Mutations In Sarcomeric Proteins Research Articles

Modeling cardiomyocyte signaling and metabolism predicts genotype-to-phenotype mechanisms in hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (HCM) is a significant precursor of heart failure and sudden cardiac death, primarily caused by mutations in sarcomeric and structural proteins. Despite the extensive research on the HCM genotype, the complex and context-specific nature of many signaling and metabolic pathways linking the HCM genotype to phenotype has hindered therapeutic advancements for patients. Here, we have developed a computational model of HCM encompassing cardiomyocyte signaling and metabolic networks and their associated interactions. Utilizing a stochastic logic-based ODE approach, we linked cardiomyocyte signaling to the metabolic network through a gene regulatory network and post-translational modifications. We validated the model against published data on activities of signaling species in the HCM context and transcriptomes of two HCM mouse models (i.e., R403Q-αMyHC and R92W-TnT). Our model predicts that HCM mutation induces changes in metabolic functions such as ATP synthase deficiency and a transition from fatty acids to carbohydrate metabolism. The model indicated major shifts in glutamine-related metabolism and increased apoptosis after HCM-induced ATP synthase deficiency. We predicted that the transcription factors STAT, SRF, GATA4, TP53, and FoxO are the key regulators of cardiomyocyte hypertrophy and apoptosis in HCM in alignment with experiments. Moreover, we identified shared (e.g., activation of PGC1α by AMPK, and FHL1 by titin) and context-specific mechanisms (e.g., regulation of Ca2+ sensitivity by titin in HCM patients) that may control genotype-to-phenotype transition in HCM across different species or mutations. We also predicted potential combination drug targets for HCM (e.g., mavacamten plus ROS inhibitors) preventing or reversing HCM phenotype (i.e., hypertrophic growth, apoptosis, and metabolic remodeling) in cardiomyocytes. This study provides new insights into mechanisms linking genotype to phenotype in familial hypertrophic cardiomyopathy and offers a framework for assessing new treatments and exploring variations in HCM experimental models.

An In Silico Cardiomyocyte Reveals the Impact of Changes in CaMKII Signalling on Cardiomyocyte Contraction Kinetics in Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is characterised by asymmetric left ventricular hypertrophy, ventricular arrhythmias, and cardiomyocyte dysfunction that may cause sudden death. HCM is associated with mutations in sarcomeric proteins and is usually transmitted as an autosomal-dominant trait. The aim of this in silico study was to assess the mechanisms that underlie the altered electrophysiological activity, contractility, regulation of energy metabolism, and crossbridge cycling in HCM at the single-cell level. To investigate this, we developed a human ventricular cardiomyocyte model that incorporates electrophysiology, metabolism, and force generation. The model was validated by its ability to reproduce the experimentally observed kinetic properties of human HCM induced by (a) remodelling of several ion channels and Ca2+-handling proteins arising from altered Ca2+/calmodulin kinase II signalling pathways and (b) increased Ca2+ sensitivity of the myofilament proteins. Our simulation showed a decreased phosphocreatine-to-ATP ratio (-9%) suggesting a negative mismatch between energy expenditure and supply. Using a spatial myofilament half-sarcomere model, we also compared the fraction of detached, weakly bound, and strongly bound crossbridges in the control and HCM conditions. Our simulations showed that HCM has more crossbridges in force-producing states than in the control condition. In conclusion, our model reveals that impaired crossbridge kinetics is accompanied by a negative mismatch between the ATP supply and demand ratio. This suggests that improving this ratio may reduce the incidence of sudden death in HCM.

Sarcomere level mechanics of the fast skeletal muscle of the medaka fish larva.

