Skeletal Myosin-Binding Protein C Isoforms Differentially Regulate Fast- and Slow-Twitch Skeletal Muscle Function

Abstract

Skeletal myosin-binding protein C (MyBP-C) is a myosin thick filament-associated protein that is encoded by two paralogous genes (slow-type (MYBPC1) and fast-type (MYBPC2)) and may regulate skeletal muscle contractility. We aimed to characterize the expression pattern, regulatory function, and phosphorylation-dependent modulation of this protein in in vitro actomyosin systems of rat slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL). By immunofluorescence imaging, slow-type MyBP-C is expressed in both EDL and SOL, while the fast-type MyBP-C is exclusive to EDL. Interestingly, these MyBP-C isoforms differentially localize within a discrete region of the sarcomere (the C-zone) that is muscle type-dependent. Mass spectrometry demonstrated expression of 3 distinct N-terminal splice variants of slow-type MyBP-C, which were present in both muscle types. When tissue-purified native thin filaments slid over tissue-purified native myosin thick filaments (containing the physiological MyBP-C compliment) in a calcium-dependent manner, only in the C-zone were thin filaments sensitized to calcium and their velocity slowed. Upon proteolysis of MyBP-C's N terminus, these modulatory capacities were attenuated, implicating this domain in MyBP-C's regulatory effects, as confirmed by in vitro motility experiments using recombinant N-terminal fragments of the various MyBP-C variant isoforms. These isoform fragments displayed differential impacts on thin filament sensitization and slowing, which were significantly reduced following PKA phosphorylation. Finally, we developed a “Monte Carlo” simulation that recapitulates thin filament trajectories observed over thick filaments by assuming that the various MyBP-C isoforms all exist within a C-zone and contribute their variant-specific modulatory impact. Thus, fast- and slow-twitch skeletal muscle contractility are differentially regulated by MyBP-C isoforms, and that MyBP-C function itself is, in turn, regulated by phosphorylation.