Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biomedical Engineering (ENAS)
First Advisor
Campbell, Stuart
Abstract
Hypertrophic Cardiomyopathy (HCM) and Dilated Cardiomyopathy (DCM) are two very divergent diseases which often have poor prognosis and are major causes of sudden cardiac death. In HCM, the left ventricular wall stiffens and thickens, making it difficult for the heart to pump blood efficiently. In DCM, the left ventricular chamber becomes enlarged while the walls become thinner and weaker, being unable to pump blood. When HCM or DCM happens as a result of an inherited mutation, it is said to be familial and can be passed down from one generation to the next. Missense mutations in alpha-tropomyosin (TPM1), a regulatory protein of the thin filament, has been shown to have the one of the highest odds ratios for both HCM and DCM. Improving our understanding of how the disease mechanism propagates in TPM1 mutations can inform potential drug targets as well as better disease management strategies. The first aim of this work focuses on studying two TPM1 variants E62Q and E54K that are known to be pathogenic. Previous studies on TPM1 mutations E62Q (HCM) and E54K (DCM) have shown divergent changes in phenotype including changes in cardiomyocyte morphology, altered calcium sensitivities, changes in isometric force production and impaired length dependent activation. However, there is a lack of understanding regarding the precise mechanisms through which these point mutations bring about observed phenotypic diversity. To address this gap in our understanding, an array of possible different mechanisms was chosen that could contribute to altered actomyosin binding and generated computational simulations of both steady-state and isometric twitch behaviors for each mechanism. It was then evaluated how well each hypothesized mechanism recapitulated previously published steady state and isometric twitch behavior. The findings suggest that distinct underlying mechanisms can explain the altered mutant phenotype in each case. In E62Q, increased calcium sensitivity and hypercontractile twitch force was explained most accurately by a reduction in tropomyosin effective stiffness and an increase in the blocked-closed equilibrium constant of tropomyosin. By contrast, in E54K the pathogenic mechanism likely propagates via long-range allosteric interactions that result in an increase in the association rate of the troponin I mobile domain to tropomyosin/actin. These results were consistent with altered atomic level interactions observed in molecular dynamics simulations. These mutation-linked molecular events produce diverging alterations in gene expression that can be observed in human engineered heart tissues. Modulators of myosin activity confirm our proposed mechanisms by rescuing normal contractile behavior in accordance with predictions. The second aim uses a multiscale investigational pipeline consisting of molecular dynamics, computational modeling, in vitro motility assay (IVMA) and EHTs to identify the pathogenic mechanisms behind a TPM1 Variant of Unknown Significance (VUS) S215L that has been reported to cause HCM in families. Molecular dynamics simulations of tropomyosin on actin suggest that the S215L mutation 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 calcium sensitivity and twitch force while slowing twitch relaxation. In vitro motility experiments with thin filaments containing TPM1 S215L revealed higher calcium sensitivity compared to wildtype. 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. The third part of this work attempts to use in silico and in vitro methods to predict the impact of 4 VUS mutations for which little or no information is available. 4 VUS mutation exhibiting the most extreme molecular signatures are selected for analysis from an array of 20 VUS mutants. IVMA and adeno-virally transfected EHTs show that aberrant calcium dependent activation drives pathologic contractile function in these mutants. Retrospective analysis reveals that interaction energy between tropomyosin and troponin-I is a strong predictor of contractile function. The final two chapters in this thesis venture out of the realm of familial cardiomyopathies to study other diseases using the EHT platform, namely non-familial HCM, and clonal hematopoiesis of indeterminate potential (CHIP). We find that while we can see a clear impact of CHIP secretome on EHTs and can rescue phenotype of CHIP treated EHTs using cytokine antagonists, the EHT platform lacks the sensitivity and specificity in identifying the contractile signatures non-familial HCM. Overall, this thesis provides an insight into the molecular, transcriptomic, and contractile signatures of TPM1 mutations associated with HCM and DCM. Combined, the studies reveal progress towards our long-term goal of computational and in vitro prediction of disease states.
Recommended Citation
Halder, Saiti Srabonti, "Identifying Mechanisms of Pathogenicity in Familial Cardiomyopathy-Linked Alpha-Tropomyosin Mutations" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1351.
https://elischolar.library.yale.edu/gsas_dissertations/1351
