Impact of Hypertrophic Cardiomyopathy mutations on cardiac thick filament function and regulation.

Abstract

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease, characterized byprogressive thickening of the left ventricular walls, impaired relaxation and potential for sudden cardiac death. This disease has been termed a “disease of the sarcomere” due to the prevelance of HCM mutations linked to sarcomeric proteins. Approximately eighty percent of pathogenic/likely pathogenic HCM mutations have been linked to two thick filament proteins, particularly in cardiac myosin binding protein-C (cMyBP-C) and beta-myosin heavy chain (β-MHC). Currently, there is no cure for HCM, only management of symptoms and disease progression, left ventricular obstruction surgery, or heart transplantation. As such, there is great need to better understand the pathological mechanisms that underly specific HCM mutations in order to better inform development of targeted therapeutics. For this project, we have chosen to study two HCM mutations and how they impact thick filament function and regulation: a) the MYBPC3-c.772G>A mutation due to its notable prevalence in the Tuscany region of Italy and b) the β-MHC R403Q mutation due to its severe disease phenotype and high penetrance. For both of these investigations, we have turned, in part, to engineering and validating human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model systems of the mutation to use for thorough mechanistic studies. The first presented work focuses on engineering a hiPSC-CM model of the MYBPC3-c.772G>A mutation, rigorously validated findings against human myectomy samples, to dissect its effects on sarcomere function, particularly via exon skipping induced cMyBP-C haploinsufficiency. The impact of cMyBP-C haploinsufficiency on myofibril mechanics revealed a complex interplay between myosin nucleotide handling, changes in cMyBP-C phospohrylation, and enhanced myosin recruitment. The second body of work focuses on how the thick filament of cardiac muscle is regulated in native porcine tissue expressing the human isoform of myosin and how this regulation can be purturbed by the R403Q HCM mutation in β-MHC, leading to altered function and disease progression. For this project, the technique of high-resolution single-molecule ATP tracking to probe the biochemical states of myosin under relaxed conditions, was newly established for cardiac myofibrils isolated from porcine and human myocardium. Using this method and a transgenic porcine model of the R403Q mutation, we were able to demonstrate that the mutation increases the ATPase activity of myosins within specific zones of the sarcomere where the regulatory protein cMyBP-C does not exist, potentially to aid in maintaining homeostasis of energetic efficiency. Using our porcine model and a new CRISPR/Cas9 gene edited hiPSC-CM model harboring the heterozygous R403Q mutation, we demonstrated impaired relaxation kinetics, greater energetic cost of contraction, and disorder within the structure of the thick filament, likely associated with modifications at the cross-bridge interface. Under intact conditions, these mechanisms were associated with hypercontractility and imapired relaxation. This work enhances our mechanistic understanding of thick filament regulation in HCM and underscores potential therapeutic targets within the thick filament for ameliorating disease progression.