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 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. Significance StatementThe functional role of the middle domains of cardiac myosin binding protein-C (cMyBP-C) are poorly understood, in part due to technical challenges inherent to in vitro methods that have mainly been used to study recombinant N-terminal domains in the absence of the whole protein. Here we overcome this barrier by using a "cut and paste" approach, selectively removing and replacing the middle domains of cMyBP-C in permeabilized cardiomyocytes. Substituting the middle domains with titin Ig-like domains resulted in a large increase in myofilament calcium sensitivity, almost instantaneous redevelopment of force, and altered response to rapid stretch. Deletion of only the C4-C5 linker (11 amino acids) qualitatively resulted in the same alterations in force mechanics, albeit to a lesser magnitude. We suggest that the middle domains directly affect the regulation of cardiac muscle function by inhibiting the activating effects of the N-terminal domains of cMyBP-C.