Myosin Binding Protein C Regulates Striated Muscle Contractility

Abstract

Myosin binding protein C (MyBP-C) is a large myosin associated protein that is frequently mutated in hypertrophic cardiomyopathy (HCM). Despite a burst of research since the early 90s, our understanding of its mechanism in muscle physiology remains elusive. Recent studies have demonstrated that the cardiac isoform of MyBP-C serves as a regulator of actomyosin contractility where it functions as both a “break” and “accelerator” in in vitro motility assays. Its absence in MYBPC3-knockout mice fibers promotes hyper-contractile function indicating that it may function to slow down actomyosin kinetics.

We begin, in Chapter 3, by identifying whether genotype-phenotype relationship exists in human HCM samples with MYBPC3 mutations scattered along its sequence. No differences in fiber contractility were observed in comparing mutation types (missense vs. nonsense), or location of mutations (N- vs. C-terminus). Our findings show HCM with MyBP-C mutants behave in a similar manner to HCM without sarcomeric mutations. Moreover, we demonstrate that HCM as a whole differs quite significantly from donors, but only in the presence of calcium.

Intriguingly, upon examination of each individual mutation, the N-terminal V219 mutation located in the C1 domain clearly deviated from mutants clustered in the C3-C9 domains. This suggests that the N-terminal portion of MyBP-C, that is known to interact with actin or myosin, plays a potentially significant role in regulating contractility. As such, in Chapter 4, our focus shifted from complex fiber contractility to a simplified in vitro motility system. The latter enables direct interrogation of how the N-terminus of MyBP-C regulates actomyosin kinetics on the molecular level.

Chapter 4 utilised N-terminal MyBP-C fragments with engineered site-directed mutagenesis that has been previously shown to ablate actin- or myosin-binding. Surprisingly, these fragments containing the actin- or myosin-binding mutations by and large prevented the thin filament from switching on at low calcium, coupled with more inhibition at higher calcium concentrations. These mutants also reduced thin filament binding affinity but myosin affinity was unaffected. Overall, these findings suggest that the N-terminus of MyBP-C interacts strongly with the thin filament, and mutations in this area will probably diminish this interaction.

The role MyBP-C plays in regulating cardiac physiology is complex. In addition to mutations, the expression of skeletal isoforms of MyBP-C was also previously identified in the failing heart. Chapter 5 investigated the role of fast and slow skeletal MyBP-C in relation to the known function of the cardiac N-terminus. In a cardiac based system, the slow isoform behaves solely as an accelerator while the fast isoform operates predominately as a brake – in stark contrast to the dual-modal regulation of the cardiac isoform. Furthermore, Chapter 6 demonstrated that PKA-phosphorylation reduced the thin filament’s sensitivity to calcium. Phosphorylation seemed to hold little to no effect on the slow MyBP-C fragment whereas inhibition was lost upon phosphorylation of the fast fragments.

Overall there is a considerable amount of work ahead of us to resolve the function of MyBP-C in muscle. The recent shift towards skeletal MyBP-C function is an interesting avenue to explore how small structural differences can ultimately alter its response to and regulation of the contractile system. Moreover, our understanding of this family of thick filament regulators poses as a potential therapeutic target for contractile-system based heart and skeletal muscle failure.