BACKGROUND:Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart ...and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations.
METHODS:We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias.
RESULTS:Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM.
CONCLUSIONS:Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.
Classic therapeutics for ischemic heart disease are less effective in individuals with the metabolic syndrome. As the prevalence of the metabolic syndrome is increasing, better understanding of ...cardiac metabolism is needed to identify potential new targets for therapeutic intervention. Thioredoxin-interacting protein (Txnip) is a regulator of metabolism and an inhibitor of the antioxidant thioredoxins, but little is known about its roles in the myocardium. We examined hearts from Txnip-KO mice by polony multiplex analysis of gene expression and an independent proteomic approach; both methods indicated suppression of genes and proteins participating in mitochondrial metabolism. Consistently, Txnip-KO mitochondria were functionally and structurally altered, showing reduced oxygen consumption and ultrastructural derangements. Given the central role that mitochondria play during hypoxia, we hypothesized that Txnip deletion would enhance ischemia-reperfusion damage. Surprisingly, Txnip-KO hearts had greater recovery of cardiac function after an ischemia-reperfusion insult. Similarly, cardiomyocyte-specific Txnip deletion reduced infarct size after reversible coronary ligation. Coordinated with reduced mitochondrial function, deletion of Txnip enhanced anaerobic glycolysis. Whereas mitochondrial ATP synthesis was minimally decreased by Txnip deletion, cellular ATP content and lactate formation were higher in Txnip-KO hearts after ischemia-reperfusion injury. Pharmacologic inhibition of glycolytic metabolism completely abolished the protection afforded the heart by Txnip deficiency under hypoxic conditions. Thus, although Txnip deletion suppresses mitochondrial function, protection from myocardial ischemia is enhanced as a result of a coordinated shift to enhanced anaerobic metabolism, which provides an energy source outside of mitochondria.
Hypertrophic cardiomyopathy is one of the most common inherited cardiomyopathies and a leading cause of sudden cardiac death in young adults. Despite profound insights into the genetics, there is ...imperfect correlation between mutation and clinical prognosis, suggesting complex molecular cascades driving pathogenesis. To investigate this, we performed an integrated quantitative multi-omics (proteomic, phosphoproteomic, and metabolomic) analysis to illuminate the early and direct consequences of mutations in myosin heavy chain in engineered human induced pluripotent stem-cell-derived cardiomyocytes relative to late-stage disease using patient myectomies. We captured hundreds of differential features, which map to distinct molecular mechanisms modulating mitochondrial homeostasis at the earliest stages of pathobiology, as well as stage-specific metabolic and excitation-coupling maladaptation. Collectively, this study fills in gaps from previous studies by expanding knowledge of the initial responses to mutations that protect cells against the early stress prior to contractile dysfunction and overt disease.
Molecular etiologies of heart failure, an emerging cardiovascular epidemic affecting 4.7 million Americans and costing 17.8 billion health-care dollars annually, remain poorly understood. Here we ...report that an inherited human dilated cardiomyopathy with refractory congestive heart failure is caused by a dominant$Arg \rightarrow Cys$missense mutation at residue 9 (R9C) in phospholamban (PLN), a transmembrane phosphoprotein that inhibits the cardiac sarcoplasmic reticular$Ca^2+-adenosine$triphosphatase (SERCA2a) pump. Transgenic$PLN^{R9C}$mice recapitulated human heart failure with premature death. Cellular and biochemical studies revealed that, unlike wild-type PLN,$PLN^{R9C}$did not directly inhibit SERCA2a. Rather,$PLN^{R9C}$trapped protein kinase A (PKA), which blocked PKA-mediated phosphorylation of wild-type PLN and in turn delayed decay of calcium transients in myocytes. These results indicate that myocellular calcium dysregulation can initiate human heart failure-a finding that may lead to therapeutic opportunities.
Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere ...content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation. We identify that β-cardiac myosin-titin-protocostamere form an essential mechanical connection that transmits forces required to direct α-actinin-2 centripetal fiber assembly and sarcomere formation. Titin propagates diastolic traction stresses from β-cardiac myosin, but not α-cardiac myosin or non-muscle myosin II, to protocostameres during sarcomerogenesis. Ablating protocostameres or decoupling titin from protocostameres abolishes sarcomere assembly. Together these results identify the mechanical and molecular components critical for human cardiac sarcomerogenesis.
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•Full-length titin is required to initiate sarcomerogenesis in human cardiomyocytes•β-cardiac myosin (MHC-β)-generated tension drives cardiac sarcomerogenesis•Protocostameres/cell-matrix adhesions are sites of sarcomere assembly initiation•Titin couples MHC-β-generated tension to protocostameres to direct sarcomerogenesis
Chopra, Kutys, Zhang, et al. identify that dilated cardiomyopathy-causing titin truncation mutations impair early-stage sarcomerogenesis in a model of dynamic human cardiac sarcomere assembly. This work uncovers fundamental mechanisms driving sarcomere assembly and reveals a critical role for titin in connecting β-cardiac myosin-generated tension to protocostameres to initiate sarcomerogenesis.
