Skeletal Myosin-Binding Protein C Modulates Actomyosin Contractility in an Isoform-Dependent Manner

Abstract

Myosin-binding protein C (MyBP-C) is a sarcomeric protein localized to discrete regions (C-zones) of myosin thick filaments in all striated muscles. Unlike cardiac muscle with its specific MyBP-C isoform, mass spectrometry shows that rat slow-twitch soleus (SOL) muscle expresses two different slow MyBP-C isoforms (sMyBP-C), having one (sMyBP-C1) or two N-terminal inserts (sMyBP-C2), while fast-twitch extensor digitorum longus (EDL) expresses these same sMyBP-C isoforms and a fast MyBP-C isoform (fMyBP-C). Do these MyBP-C isoforms differentially regulate skeletal actomyosin contractility in a muscle-specific manner? To address this, we characterized Ca2+-regulated motion of SOL and EDL native actin-thin filaments (NTFs) over their corresponding native myosin-thick filaments, having the physiological complement of MyBP-C in the C-zone. At low calcium (pCa7.5) where NTFs should be inhibited, motion occurred within the C-zone of both SOL and EDL native thick filaments. Whereas, at high Ca2+ (pCa5), SOL and EDL NTFs had initial fast velocities followed by ∼30% slower velocities within the C-zone. These data suggest that one or more of these MyBP-C can sensitize NTFs to Ca2+ and modulate contractility at high Ca2+. To determine how each isoform contributes to these modulatory capacities, we bacterially-expressed N-terminal fragments (sMyBP-C1, sMyBP-C2, fMyBP-C) and assessed their individual impact on SOL or EDL NTF motility over their respective monomeric myosins. For the EDL where all three MyBP-Cs are expressed, at pCa7.5 sMyBP-C1 was most effective at activating NTFs, while at pCa5 all three reduced velocity by ∼50%. For SOL, where only sMyBP-C isoforms are expressed, at pCa7.5 sMyBP-C1 activated NTFs while at pCa5 sMyBP-C2 reduced velocity ∼40%. Our data suggest that the distinctive mixture of MyBP-C isoforms expressed in the SOL and EDL is matched to the muscle's physiological demands. Each MyBP-C isoform possesses specific modulatory capacities that may be called upon under different contractile states. Specifically, to fine-tune actomyosin contractility within the C-zone by Ca2+-sensitizing the thin filament and/or modulating velocity at maximal activation.