Complete mitochondrial genomes of patients from Thailand with cardiovascular diseases.

Several previous studies have reported that both variation and haplogroups of mitochondrial (mt) DNA were associated with various kinds of diseases, including cardiovascular diseases, in different populations, but such studies have not been carried out in Thailand. Here, we sequenced complete mtDNA genomes from 82 patients diagnosed with three types of cardiovascular disease, i.e., Hypertrophic Cardiomyopathy (HCM) (n = 26), Long Q-T Syndrome (LQTS) (n = 7) and Brugada Syndrome (BrS) (n = 49) and compared these with 750 previously published mitogenome sequences from interviewed normal individuals as a control group. Both patient and control groups are from the same geographic region of northeastern Thailand. We found 9, 2, and 5 novel mutations that were not both damaging and deleterious in HCM, LQTS, and BrS patients, respectively. Haplogroup R9c was significantly associated with HCM (P = 0.0032; OR = 62.42; 95%CI = 6.892-903.4) while haplogroup M12b was significantly associated with LQTS (P = 0.0039; OR = 32.93; 95% CI = 5.784-199.6). None of the haplogroups was found to be significantly associated with BrS. A significantly higher density of mtDNA variants in the rRNA genes was found in patients with HCM and BrS (P < 0.001) than in those with LQTS or the control group. Effects of detected SNPs in either protein coding or tRNA genes of all the mitogenome sequences were also predicted. Interestingly, three SNPs in two tRNA genes (MT-TA m.5618T>C and m.5631G>A heteroplasmic variants in two BrS patients and MT-TQ m.4392C>T novel homoplasmic variant in a HCM patient) were predicted to alter tRNA secondary structure, possibly leading to abnormal tRNA function.

Metabolic remodeling and calcium handling abnormality in induced pluripotent stem cell-derived cardiomyocytes in dilated phase of hypertrophic cardiomyopathy with MYBPC3 frameshift mutation.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder characterized by left ventricular hypertrophy and diastolic dysfunction, and increases the risk of arrhythmias and heart failure. Some patients with HCM develop a dilated phase of hypertrophic cardiomyopathy (D-HCM) and have poor prognosis; however, its pathogenesis is unclear and few pathological models exist. This study established disease-specific human induced pluripotent stem cells (iPSCs) from a patient with D-HCM harboring a mutation in MYBPC3 (c.1377delC), a common causative gene of HCM, and investigated the associated pathophysiological mechanisms using disease-specific iPSC-derived cardiomyocytes (iPSC-CMs). We confirmed the expression of pluripotent markers and the ability to differentiate into three germ layers in D-HCM patient-derived iPSCs (D-HCM iPSCs). D-HCM iPSC-CMs exhibited disrupted myocardial sarcomere structures and an increased number of damaged mitochondria. Ca2+ imaging showed increased abnormal Ca2+ signaling and prolonged decay time in D-HCM iPSC-CMs. Cell metabolic analysis revealed increased basal respiration, maximal respiration, and spare-respiratory capacity in D-HCM iPSC-CMs. RNA sequencing also showed an increased expression of mitochondrial electron transport system-related genes. D-HCM iPSC-CMs showed abnormal Ca2+ handling and hypermetabolic state, similar to that previously reported for HCM patient-derived iPSC-CMs. Although further studies are required, this is expected to be a useful pathological model for D-HCM.

MYH7 R453C induced cardiac remodelling via activating TGF-ß/Smad2/3, ERK1/2 and Nox4/ROS/NF-κB signalling pathways.

Hypertrophic cardiomyopathy (HCM) is a monogenic cardiac disorder commonly induced by sarcomere gene mutations. However, the mechanism for HCM is not well defined. Here, we generated transgenic MYH7 R453C and MYH6 R453C piglets and found both developed typical cardiac hypertrophy. Unexpectedly, we found serious fibrosis and cardiomyocyte loss in the ventricular of MYH7 R453C, not MYH6 R453C piglets, similar to HCM patients. Then, RNA-seq analysis and western blotting identified the activation of ERK1/2 and PI3K-Akt pathways in MYH7 R453C. Moreover, we observed an increased expression of fetal genes and an excess of reactive oxygen species (ROS) in MYH7 R453C piglet models, which was produced by Nox4 and subsequently induced inflammatory response. Additionally, the phosphorylation levels of Smad2/3, ERK1/2 and NF-kB p65 proteins were elevated in cardiomyocytes with the MYH7 R453C mutation. Furthermore, epigallocatechin gallate, a natural bioactive compound, could be used as a drug to reduce cell death by adjusting significant downregulation of the protein expression of Bax and upregulated Bcl-2 levels in the H9C2 models with MYH7 R453C mutation. In conclusion, our study illustrated that TGF-ß/Smad2/3, ERK1/2 and Nox4/ROS pathways have synergistic effects on cardiac remodelling and inflammation in MYH7 R453C mutation.

