Search for: All records

Abstract Hypertrophic cardiomyopathy (HCM) is an inherited disorder often caused by mutations to sarcomeric genes. Many different HCM-associated TPM1 mutations have been identified but they vary in their degrees of severity, prevalence, and rate of disease progression. The pathogenicity of many TPM1 variants detected in the clinical population remains unknown. Our objective was to employ a computational modeling pipeline to assess pathogenicity of one such variant of unknown significance, TPM1 S215L, and validate predictions using experimental methods. Molecular dynamic simulations of tropomyosin on actin suggest that the S215L significantly destabilizes the blocked regulatory state while increasing flexibility of the tropomyosin chain. These changes were quantitatively represented in a Markov model of thin-filament activation to infer the impacts of S215L on myofilament function. Simulations of in vitro motility and isometric twitch force predicted that the mutation would increase Ca2+ sensitivity and twitch force while slowing twitch relaxation. In vitro motility experiments with thin filaments containing TPM1 S215L revealed higher Ca2+ sensitivity compared with wild type. Three-dimensional genetically engineered heart tissues expressing TPM1 S215L exhibited hypercontractility, upregulation of hypertrophic gene markers, and diastolic dysfunction. These data form a mechanistic description of TPM1 S215L pathogenicity that starts with disruption of the mechanical and regulatory properties of tropomyosin, leading thereafter to hypercontractility and finally induction of a hypertrophic phenotype. These simulations and experiments support the classification of S215L as a pathogenic mutation and support the hypothesis that an inability to adequately inhibit actomyosin interactions is the mechanism whereby thin-filament mutations cause HCM. 

Engineered heart tissues (EHTs) have emerged as a robust in vitro model to study cardiac physiology. Although biomimetic culture environments have been developed to better approximate in vivo conditions, currently available methods do not permit full recapitulation of the four phases of the cardiac cycle. We have developed a bioreactor which allows EHTs to undergo cyclic loading sequences that mimic in vivo work loops. EHTs cultured under these working conditions exhibited enhanced concentric contractions but similar isometric contractions compared with EHTs cultured isometrically. EHTs that were allowed to shorten cyclically in culture had increased capacity for contractile work when tested acutely. Increased work production was correlated with higher levels of mitochondrial proteins and mitochondrial biogenesis; this effect was eliminated when tissues were cyclically shortened in the presence of a myosin ATPase inhibitor. Leveraging our novel in vitro method to precisely apply mechanical loads in culture, we grew EHTs under two loading regimes prescribing the same work output but with different associated afterloads. These groups showed no difference in mitochondrial protein expression. In loading regimes with the same afterload but different work output, tissues subjected to higher work demand exhibited elevated levels of mitochondrial protein. Our findings suggest that regulation of mitochondrial mass in cultured human EHTs is potently modulated by the mechanical work the tissue is permitted to perform in culture, presumably communicated through ATP demand. Precise application of mechanical loads to engineered heart tissues in culture represents a novel in vitro method for studying physiological and pathological cardiac adaptation. NEW & NOTEWORTHY In this work, we present a novel bioreactor that allows for active length control of engineered heart tissues during extended tissue culture. Specific length transients were designed so that engineered heart tissues generated complete cardiac work loops. Chronic culture with various work loops suggests that mitochondrial mass and biogenesis are directly regulated by work output.