Titin, an important protein in the sarcomere, is the largest human protein. This study identified mutations in the titin gene that result in a truncated protein as important causes of dilated ...cardiomyopathy.
Gene mutation is an important cause of cardiomyopathy. Mutations in eight sarcomere-protein genes cause hypertrophic cardiomyopathy, detected in 40 to 70% of patients.
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Variations in more than 40 genes, most of which encode components of the sarcomere, the cytoskeleton, or the nuclear lamina, have been shown or posited to cause dilated cardiomyopathy.
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Clinical evaluation identifies 30 to 50% of patients with dilated cardiomyopathy as having a relative who is affected or likely to be affected,
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implicating a genetic cause. However, pathogenic mutations have been found in only 20 to 30% of patients.
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TTN,
the gene encoding titin, . . .
Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein ...interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease.
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•GATA4:TBX5 interactome in CPs is enriched in de novo variants associated with CHD•A method for scoring interactome variants identified GLYR1 as a candidate CHD gene•GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes•The GLYR1 CHD variant disrupted interaction with GATA4 and impaired cardiogenesis
The integration of human protein-protein interactome networks of endogenous transcription factors known to be involved in cardiac malformations with the largest whole-exome-sequencing dataset of trios with congenital heart disease identified an enrichment of rare de novo variants among interactome proteins, pointing to candidate disease genes.
Peripartum cardiomyopathy shares clinical features with idiopathic dilated cardiomyopathy, a disorder associated with mutations in more than 40 genes. This study shows that mutations in some of these ...genes, notably
TTN,
also have a strong association with this condition.
Peripartum cardiomyopathy is marked by the development of maternal systolic heart failure late in pregnancy or early in the postpartum period.
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The incidence varies from 1 in 100 to 1 in 300 in geographic hot spots, including Nigeria and Haiti, to 1 in 1000 to 1 in 4000 in Europe and the United States. The strongest known risk factors are the presence of preeclampsia, twin gestation, and advanced maternal age. Among patients with peripartum cardiomyopathy, heart failure can resolve but often does not: rates of death of 5 to 10% are common, and 4% of cardiac transplantations in the . . .
Substantial progress has been made recently in understanding the genetic basis of cardiomyopathy. Cardiomyopathies with known genetic cause include hypertrophic (HCM), dilated (DCM), restrictive ...(RCM), arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) and left ventricular noncompaction (LVNC). HCM, DCM, and RCM have been recognized as distinct clinical entities for decades, whereas ARVD/C and LVNC are relative newcomers to the field. Hence the clinical and genetic knowledge for each cardiomyopathy varies, as do the recommendations and strength of evidence.
RATIONALE:Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) in combination with CRISPR/Cas9 genome editing provide unparalleled opportunities to study cardiac biology and ...disease. However, sarcomeres, the fundamental units of myocyte contraction, are immature and nonlinear in hiPSC-CMs, which technically challenge accurate functional interrogation of contractile parameters in beating cells. Furthermore, existing analysis methods are relatively low-throughput, indirectly assess contractility, or only assess well-aligned sarcomeres found in mature cardiac tissues.
OBJECTIVE:We aimed to develop an analysis platform that directly, rapidly, and automatically tracks sarcomeres in beating cardiomyocytes. The platform should assess sarcomere content, contraction and relaxation parameters, and beat rate.
METHODS AND RESULTS:We developed SarcTrack, a MatLab software that monitors fluorescently tagged sarcomeres in hiPSC-CMs. The algorithm determines sarcomere content, sarcomere length, and returns rates of sarcomere contraction and relaxation. By rapid measurement of hundreds of sarcomeres in each hiPSC-CM, SarcTrack provides large data sets for robust statistical analyses of multiple contractile parameters. We validated SarcTrack by analyzing drug-treated hiPSC-CMs, confirming the contractility effects of compounds that directly activate (CK-1827452) or inhibit (MYK-461) myosin molecules or indirectly alter contractility (verapamil and propranolol). SarcTrack analysis of hiPSC-CMs carrying a heterozygous truncation variant in the myosin-binding protein C (MYBPC3) gene, which causes hypertrophic cardiomyopathy, recapitulated seminal disease phenotypes including cardiac hypercontractility and diminished relaxation, abnormalities that normalized with MYK-461 treatment.
CONCLUSIONS:SarcTrack provides a direct and efficient method to quantitatively assess sarcomere function. By improving existing contractility analysis methods and overcoming technical challenges associated with functional evaluation of hiPSC-CMs, SarcTrack enhances translational prospects for sarcomere-regulating therapeutics and accelerates interrogation of human cardiac genetic variants.