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.

Genetic Mutations and Mitochondrial Redox Signaling as Modulating Factors in Hypertrophic Cardiomyopathy: A Scoping Review.

Hypertrophic cardiomyopathy (HCM) is a heart condition characterized by cellular and metabolic dysfunction, with mitochondrial dysfunction playing a crucial role. Although the direct relationship between genetic mutations and mitochondrial dysfunction remains unclear, targeting mitochondrial dysfunction presents promising opportunities for treatment, as there are currently no effective treatments available for HCM. This review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Extension for Scoping Reviews guidelines. Searches were conducted in databases such as PubMed, Embase, and Scopus up to September 2023 using "MESH terms". Bibliographic references from pertinent articles were also included. Hypertrophic cardiomyopathy (HCM) is influenced by ionic homeostasis, cardiac tissue remodeling, metabolic balance, genetic mutations, reactive oxygen species regulation, and mitochondrial dysfunction. The latter is a common factor regardless of the cause and is linked to intracellular calcium handling, energetic and oxidative stress, and HCM-induced hypertrophy. Hypertrophic cardiomyopathy treatments focus on symptom management and complication prevention. Targeted therapeutic approaches, such as improving mitochondrial bioenergetics, are being explored. This includes coenzyme Q and elamipretide therapies and metabolic strategies like therapeutic ketosis. Understanding the biomolecular, genetic, and mitochondrial mechanisms underlying HCM is crucial for developing new therapeutic modalities.

Clinical exome sequencing unravels the diverse spectrum of genetic heterogeneity and genotype-phenotype correlations in hypertrophic cardiomyopathy.

BACKGROUND: Catalogues of pathogenic genetic mutations in hypertrophic cardiomyopathy (HCM) are disproportionately small when compared to that of the size of the population with South Asian ancestry and their collective increased risk of heart disease. METHODS: We conducted clinical exome sequencing of 200 HCM patients to identified cardiomyopathy-associated genetic mutations. The clinical and echocardiographic characteristics of genotype-positive and genotype-negative patients were compared, and the likelihood of detecting a positive genetic test result was evaluated. Allelic burden analysis was done to compare the minor allele frequencies (MAF) of the pathogenic or likely pathogenic (P/LP) variants and variants of uncertain significance (VUSs) identified in the cohort against various population genomics databases. RESULTS: The genetic yield was 40% for P/LP variants, with MYBPC3 and MYH7 as the predominant sarcomere genes. Younger age-at-diagnosis, family history of HCM, asymmetric hypertrophic (ASH) pattern, the ratio of the interventricular septum to posterior wall thickness (IVS/PW ratio), left atrial (LA) dimensions, severe mitral regurgitation grade (MR grade), late gadolinium enhancement (LGE) detected fibrosis and absence of hypertension were associated with an increased likelihood of HCM-associated variants. Patients who experienced ventricular tachycardia and premature cardiovascular death were significantly likely to carry MYBPC3 or loss-of-function variants. LA and interventricular septal (IVS) dimensions were associated with MYH7 variants. The rare variant burden for P/LP variants and VUSs was significantly enriched in HCM cases compared to population controls. CONCLUSION: Our study provides a comprehensive evaluation of HCM-associated genetic mutations from an Indian population. The identified genotype-phenotype associations could improve the yield of targeted genetic testing in HCM.

Viral myocarditis in combination with genetic cardiomyopathy as a cause of sudden death. An autopsy series.