The medaka fish (Oryzias latipes) is a vertebrate model used in developmental biology and genetics. Here we explore its suitability as a model for investigating the molecular mechanisms of human myopathies caused by mutations in sarcomeric proteins. To this end, the relevant mechanical parameters of the intact skeletal muscle of wild-type medaka are determined using the transparent tail at larval stage 40. Tails were mounted at sarcomere length of 2.1 μm in a thermoregulated trough containing physiological solution. Tetanic contractions were elicited at physiological temperature (10°C-30°C) by electrical stimulation, and sarcomere length changes were recorded with nanometer-microsecond resolution during both isometric and isotonic contractions with a striation follower. The force output has been normalized for the actual fraction of the cross section of the tail occupied by the myofilament lattice, as established with transmission electron microscopy (TEM), and then for the actual density of myofilaments, as established with X-ray diffraction. Under these conditions, the mechanical performance of the contracting muscle of the wild-type larva can be defined at the level of the half-thick filament, where ∼300 myosin motors work in parallel as a collective motor, allowing a detailed comparison with the established performance of the skeletal muscle of different vertebrates. The results of this study point out that the medaka fish larva is a suitable model for the investigation of the genotype/phenotype correlations and therapeutic possibilities in skeletal muscle diseases caused by mutations in sarcomeric proteins.NEW & NOTEWORTHY The suitability of the medaka fish as a model for investigating the molecular mechanisms of human myopathies caused by mutations of sarcomeric proteins is tested by combining structural analysis and sarcomere-level mechanics of the skeletal muscle of the tail of medaka larva. The mechanical performance of the medaka muscle, scaled at the level of the myosin-containing thick filament, together with its reduced genome duplication makes this model unique for investigations of the genotype/phenotype correlations in human myopathies.

Influence of sarcomeric protein mutations on myocardial function in a 3D-disease model of human iPSC-derived bioartificial cardiac tissue

Abstract Background Peripartum cardiomyopathy (PPCM) is a rare type of cardiomyopathy that occurs in women without prior history of heart disease at later stages of pregnancy or several months after childbirth (1). The disease is characterized by lowered left ventricular ejection fraction (LVEF<45%) and can lead to heart failure if left untreated (1). Approximately 20% of PPCM patients harbor sarcomeric mutations, from which titin truncating variants (TTNtv-s) have the highest pathogenic potential (2). However, the role of such genetic defects of the contractile apparatus on PPCM pathogenesis is not well understood. Purpose The study aims to establish an in vitro 3D disease model of PPCM to elucidate the role of sarcomeric mutations, including titin mutations, in its etiology. We hypothesize, that the disease phenotype can be uncovered in vitro via the stimulation of PPCM patient-derived Bioartificial Cardiac Tissues (BCTs), utilizing pregnancy-related factors in our custom-made bioreactors (3). Methods We selected three patients carrying previously unstudied mutations in the TTN gene for the study. The iPSC reprogramming was performed using a commercial, Sendai virus-based kit. Pluripotency was confirmed via flow cytometry and immunofluorescence stainings. The presence of mutations was confirmed by exome sequencing. Cardiomyocyte differentiation (4) and BCT generation (3) were carried out via established protocols. Following maturation, BCTs were stimulated with the pregnancy-related factors, and tissue forces were measured in the bioreactors. These forces include intrinsically generated (spontaneous) and electrically induced (paced) contraction forces (mN), as well as passive forces, which correlate with tissue stiffness. Results We successfully generated two clonal induced pluripotent stem cell (iPSC) lines from each patient. We confirmed that all of the cell lines were >90% positive for pluripotency markers. After differentiation, CMs contracted spontaneously from day 7 onwards and were >95% positive for cardiac markers (SA, MF20, cTnT). The BCTs remodeled and started contracting on days 1-2 after generation, similarly to wild-type tissues in the baseline conditions. Prolactin treatment of Patient A, harboring a missense mutation in titin, did not show significant functional effects. We confirmed, that patients B and C harbor TTNtvs, and tissues from these patients show a trend of altered stiffness after stimulation with repeated mechanical stretch cycles or β-adrenergic agonist isoprenaline. Further analysis of the underlying mechanisms investigating metabolic changes, signaling pathways, and transcriptomics are ongoing. Conclusion The established iPSC and tissue engineering technologies offer a valuable tool for the development of an iPS-CM-based 3D in vitro disease model for PPCM, which can be used for risk stratification of individual and combined sarcomeric mutations and the development of tailored therapeutic strategies.

