Regulation of the Super-Relaxed State of Myosin by Cardiac Myosin Binding Protein-C

In this thesis, I investigate the role the sarcomeric protein, cardiac myosin binding protein-C (cMyBP-C) plays in maintaining the little-known state of relaxed myosin, the super-relaxed state (SRX). The SRX represents a population of myosin that has greatly reduced enzymatic activity. Structurally, myosins in the SRX helically fold back onto the surface of the thick filament, and are sterically inhibited from binding actin. When myosin heads are released from this interaction with the thick filament, they enter into what we term the disordered relaxed (DRX) state, where they can interact with the actin filament. Therefore, the relative populations of the SRX and DRX states are important for both modulating contractility and the energetic requirements of the sarcomere. Experiments were carried out in genetic mouse models, as well as human cardiac tissues. In Chapter 3, I show that the SRX is significantly reduced in mice that express no cMyBP-C, through genetic ablation. Chapter 4 builds on these findings, using human hypertrophic cardiomyopathy (HCM) samples containing mutations in the cMyBP-C gene. These patients exhibited a decreased SRX, while HCM samples with no mutation were unaffected compared to donor hearts, further implicating the importance of cMyBP-C in maintaining the helical order of myosin. Chapter 5 investigates the role of the N-terminus of cMyBP-C in maintaining the SRX. The first model had the first 271 amino acids removed. While the actin binding domain(s) reside within this fragment, the location of myosin binding domains is unknown. There was no change in the SRX state in these mice, suggesting that the domains for myosin binding are downstream from this deleted fragment of cMyBP-C. The other models investigated the role of cMyBP-C phosphorylation, by replacement of phosphorylatable serines with phospho-mimetic or –ablated amino acids. Results show that phosphorylation of cMyBP-C releases myosin heads from the thick filament, showing that the N-terminal of cMyBP-C regulates the SRX. Overall, the work presented in this thesis provide a substantial contribution to understanding the SRX state – a topic that has gained significant interest of recent times. However, considerable work remains to elucidate the complete role of this novel state of myosin in normal physiological functioning