investigated using different biophysical techniques in left ventricular samples from 4 patients who underwent myectomy for refractory outflow obstruction, compared with samples from non-failing non-hypertrophic surgical patients and healthy donors. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and engineered heart tissues (EHTs) were also investigated. RESULTS: Haplotype analysis revealed MYBPC3:c772G>A as a founder mutation in Tuscany. In ventricular myocardium, the mutation leads to reduced cMyBP-C (cardiac myosin binding protein-C) expression, supporting haploinsufficiency as the main primary disease mechanism. Mechanical studies in single myofibrils and permeabilized muscle strips highlighted faster cross-bridge cycling, and higher energy cost of tension generation. A novel approach based on tissue clearing and advanced optical microscopy supported the idea that the sarcomere energetics dysfunction is intrinsically related with the reduction in cMyBP-C. Studies in single cardiomyocytes (native and hiPSC-derived), intact trabeculae and hiPSC-EHTs revealed prolonged action potentials, slower Ca2+ transients and preserved twitch duration, suggesting that the slower excitation-contraction coupling counterbalanced the faster sarcomere kinetics. This conclusion was strengthened by in silico simulations. CONCLUSIONS: HCM-related MYBPC3:c772G>A mutation invariably impairs sarcomere energetics and cross-bridge cycling. Compensatory electrophysiological changes (eg, reduced potassium channel expression) appear to preserve twitch contraction parameters, but may expose patients to greater arrhythmic propensity and disease progression. Therapeutic approaches correcting the primary sarcomeric defects may prevent secondary cardiomyocyte remodeling.

EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.

BACKGROUND: Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. METHODS: We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. RESULTS: Screening of 157 kinase inhibitors in wild-type (N=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (N=24 HCM and N=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (N=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. CONCLUSIONS: We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.

A novel gene-trap line reveals the dynamic patterns and essential roles of cysteine and glycine-rich protein 3 in zebrafish heart development and regeneration.

Mutations in cysteine and glycine-rich protein 3 (CSRP3)/muscle LIM protein (MLP), a key regulator of striated muscle function, have been linked to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, the roles of CSRP3 in heart development and regeneration are not completely understood. In this study, we characterized a novel zebrafish gene-trap line, gSAIzGFFM218A, which harbors an insertion in the csrp3 genomic locus, heterozygous fish served as a csrp3 expression reporter line and homozygous fish served as a csrp3 mutant line. We discovered that csrp3 is specifically expressed in larval ventricular cardiomyocytes (CMs) and that csrp3 deficiency leads to excessive trabeculation, a common feature of CSRP3-related HCM and DCM. We further revealed that csrp3 expression increased in response to different cardiac injuries and was regulated by several signaling pathways vital for heart regeneration. Csrp3 deficiency impeded zebrafish heart regeneration by impairing CM dedifferentiation, hindering sarcomere reassembly, and reducing CM proliferation while aggravating apoptosis. Csrp3 overexpression promoted CM proliferation after injury and ameliorated the impairment of ventricle regeneration caused by pharmacological inhibition of multiple signaling pathways. Our study highlights the critical role of Csrp3 in both zebrafish heart development and regeneration, and provides a valuable animal model for further functional exploration that will shed light on the molecular pathogenesis of CSRP3-related human cardiac diseases.