P21-Activated Kinases Present a New Drug Target for Hypertrophic Cardiomyopathy

Expert review p21-Activated Kinases Present a New Drug Target for Hypertrophic Cardiomyopathy He Yu , and Lei Ming , * Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK * Correspondence: ming.lei@pharm.ox.ac.uk Received: 17 February 2023 Accepted: 26 March 2023 Published: 21 August 2023 Abstract: Hypertrophic cardiomyopathy (HCM), primarily involving mutations in sarcomeric proteins, is the most common form of inherited heart disease and a leading cause of sudden death in young adults and athletes. HCM patients present with cardiac hypertrophy, fibrosis, and diastolic dysfunction often in a progressive manner. Despite significant progress made in understanding the molecular genetic basis of HCM, there remains a lack of effective and specific treatment for preventing disease progression in HCM. This article first provides an overview of recent progress in understanding the pathogenic basis of disease progression in HCM, in particular dysfunctional calcium handling, mitochondrial impairment, and endoplasmic reticulum stress. This article then analyses the evidence for critical roles of the multifunctional enzymes P21-activated kinase-1 and 2 (Pak1/2) in the heart and our opinion on their therapeutic value as a promising druggable target in pathological hypertrophy and associated ventricular arrhythmias.

Abstract P3123: Sex-related Differences In Cardiac Biomarkers And Ventricular Fibrosis In A Minipig Model Of Hypertrophic Cardiomyopathy

Hypertrophic Cardiomyopathy (HCM) is characterized by ventricular hypertrophy and stiffness resulting in part from interstitial fibrosis. While associated with autosomal dominant mutations in sarcomere proteins, it has been reported that males and females may have different clinical presentations and disease courses. We have previously shown that minipigs carrying the R403Q mutation in MYH7 demonstrate a phenotype that recapitulates human non-obstructive HCM with increased hypertrophy, markers of cardiac stress, and diastolic dysfunction. Under the hypothesis that sex-dependent differences in cardiac physiology can independently modulate disease phenotype, we evaluated ventricular fibrosis and cardiac biomarkers in both male and female mutant pigs. Purpose-bred Yucatan R403Q MYH7 heterozygous mutant minipigs of either sex (M: n=8, F: n=6) and wild-type littermates (M: n=4, F: n=14) were used; for the purposes of these comparisons, cloned male pigs were also included (R403Q: 8). Males were castrated, and all pigs received soybean-based feed. Circulating levels of both cardiac troponin T (cTnT) and brain natriuretic peptide (BNP), markers of cardiac injury and stress, were assessed. Interstitial fibrosis was assessed in left ventricular free wall (LV-FW) and apical tissue samples utilizing picrosirius red staining and semi-automated quantification (HALO). Male HCM pigs died earlier (<9M of age, P<0.05) and had higher serum biomarkers than females (cTnT levels (M: 143±17 vs F: 44±7 ng/L, P<0.05; BNP 1536±335 vs 4881±414 ng/L, P<0.05) Consistent with the higher cTnT levels, male HCM pigs had greater fibrosis areas than females in both LV-FW (11±3% vs 7±2%, P<0.05) and apical regions (13±1% vs. 8±2%, respectively, P<0.05). All HCM pigs had significantly increased fibrotic area vs WT controls (R403Q: 10±3% vs WT: 3±2%, P<0.05). Our findings demonstrate that despite a common genotype, female and male carriers of the HCM pathogenic R403Q mutation present different disease phenotypes, with castrated males showing greater fibrotic burden. Future studies to understand the mechanistic drivers of these differences should not only help elucidate the role of sex in HCM disease progression, but also the development of sex-informed therapies.

PO-01-158 MODELING AN ATRIAL FIBRILLATION-CAUSING TITIN MUTATION USING HUMAN IPSC-DERIVED ATRIAL CARDIOMYOCYTES

Atrial fibrillation (AF) affects more than 33 million people worldwide and, if left untreated, can lead to increased morbidity and mortality. Although AF has been classified as an ‘ion channelopathy’, next-generation sequencing has identified mutations in sarcomeric proteins like TTN, the gene encoding titin, that are associated with AF. Despite progress in understanding the genetic basis of AF, the translation of these discoveries to the bedside care of patients has been limited due to challenges in understanding the myocardial substrate for AF, especially that develops from the mutation in the structural protein. As human atrial tissue is rarely available and animal models are mechanistically limited, our goal is to model AF using mature human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) by a CRISPR-Cas9-mediated single amino acid substitution of a rare missense ethnic-specific mutation (TTN_T32756I) in an immunoglobulin (Ig)-like domain of the A-band of titin that is enriched in AF-causing pathogenic variants. Isogenic TTN_T32756I hiPSC-aCM mutants were generated using the CRISPR-Cas9 technique. Cells were matured either with metabolic conditioning or co-culturing with atrial fibroblasts in a micropatterned platform. We used confocal microscopy to assess sarcomeric integrity, MuscleMotion to assess contractility, and optical voltage mapping to assess the electrophysiological properties of TTN_T32756I hiPSC-aCMs. T32756I results in a non-conservative amino acid change, which in-silico tools have previously predicted results in an impairment in titin function. Here, we show that TTN_T32756I hiPSC-aCM mutants display aberrant contractility (Fig. 1A-C) and Ca2+ homeostasis (Fig. 1D) leading to abnormal electrophysiology (Fig. 1E-F) at the cellular level without compromising the sarcomeric integrity of the atrial cardiomyocytes (Fig. 1G-H). Our results suggest that a single amino acid change could have a profound effect on titin function, further establishing titin as a major signaling hub for contractility. Ongoing studies will not only identify the key pathways and transcription factors involved in the pathogenesis of TTN_ T32756I mutant but also uncover the impact of the mutation on atrial action potential and cardiac ion channel activity. Our study will shed new light on the mechanisms by which TTN mutation cause AF and identify novel therapeutic targets for patients carrying AF-associated rare variants in the structural genes.