Sudden cardiac death (SCD) is a major public health issue worldwide. In the young (< 40 years of age), genetic cardiomyopathies and viral myocarditis, sometimes in combination, are the most frequent, but underestimated, causes of SCD. Molecular autopsy is essential for prevention. Several studies have shown an association between genetic cardiomyopathies and viral myocarditis, which is probably underestimated due to insufficient post-mortem investigations. We report on four autopsy cases illustrating the pathogenesis of these combined pathologies. In two cases, a genetic hypertrophic cardiomyopathy was diagnosed in combination with Herpes Virus Type 6 (HHV6) and/or Parvovirus-B19 (PVB19) in the heart. In the third case, autopsy revealed a dilated cardiomyopathy and virological analyses revealed acute myocarditis caused by three viruses: PVB19, HHV6 and Epstein-Barr virus. Genetic analyses revealed a mutation in the gene coding for desmin. The fourth case illustrated a channelopathy and a PVB19/HHV6 coinfection. Our four cases illustrate the highly probable deleterious role of cardiotropic viruses in the occurrence of SCD in subjects with genetic cardiomyopathies. We discuss the pathogenetic link between viral myocarditis and genetic cardiomyopathy. Molecular autopsy is essential in prevention of these SCD, and a close collaboration between cardiologists, pathologists, microbiologists and geneticians is mandatory.

Survival analysis and gender differences in hypertrophic cardiomyopathy proband patients referred for genetic testing.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is believed to have low overall mortality rate, that could be influenced by gender, particularly among probands. We aimed to evaluate the survival rates and possible gender differences in a homogeneous cohort of HCM proband patients, referred for genetic testing, from the same geographical area, without differences in medical care access nor clinical referral pathways. METHODS: we compared the mortality rates of a cohort of consecutive HCM probands referred for genetic testing (2000-2022), from a Spanish region (xxx1) with a centralized genetic testing pathway, with its control reference population by Ederer II method. Gender differences were analyzed. RESULTS: Among the 649 HCM probands included in this study, there were significantly more men than women (61.3% vs 38.7, p < 0.05), with an earlier diagnosis (53.5 vs 61.1 years old, p < 0.05). Clinical evolution or arrhythmogenic HCM profile did no show no significant gender differences. Mean follow up was 9,8 years ±6,6 SD (9,9 ± 7 vs 9,6 ± 6,1, p = 0.59). No statistically significant differences in observed mortality, expected survival and excess mortality were found in the general HCM proband cohort. However, we found a significant excess mortality in female probands with HCM. No additional differences in analysis by genetic status were identified. CONCLUSION: Expected survival in our HCM probands did not differ from its reference population. However, despite no gender differences in phenotype severity were identified, proband HCM women did present a diagnosis delay and worse mortality outcomes.

A therapeutic leap: how myosin inhibitors moved from cardiac interventions to skeletal muscle myopathy solutions.

The myosin inhibitor mavacamten has transformed the management of obstructive hypertrophic cardiomyopathy (HCM) by targeting myosin ATPase activity to mitigate cardiac hypercontractility. This therapeutic mechanism has proven effective for patients with HCM independent of having a primary gene mutation in myosin. In this issue of the JCI, Buvoli et al. report that muscle hypercontractility is a mechanism of pathogenesis underlying muscle dysfunction in Laing distal myopathy, a disorder characterized by mutations altering the rod domain of ß myosin heavy chain. The authors performed detailed physiological, molecular, and biomechanical analyses and demonstrated that myosin ATPase inhibition can correct a large extent of muscle abnormalities. The findings offer a therapeutic avenue for Laing distal myopathy and potentially other myopathies. This Commentary underscores the importance of reevaluating myosin activity's role across myopathies in general for the potential development of targeted myosin inhibitors to treat skeletal muscle disorders.

Relationship Between Genotype Status and Clinical Outcome in Hypertrophic Cardiomyopathy.

BACKGROUND: The genetic basis of hypertrophic cardiomyopathy (HCM) is complex, and the relationship between genotype status and clinical outcome is incompletely resolved. METHODS AND RESULTS: We assessed a large international HCM cohort to define in contemporary terms natural history and clinical consequences of genotype. Consecutive patients (n=1468) with established HCM diagnosis underwent genetic testing. Patients with pathogenic (or likely pathogenic) variants were considered genotype positive (G+; n=312; 21%); those without definite disease-causing mutations (n=651; 44%) or variants of uncertain significance (n=505; 35%) were considered genotype negative (G-). Patients were followed up for a median of 7.8 years (interquartile range, 3.5-13.4 years); HCM end points were examined by cumulative event incidence. Over follow-up, 135 (9%) patients died, 33 from a variety of HCM-related causes. After adjusting for age, all-cause and HCM-related mortality did not differ between G- versus G+ patients (hazard ratio [HR], 0.78 [95% CI, 0.46-1.31]; P=0.37; HR, 0.93 [95% CI, 0.38-2.30]; P=0.87, respectively). Adverse event rates, including heart failure progression to class III/IV, heart transplant, or heart failure death, did not differ (G- versus G+) when adjusted for age (HR, 1.20 [95% CI, 0.63-2.26]; P=0.58), nor was genotype independently associated with sudden death event risk (HR, 1.39 [95% CI, 0.88-2.21]; P=0.16). In multivariable analysis, age was the only independent predictor of all-cause and HCM-related mortality, heart failure progression, and sudden death events. CONCLUSIONS: In this large consecutive cohort of patients with HCM, genotype (G+ or G-) was not a predictor of clinical course, including all-cause and HCM-related mortality and risk for heart failure progression or sudden death. G+ status should not be used to dictate clinical management or predict outcome in HCM.

