Protein Turnover and Quality Control of Cardiac Myosin Binding Protein C in Hypertrophic Cardiomyopathy

Abstract

The cardiac sarcomere is a complex matrix of molecular machinery responsible for contraction of the heart. Proper contractile function relies on maintenance of rigid stoichiometry of myofilament proteins and multiple protein quality control (PQC) pathways. Though PQC is undeniably essential to sarcomere protein homeostasis (proteostasis), there are many gaps in our knowledge of factors that regulate turnover of sarcomere proteins. Mutations in many of these proteins cause hypertrophic cardiomyopathy (HCM), which is often designated a “sarcomeropathy.” HCM and is characterized by thickening of the left ventricular free wall and intraventricular septum, myocardial fibrosis, and diastolic dysfunction. The most commonly mutated gene in HCM is cardiac myosin binding protein C (MYBPC3). The primary pathogenic mechanisms associated with MYBPC3 mutations remain unresolved. PQC and protein turnover are central to two hypotheses with the most supportive evidence: haploinsufficiency of MYBPC3 in the sarcomere, and proteotoxicity of truncated MYBPC3 protein products. However, very little is known about how MYBPC3 interacts with PQC networks in either physiological or pathological conditions. The goal of this thesis was to investigate pathogenic mechanisms associated with different MYBPC3 mutations, explore the roles of protein homeostasis and turnover with respect to these mechanisms, and identify PQC factors which are involved in MYBPC3 turnover. Using affinity purification-mass spectrometry, we identified several molecular chaperones as potential novel interactors with MYBPC3, including αB-crystallin and HSP27, and the inducible and constitutive isoforms of the ubiquitous heat shock protein 70kDa family (HSP70 and HSC70, respectively). We then confirmed that HSP70 chaperones regulate proteasomal degradation of MYBPC3 by modulating their expression and activity and observing effects on MYBPC3 protein half-life in a primary cardiomyocyte culture system. This represents to our knowledge the first identification of a chaperone associated with MYBPC3. To determine the extent to which proteotoxicity of truncated MYBPC3 contributes to HCM pathogenesis in isolation from haploinsufficiency, we explored the effects of acute and chronic expression of truncated MYBPC3 on cardiomyocyte PQC using primary cell culture and a transgenic mouse model, respectively. We reported no deleterious effects of truncated MYBPC3 expression on proteostasis in vitro or in vivo. Further, chronic expression of a truncating MYBPC3 transgene in mice up to 12 months of age was not sufficient to elicit hypertrophic remodeling. These results challenge the hypothesis that truncated MYBPC3 is directly proteotoxic and suggest a “poison peptide” mechanism may not be relevant to HCM pathogenesis without concurrent MYBPC3 haploinsufficiency. Lastly, we investigated the locus-dependency of protein stability in non-truncating MYBPC3 mutations. Novel analysis of genotypes using the Sarcomeric Human Cardiomyopathy Registry (SHaRe) of HCM patients uncovered putative clusters of missense mutations in the C3, C6 and C10 domains of MYBPC3. We identified a consistent pattern in C10 mutants of lack of sarcomere incorporation and markedly rapid degradation. This was in contrast to C3 and C6 mutants, which were generally equally stable as WT MYBPC3 and localized correctly within the sarcomere. These findings demonstrate mutation locus significantly influences protein stability and turnover, and further dissect pathogenic mechanisms associated with non-truncating mutations. With these studies, we have moved toward clarifying pathogenic mechanisms in MYBPC3-linked HCM, which will inform future development of targeted interventions for patients with different genotypes. Furthermore, we have identified new potential therapeutic targets to restore normal stoichiometry to haploinsufficient sarcomeres, and contributed to our understanding of the enigmatic process of sarcomere protein quality control.PHDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/149802/1/glaziera_1.pd