Altered coronary artery function, arteriogenesis and endothelial YAP signaling in postnatal hypertrophic cardiomyopathy.

Introduction: Hypertrophic cardiomyopathy (HCM) is a cardiovascular genetic disease caused largely by sarcomere protein mutations. Gaps in our understanding exist as to how maladaptive sarcomeric biophysical signals are transduced to intra- and extracellular compartments leading to HCM progression. To investigate early HCM progression, we focused on the onset of myofilament dysfunction during neonatal development and examined cardiac dynamics, coronary vascular structure and function, and mechano-transduction signaling in mice harboring a thin-filament HCM mutation. Methods: We studied postnatal days 7-28 (P7-P28) in transgenic (TG) TG-cTnT-R92Q and non-transgenic (NTG) mice using skinned fiber mechanics, echocardiography, biochemistry, histology, and immunohistochemistry. Results: At P7, skinned myofiber bundles exhibited an increased Ca2+-sensitivity (pCa50 TG: 5.97 ± 0.04, NTG: 5.84 ± 0.01) resulting from cTnT-R92Q expression on a background of slow skeletal (fetal) troponin I and α/β myosin heavy chain isoform expression. Despite the transition to adult isoform expressions between P7-P14, the increased Ca2+- sensitivity persisted through P28 with no apparent differences in gross morphology among TG and NTG hearts. At P7 significant diastolic dysfunction was accompanied by coronary flow perturbation (mean diastolic velocity, TG: 222.5 ± 18.81mm/s, NTG: 338.7 ± 28.07mm/s) along with localized fibrosis (TG: 4.36% ± 0.44%, NTG: 2.53% ± 0.47%). Increased phosphorylation of phospholamban (PLN) was also evident indicating abnormalities in Ca2+ homeostasis. By P14 there was a decline in arteriolar cross-sectional area along with an expansion of fibrosis (TG: 9.72% ± 0.73%, NTG: 2.72% ± 0.2%). In comparing mechano-transduction signaling in the coronary arteries, we uncovered an increase in endothelial YAP expression with a decrease in its nuclear to cytosolic ratio at P14 in TG hearts, which was reversed by P28. Conclusion: We conclude that those early mechanisms that presage hypertrophic remodeling in HCM include defective biophysical signals within the sarcomere that drive diastolic dysfunction, impacting coronary flow dynamics, defective arteriogenesis and fibrosis. Changes in mechano-transduction signaling between the different cellular compartments contribute to the pathogenesis of HCM.

Changes in myosin biomechanics influence growth and maturation of iPSC-cardiomyocytes.