Myosin-Catalyzed ATP Hydrolysis in the Presence of Disease-Causing Mutations: Mavacamten as a Way to Repair Mechanism.

Hypertrophic cardiomyopathy is one of the most common forms of genetic cardiomyopathy. Mavacamten is a first-in-class myosin modulator that was identified via activity screening on the wild type, and it is FDA-approved for the treatment of obstructive hypertrophic cardiomyopathy (HCM). The drug selectively binds to the cardiac ß-myosin, inhibiting myosin function to decrease cardiac contractility. Though the drug is thought to affect multiple steps of the myosin cross-bridge cycle, its detailed mechanism of action is still under investigation. Individual steps in the overall cross-bridge cycle must be queried to elucidate the full mechanism of action. In this study, we utilize the rare-event method of transition path sampling to generate reactive trajectories to gain insights into the action of the drug on the dynamics and rate of the ATP hydrolysis step for human cardiac ß-myosin. We study three known HCM causative myosin mutations: R453C, P710R, and R712L to observe the effect of the drug on the alterations caused by these mutations in the chemical step. Since the crystal structure of the drug-bound myosin was not available at the time of this work, we created a model of the drug-bound system utilizing a molecular docking approach. We find a significant effect of the drug in one case, where the actual mechanism of the reaction is altered from the wild type by mutation. The drug restores both the rate of hydrolysis to the wildtype level and the mechanism of the reaction. This is a way to check the effect of the drug on untested mutations.

Characteristics and outcomes associated with sarcomere mutations in patients with hypertrophic cardiomyopathy: A systematic review and meta-analysis.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is an inherited heart disease that can lead to sudden cardiac death. Impact of genetic testing for the prognosis and treatment of patients with HCM needs to be improved. We conducted a systematic review and meta-analysis to investigate the characteristics and outcomes associated with sarcomere genotypes in index patients with HCM. METHODS: A systematic search was conducted in Medline, Embase, and Cochrane Library up to Dec 31, 2023. Data on clinical characteristics, morphological and imaging features, outcomes and interventions were collected from published studies and pooled using a random-effects meta-analysis. RESULTS: A total of 30 studies with 10,825 HCM index patients were included in the pooled analyses. The frequency of sarcomere genes in HCM patients was 41%. Sarcomere mutations were more frequent in women (p < 0.00001), and were associated with lower body mass index (26.1 ± 4.7 versus 27.5 ± 4.3; p = 0.003) and left ventricular ejection fraction (65.7% ± 10.1% vs. 67.1% ± 8.6%; p = 0.03), less apical hypertrophy (6.5% vs. 20.1%; p < 0.0001) and left ventricular outflow tract obstruction (29.1% vs. 33.2%; p = 0.03), greater left atrial volume index (43.6 ± 21.1 ml/m2 vs. 37.3 ± 13.0 ml/m2; p = 0.02). Higher risks of ventricular tachycardia (23.4% vs. 14.1%; p < 0.0001), syncope (18.3% vs. 10.9%; p = 0.01) and heart failure (17.3% vs. 14.6%; p = 0.002) were also associated with sarcomere mutations. CONCLUSIONS: Sarcomere mutations are more frequent in women, and are associated with worse clinical characteristics and poor outcomes.

Target population for a selective cardiac myosin inhibitor in hypertrophic obstructive cardiomyopathy: Real-life estimation from the French register of hypertrophic cardiomyopathy (REMY).

