Protein kinase C‐site phosphorylation of cardiac myosin binding protein‐C decreases cross‐bridge kinetics (1081.5)

Abstract

Cardiac myosin binding protein‐C (cMyBP‐C) phosphorylation is necessary for the regulation of sarcomeric structure and myocardial contractility in vivo. Different from skeletal isoforms, cMyBP‐C has M‐domain phosphorylation motifs in which three serines, Ser273, 282 and 302, are known to be substrates for several kinases. Phosphorylation of these M‐domain sites regulates the interaction of cMyBP‐C with thick and thin myofilaments during muscle contraction. While protein kinase C (PKC) is known to phosphorylate Ser273 and Ser302, the precise functional consequences of PKC‐mediated cMyBP‐C phosphorylation are not fully characterized. Therefore, the objective of this study was to determine how PKC‐mediated phosphorylation at Ser273/302 might control the function of cMyBP‐C in the context of thick and thin myofilament regulation and sarcomere contraction. Using recombinant proteins and yeast‐2‐hybrid (Y2H) clones expressing Ser273‐Ser282‐Ser302 (cMyBP‐CWT), Ala273‐Asp282‐Ala302 (cMyBP‐CADA) or Asp273‐Ala282‐Asp302 (cMyBP‐CDAD), cosedimentation of C0C2 and Y2H of C1C2 assays with either cardiac myosin S2 region or sarcomeric actin revealed that PKC phosphorylation of Ser273/Ser302 in cMyBP‐C M‐domain is sufficient to maintain its interaction with myosin S2 region, but diminish its interaction with actin in vitro. Furthermore, mechanical studies using skinned ventricular fibers from cMyBP‐CDAD revealed that PKC phospho‐mimetic exhibits a significant decrease in active force, cross‐bridge kinetics and Mg2+‐ATPase activity and myofilament Ca2+ sensitivity in vitro, compared to the controls. Taken together, our studies demonstrated that two phosphorylatable (by PKC) serines in cMyBP‐C M‐domain enable the interaction between cMyBP‐C protein and myosin S2, but diminishes its interaction with actin, thus affecting the ability of this protein to regulate sarcomere contractility