Hypertrophic cardiomyopathy (HCM) is characterized by hypercontractility, cardiomyocyte hypertrophy, myofibril disarray, and altered energetics. Despite the identification of >1000 mutations in sarcomeric proteins, one-third of which are in β-cardiac myosin (MYH7), the mechanism by which altered force at the level of the sarcomere is transduced into cellular hypertrophy and other phenotypes is still incompletely understood. Our biochemical studies reveal surprising heterogeneity in the impact of different MYH7 mutations on myosin force generation and ATPase activity with some mutations reducing activity at the molecular level. Using CRISPR-edited hiPSC-cardiomyocytes (hiPSC-CMs), we studied the impact of myosin HCM mutations on the generation of contractile forces, hypertrophy, myofibril organization, and mitochondrial function, and compared cellular phenotypes with the effects of the mutations on myosin biomechanics. Our studies reveal a critical role for alterations in the myosin super-relaxed state (SRX) as a mechanism for cellular hypercontractility. To validate the fidelity of our findings to human disease, we compared hiPSC-CM results to those in 26 septal myectomy samples from HCM patients and 13 donor heart controls. In both hiPSC-CMs and myectomy samples we observed fiber disarray, altered mitochondrial metabolism, and widening of the Z-disc. A genome-wide key driver analysis revealed altered expression of 52 Z-disc-related genes in patient samples; 24 Z-disc genes in hiPSC-CMs; with 14 Z-disc genes shared between the two platforms. Activation of calcineurin pro-hypertrophic signaling provides a potential link between Z-disc mechano-sensation of sarcomeric forces and downstream hypertrophic signaling, which we explore using a vinculin FRET-tension sensor to directly measure intracellular force at the Z-disc. Our multi-scale approach, combined with both patient samples and CRISPR-edited hiPSC-CMs, confirms the fidelity of our in vitro model in recapitulating human disease and increases the potential translation of our findings to develop new precision-based HCM therapies.

The β-myosin G256E mutation impairs relaxation by altering transducer stiffness.

Associations between mutations in sarcomeric proteins and familial cardiomyopathies are myriad. However, detailed structural and functional studies are required to describe the mechanistic relationship between a mutation and its pathology. To accelerate such studies, we utilize a combined computational and experimental platform to probe how mutations to b-myosin lead to familial cardiomyopathies. Here, we report results on the HCN-associated MYH7 mutation G256E, which is located in the myosin transducer. Experimentally, the specific force generation of mutant G256E myofibrils was ∼2x greater than isogenic control myofibrils. The activation rate was similarly increased. The initial slow phase relaxation kinetics of G256E myosin were significantly reduced relative to isogenic controls, indicating a slower cross-bridge detachment rate. Using stopped flow kinetics, the ATP binding rate was reduced in G256E relative to WT. Molecular dynamics simulations of the rigor and myosin.ATP states showed that G256E led to reorganization of hydrogen bonding networks within the transducer region. Enhanced sampling MD simulations modeled the rigor (nucleotide free) to post-rigor (ATP-bound) structural transition along multiple reaction coordinates. The transition required that G256E myosins surmount a greater energetic barrier that describes the binding of switch 1 to ATP, rotation of the transducer backbone, and opening of the actin binding cleft. This increased barrier is attributed to salt bridges formed by the mutant Glu residue. Based on our combined computational and experimental results, we hypothesize that G256E leads to a hypercontractile and delayed relaxation phenotype via modulation of the transducer structure. This alters communication between the nucleotide binding pocket and the actin binding surface during steps in the chemo-mechanical cycle that involve transducer motion during nucleotide release-induced strong actin binding and ATP binding-induced release from actin. Other effects on myosin structure and its activation during contraction are being investigated.

Towards automated assessment of drug efficacy on cardiomyopathic human heart function.

Structural and functional abnormalities of ventricular myocardium related to Hypertrophic and Dilated Cardiomyopathies (HCM and DCM) are commonly caused by inherited mutations in sarcomeric proteins. Automating predictions of mutation and drug effects on human cardiac muscle function could enhance early diagnosis and evaluation of response to treatments for cardiomyopathies. However, the development of automation tools that integrate results from multiple experiments, conducted under various experimental conditions at different time and spatial scales from multiple species, is a significant challenge. Such automation tools usually require large sets of training data, which are extremely limited or even nonexistent from humans, thus any information generated could be significantly inaccurate or even misleading. Recent advances in multiscale computational tools, coupled with multiple experiments, for the translation of data concerning sarcomere level molecular interactions, including the effect of drugs, to the level of cardiac muscle fibers and tissues, might provide the most relevant approach for generating databases for automation algorithms. Using the MUSICO platform for precise modeling of protein-protein interactions and Ca2+ regulation in cardiac muscle cells, coupled with the finite element (FE) solver Mexie via Mijailovich-Prodanovic surrogate model, we can simulate experimental data from various sources. These tools collectively enable quantitative assessment of the changes in human cardiac muscle contractile responses caused by HCM and DCM extending from the molecular level to whole organ function, as well as prediction of improvements of cardiac muscle function by drugs. Our simulations successfully predicted pressure and volume changes due to troponin-C mutations and the effect of drugs, disopyramide and digoxin, in improving heart function in HCM and DCM, respectively, during multiple heartbeats in human heart. These simulations can potentially provide sufficient information for physicians to evaluate the progress of disease and the efficacy of administrated therapeutics.