BACKGROUND: The efficacy of current pharmacological therapies in hypertrophic cardiomyopathy is limited. A cardiac myosin inhibitor, mavacamten, has recently been approved as a first-in-class treatment for symptomatic hypertrophic obstructive cardiomyopathy. AIMS: To assess the profile and burden of cardiac myosin inhibitor candidates in the hypertrophic cardiomyopathy prospective Register of hypertrophic cardiomyopathy (REMY) held by the French Society of Cardiology. METHODS: Data were collected at baseline and during follow-up from patients with hypertrophic cardiomyopathy enrolled in REMY by the three largest participating centres. RESULTS: Among 1059 adults with hypertrophic cardiomyopathy, 461 (43.5%) had obstruction; 325 (30.7%) of these were also symptomatic, forming the "cardiac myosin inhibitor candidates" group. Baseline features of this group were: age 58±15years; male sex (n=196; 60.3%); diagnosis-to-inclusion delay 5 (1-12)years; maximum wall thickness 20±6mm; left ventricular ejection fraction 69±6%; family history of hypertrophic cardiomyopathy or sudden cardiac death (n=133; 40.9%); presence of a pathogenic sarcomere gene mutation (n=101; 31.1%); beta-blocker or verapamil treatment (n=304; 93.8%), combined with disopyramide (n=28; 8.7%); and eligibility for septal reduction therapy (n=96; 29%). At the end of a median follow-up of 66 (34-106) months, 319 (98.2%) were treated for obstruction (n=43 [13.2%] received disopyramide), 46 (14.2%) underwent septal reduction therapy and the all-cause mortality rate was 1.9/100 person-years (95% confidence interval 1.4-2.6) (46 deaths). Moreover, 41 (8.9%) patients from the initial hypertrophic obstructive cardiomyopathy group became eligible for a cardiac myosin inhibitor. CONCLUSIONS: In this cohort of patients with hypertrophic cardiomyopathy selected from the REMY registry, one third were eligible for a cardiac myosin inhibitor.

Generation of induced pluripotent stem cells from an individual with early onset and severe hypertrophic cardiomyopathy linked to MYBPC3: c.772G > A mutation.

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in the MYPBC3 gene, which encodes the cardiac myosin-binding protein C (cMyBP-C). Most pathogenic variants in MYPBC3 are either nonsense mutations or result in frameshifts, suggesting that the primary disease mechanism involves reduced functional cMyBP-C protein levels within sarcomeres. However, a subset of MYPBC3 variants are missense mutations, and the molecular mechanisms underlying their pathogenicity remain elusive. Upon in vitro differentiation into cardiomyocytes, induced pluripotent stem cells (iPSCs) derived from HCM patients represent a valuable resource for disease modeling. In this study, we generated two iPSC lines from peripheral blood mononuclear cells (PBMCs) of a female with early onset and severe HCM linked to the MYBPC3: c.772G > A variant. Although this variant was initially classified as a missense mutation, recent studies indicate that it interferes with splicing and results in a frameshift. The generated iPSC lines exhibit a normal karyotype and display hallmark characteristics of pluripotency, including the ability to undergo trilineage differentiation. These novel iPSCs expand the existing repertoire of MYPBC3-mutated cell lines, broadening the spectrum of resources for exploring how diverse mutations induce HCM. They additionally offer a platform to study potential secondary genetic elements contributing to the pronounced disease severity observed in this individual.

Unveiling MiRNA-124 as a biomarker in hypertrophic cardiomyopathy: An innovative approach using machine learning and intelligent data analysis.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a widespread hereditary cardiac pathology characterized by thickened heart walls and rearrangement of cardiomyocytes. Despite extensive research, the mechanisms underlying HCM development remain poorly understood, impeding the development of effective therapeutic and diagnostic strategies. Recent studies have suggested a polygenic nature of HCM development alongside monogenic forms. Transcriptomic profiling is a valuable tool for investigating such diseases. In this study, we propose a novel approach to study regulatory microRNAs (miRNAs) in the context of HCM, utilizing state-of-the-art data analysis tools. METHODS AND RESULTS: Our method involves applying the Monte Carlo simulation and machine learning algorithm to transcriptomic data to generate high-capacity classifiers for HCM. From these classifiers, we extract key genes crucial for their performance, resulting in the identification of 16 key genes. Subsequently, we narrow down the pool of miRNAs by selecting those that may target the greatest number of key genes within the best models. We particularly focused on miR-124-3p, which we validated to have an association with HCM on an independent dataset. Subsequent investigation of its function revealed involvement of miR-124-3p in the RhoA signaling pathway. CONCLUSIONS: In this study we propose a new approach to analyze transcriptomic data to search for microRNAs associated with a disease. Using this approach for transcriptomic profiling data of patients with HCM, we identified miR-124-3p as a potential regulator of the RhoA signaling pathway in the pathogenesis of HCM.

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.