Autoinhibition of cMyBP-C by its middle domains

Cardiac myosin binding protein-C (cMyBP-C) is a sarcomere regulatory protein consisting of 11 well-folded immunoglobulin-like (Ig-like) and fibronectin type-III domains with the individual domains numbered C0-C10. Despite progress in understanding the functions of the N' and C'-terminal ends of the protein, our understanding of the functional effects of the middle domains (C3-C4-C5-C6-C7) is still limited. Here we aimed to determine the functional significance of the middle domains by replacing endogenous cMyBP-C with recombinant proteins with and without the middle domains using our "cut and paste" SpyC3 mouse model. Specifically, we deleted domains C3-C7 or substituted these domains with unrelated Ig-like domains from titin to behave as inert "spacer" domains. Replacement with the spacer constructs resulted in a significant increase in myofilament calcium sensitivity, an almost instantaneous redevelopment of tension after a slack re-stretch protocol, and altered stretch activation responses, suggesting that the middle domains are functionally relevant and normally exert inhibitory effects on force development. We also investigated the significance of a potentially flexible linker between domains C4 and C5 and a unique 28 amino acid loop insertion in C5. Whereas deletion of the C5 loop had no effect on force, deletion of the linker between C4 and C5 had comparable effects to deletion of domains C3-C7. Taken together, these data indicate that the middle domains play an important role in limiting the activating effects of the C0-C2 domains and that the C4C5 linker contributes to these effects.