HCM and DCM associated TNNT2 sarcomeric variants induce nucleus remodeling in human iPSC-CMs.

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are associated with sarcomeric protein mutations in thick- and thin-filament genes, particularly in the gene TNNT2, which encodes cardiac troponin T (cTnT). Mutations in cTnT can lead to altered contractility dynamics and a high incidence of sudden cardiac death. Human-induced pluripotent stem cells cardiomyocytes (hiPSC-CMs) model offers an effective method to investigate cardiac diseases in vitro. We analyze morphological and functional alterations in two common mutations associated with HCM and DCM, cTnT I79N and cTnT R141W, respectively. We performed experiments in physiologically relevant microenvironment and in the presence of two clinically relevant drugs to treat cardiomyopathies, mavacamten and omencativ mercabil. Cellular characterization confirmed successful maturation of cardiomyocytes and altered contractility consistent with human phenotypes. Stiffer microenvironment determined potential physiological conditions of our model to the human system. In addition, cardiomyocytes under constant physical influence respond to microenvironmental changes, such as energetic metabolism, signal transduction and propagation of electrical stimuli. Through mechanotransduction, the sarcomeric contractility is translated intracellularly to other organelles, including the nucleus. We analyze the mechanical effect of altered sarcomeric contraction, due to TNNT2 mutations, on the nuclear morphology and stiffness of hiPSC-CMs. We observed altered nuclear circularity, aspect ratio and area. We also observed alterations in the nucleus stiffness and protein expression for both HCM and DCM, with opposite effects. Drug treatment showed promising results for the model to correct both contractility and nuclear stiffness. Increased or diminished contractility imposed by TNNT2 pathogenic variants can trigger nuclear remodeling, potentially compromising cellular structure and composition. This supports that sarcomeric variants associated with cardiomyopathy plays a role in cardiomyocyte mechanotransduction.

Intrafamilial Phenotypical Variability Linked to PRKAG2 Mutation-Family Case Report and Review of the Literature.

PRKAG2 syndrome (PS) is a rare, early-onset autosomal dominant phenocopy of sarcomeric hypertrophic cardiomyopathy (HCM), that mainly presents with ventricular pre-excitation, cardiac hypertrophy and progressive conduction system degeneration. Its natural course, treatment and prognosis are significantly different from sarcomeric HCM. The clinical phenotypes of PRKAG2 syndrome often overlap with HCM due to sarcomere protein mutations, causing this condition to be frequently misdiagnosed. The syndrome is caused by mutations in the gene encoding for the γ2 regulatory subunit (PRKAG2) of 5' Adenosine Monophosphate-Activated Protein Kinase (AMPK), an enzyme that modulates glucose uptake and glycolysis. PRKAG2 mutations (OMIM#602743) are responsible for structural changes of AMPK, leading to an impaired myocyte glucidic uptake, and finally causing storage cardiomyopathy. We describe the clinical and investigative findings in a family with several affected members (NM_016203.4:c.905G>A or p.(Arg302Gln), heterozygous), highlighting the various phenotypes even in the same family, and the utility of genetic testing in diagnosing PS. The particularity of this family case is represented by the fact that the index patient was diagnosed at age 16 with cardiac hypertrophy and ventricular pre-excitation while his mother, by age 42, only had Wolff-Parkinson-White syndrome, without left ventricle hypertrophy. Both the grandmother and the great-grandmother underwent pacemaker implantation at a young age because of conduction abnormalities. Making the distinction between PS and sarcomeric HCM is actionable, given the early-onset of the disease, the numerous life-threatening consequences and the high rate of conduction disorders. In patients who exhibit cardiac hypertrophy coexisting with ventricular pre-excitation, genetic screening for PRKAG2 mutations should be considered.

Hypertrophic cardiomyopathy: Mutations to mechanisms to therapies.

