Skeletal Myosin Binding Protein-C Isoforms Modulate Actomyosin Contractility and are Regulated by Phosphorylation

Abstract

Myosin binding protein C (MyBP-C) is a thick filament-associated protein found in striated muscle and may regulate muscle contractility. Separate genes encode the fast and slow skeletal isoforms, and there are potential PKA phosphorylation sites in their functionally important N-terminal regions. Here, we compare mouse N-terminal fast (fC1C2) and slow (sC1C2) skeletal fragments containing the initial ∼50 aa Pro/Ala-rich domain and the C1 and C2 Ig-domains that are linked by the ∼100 aa M-domain. Of the known slow skeletal splice variants, we chose a highly expressed variant lacking the N-terminal 34-59 residue insert. To define the potential mechanisms by which skeletal MyBP-Cs affect contractility and whether these effects are modulated by PKA phosphorylation, we assessed the Ca2+-dependent motility of rabbit skeletal native thin filaments over a surface of rabbit psoas myosin in the presence of C1C2 fragments. While thin filaments were fully regulated, with no motion observed at pCa > 7 in the absence of fragments, the addition of either 0.50 μM fC1C2 or sC1C2 resulted in significant motility. This suggests that skeletal MyBP-C isoforms effectively sensitize the thin filament. Under fully activating conditions (pCa ≤ 5), sC1C2 had little effect on motility whereas fC1C2 inhibited sliding velocities by nearly 50%. Thus, these fragments differ in their modulatory capacities with fC1C2 sensitizing the thin filament to Ca2+ and inhibiting maximal velocities, while the sC1C2 variant exhibits only a single mode of contractile modulation; i.e. thin filament sensitization. Interestingly, PKA phosphorylation in the Pro/Ala (sC1C2) and M-domains (sC1C2 and fC1C2), as confirmed by mass spectrometry, reduced both fragments’ Ca2+ sensitization of the thin filament. Thus, the function of MyBP-C isoforms may be tuned to match the physiological demands of the muscle in which they are expressed.