Hypertrophic cardiomyopathy (HCM) affects more than 1 in 500 people in the general population with an extensive burden of morbidity in the form of arrhythmia, heart failure, and sudden death. More than 25 years since the discovery of the genetic underpinnings of HCM, the field has unveiled significant insights into the primary effects of these genetic mutations, especially for the myosin heavy chain gene, which is one of the most commonly mutated genes. Our group has studied the molecular effects of HCM mutations on human β-cardiac myosin heavy chain using state-of-the-art biochemical and biophysical tools for the past 10 years, combining insights from clinical genetics and structural analyses of cardiac myosin. The overarching hypothesis is that HCM-causing mutations in sarcomere proteins cause hypercontractility at the sarcomere level, and we have shown that an increase in the number of myosin molecules available for interaction with actin is a primary driver. Recently, two pharmaceutical companies have developed small molecule inhibitors of human cardiac myosin to counteract the molecular consequences of HCM pathogenesis. One of these inhibitors (mavacamten) has recently been approved by the FDA after completing a successful phase III trial in HCM patients, and the other (aficamten) is currently being evaluated in a phase III trial. Myosin inhibitors will be the first class of medication used to treat HCM that has both robust clinical trial evidence of efficacy and that targets the fundamental mechanism of HCM pathogenesis. The success of myosin inhibitors in HCM opens the door to finding other new drugs that target the sarcomere directly, as we learn more about the genetics and fundamental mechanisms of this disease.

Genotype and Phenotype Analysis Using a Hypertrophic Cardiomyopathy-Associated Gene Panel in Turkish Cardiomyopathy Patients

Objective: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder caused by mutations in sarcomeric proteins and characterized by hypertrophy of the heart muscle. 
 Materials and Methods: In the present study, 21 patients with HCM and some of their parents were evaluated via next-generation sequencing (NGS) using a targeted panel of 17 genes. 
 Results: Pathogenic or likely pathogenic variants were detected in six patients in the genes MYH7 (p.R663C, p.A423V), MYBPC3 (p.P955fs*95, p.K301fs*31), TNNT2 (p.R154Q), and TNNI3 (p.R204C).
 Conclusion: The genotype-phenotype correlations of these variants were discussed by comparing the clinical findings with the literature. p.R204C variant in the TNNI3 gene was found to be caused restrictive cardiomyopathy for the first time in the literature.

Exploring the Potential of Symmetric Exon Deletion to Treat Non-Ischemic Dilated Cardiomyopathy by Removing Frameshift Mutations in TTN.

Non-ischemic dilated cardiomyopathy (DCM) is one of the most frequent pathologies requiring cardiac transplants. Even though the etiology of this disease is complex, frameshift mutations in the giant sarcomeric protein Titin could explain up to 25% of the familial and 18% of the sporadic cases of DCM. Many studies have shown the potential of genome editing using CRISPR/Cas9 to correct truncating mutations in sarcomeric proteins and have established the grounds for myoediting. However, these therapies are still in an immature state, with only few studies showing an efficient treatment of cardiac diseases. This publication hypothesizes that the Titin (TTN)-specific gene structure allows the application of myoediting approaches in a broad range of locations to reframe TTNtvvariants and to treat DCM patients. Additionally, to pave the way for the generation of efficient myoediting approaches for DCM, we screened and selected promising target locations in TTN. We conceptually explored the deletion of symmetric exons as a therapeutic approach to restore TTN’s reading frame in cases of frameshift mutations. We identified a set of 94 potential candidate exons of TTN that we consider particularly suitable for this therapeutic deletion. With this study, we aim to contribute to the development of new therapies to efficiently treat titinopathies and other diseases caused by mutations in genes encoding proteins with modular structures, e.g., Obscurin.

Effects of Sarcomere Activators and Inhibitors Targeting Myosin Cross-Bridges on Ca2+-Activation of Mature and Immature Mouse Cardiac Myofilaments.

We tested the hypothesis that isoform shifts in sarcomeres of the immature heart modify the effect of cardiac myosin-directed sarcomere inhibitors and activators. Omecamtiv mecarbil (OM) activates tension and is in clinical trials for the treatment of adult acute and chronic heart failure. Mavacamten (Mava) inhibits tension and is in clinical trials to relieve hypercontractility and outflow obstruction in advanced genetic hypertrophic cardiomyopathy (HCM), which is often linked to mutations in sarcomeric proteins. To address the effect of these agents in developing sarcomeres, we isolated heart fiber bundles, extracted membranes with Triton X-100, and measured tension developed over a range of Ca2+ concentrations with and without OM or Mava treatment. We made measurements in fiber bundles from hearts of adult nontransgenic (NTG) controls expressing cardiac troponin I (cTnI), and from hearts of transgenic (TG-ssTnI) mice expressing the fetal/neonatal form, slow skeletal troponin I (ssTnI). We also compared fibers from 7- and 14-day-old NTG mice expressing ssTnI and cTnI. These studies were repeated with 7- and 14-day-old transgenic mice (TG-cTnT-R92Q) expressing a mutant form of cardiac troponin T (cTnT) linked to HCM. OM increased Ca2+-sensitivity and decreased cooperative activation in both ssTnI- and cTnI-regulated myofilaments with a similar effect: reducing submaximal tension in immature and mature myofilaments. Although Mava decreased tension similarly in cTnI- and ssTnI-regulated myofilaments controlled either by cTnT or cTnT-R92Q, its effect involved a depressed Ca2+-sensitivity in the mature cTnT-R92 myofilaments. Our data demonstrate an influence of myosin and thin-filament associated proteins on the actions of myosin-directed agents such as OM and Mava. SIGNIFICANCE STATEMENT: The effects of myosin-targeted activators and inhibitors on Ca2+-activated tension in developing cardiac sarcomeres presented here provide novel, ex vivo evidence as to their actions in early-stage cardiac disorders. These studies advance understanding of the molecular mechanisms of these agents, which are important in preclinical studies employing sarcomere Ca2+-response as a screening approach. The data also inform the use of commonly immature cardiac myocytes generated from human-inducible pluripotent stem cells in screening for sarcomere activators and inhibitors.

Critical Evaluation of Current Hypotheses for the Pathogenesis of Hypertrophic Cardiomyopathy.

Hereditary hypertrophic cardiomyopathy (HCM), due to mutations in sarcomere proteins, occurs in more than 1/500 individuals and is the leading cause of sudden cardiac death in young people. The clinical course exhibits appreciable variability. However, typically, heart morphology and function are normal at birth, with pathological remodeling developing over years to decades, leading to a phenotype characterized by asymmetric ventricular hypertrophy, scattered fibrosis and myofibrillar/cellular disarray with ultimate mechanical heart failure and/or severe arrhythmias. The identity of the primary mutation-induced changes in sarcomere function and how they trigger debilitating remodeling are poorly understood. Support for the importance of mutation-induced hypercontractility, e.g., increased calcium sensitivity and/or increased power output, has been strengthened in recent years. However, other ideas that mutation-induced hypocontractility or non-uniformities with contractile instabilities, instead, constitute primary triggers cannot yet be discarded. Here, we review evidence for and criticism against the mentioned hypotheses. In this process, we find support for previous ideas that inefficient energy usage and a blunted Frank–Starling mechanism have central roles in pathogenesis, although presumably representing effects secondary to the primary mutation-induced changes. While first trying to reconcile apparently diverging evidence for the different hypotheses in one unified model, we also identify key remaining questions and suggest how experimental systems that are built around isolated primarily expressed proteins could be useful.

Understanding the molecular basis of HCM-causing mutations in cardiac myosin and cardiac myosin binding protein-C

Hypertrophic cardiomyopathy (HCM), a heritable cardiovascular disease marked by hypercontractility and abnormal thickening of heart muscle, affects up to 1 in 200 individuals. Mutations in sarcomere proteins result in HCM, but the mechanism by which these mutant proteins lead to hypercontractility and heart failure remains poorly understood, limiting clinical interventions for the same. We are currently focusing on two sarcomere proteins that together comprise ∼70% of all known HCM mutations: β-cardiac myosin and cardiac myosin binding protein-C, cMyBPC (a regulatory protein in the thick filament).

Allosteric destabilization of the super-relaxed state of cardiac myosin by hypertrophic cardiomyopathy-causing mutations

Hypertrophic cardiomyopathy (HCM), a leading cause of heart failure and sudden death, results from mutations in sarcomeric proteins. ∼35% of causative mutations affect β-cardiac myosin. It is now recognized that a large fraction of these may be affecting the conformational dynamics of myosin, and not its catalytic activity. Studies over the past decade have shifted the focus of HCM research to the evolutionarily conserved ‘super-relaxed’ (SRX) state of myosin, which is an important modulator of cardiac contractile output and energy consumption.