Protein kinase A-mediated phosphorylation of cMyBP-C increases proximity of myosin heads to actin in resting myocardium

Protein kinase A-mediated (PKA) phosphorylation of cardiac myosin binding protein C (cMyBP-C) accelerates the kinetics of cross-bridge cycling and may relieve the tether-like constraint of myosin heads imposed by cMyBP-C. We favor a mechanism in which cMyBP-C modulates cross-bridge cycling kinetics by regulating the proximity and interaction of myosin and actin. To test this idea, we used synchrotron low-angle x-ray diffraction to measure interthick filament lattice spacing and the equatorial intensity ratio, I-11/I-10, in skinned trabeculae isolated from wild-type and cMyBP-C null (cMyBP-C-/-) mice. In wild-type myocardium, PKA treatment appeared to result in radial or azimuthal displacement of cross-bridges away from the thick filaments as indicated by an increase ( approximately 50%) in I-11/I-10 (0.22 +/- 0.03 versus 0.33 +/- 0.03). Conversely, PKA treatment did not affect cross-bridge disposition in mice lacking cMyBP-C, because there was no difference in I-11/I-10 between untreated and PKA-treated cMyBP-C-/- myocardium (0.40 +/- 0.06 versus 0.42 +/- 0.05). Although lattice spacing did not change after treatment in wild-type ( 45.68 +/- 0.84 nm versus 45.64 +/- 0.64 nm), treatment of cMyBP-C-/- myocardium increased lattice spacing (46.80 +/- 0.92 nm versus 49.61 +/- 0.59 nm). This result is consistent with the idea that the myofilament lattice expands after PKA phosphorylation of cardiac troponin I, and when present, cMyBP-C, may stabilize the lattice. These data support our hypothesis that tethering of cross- bridges by cMyBP-C is relieved by phosphorylation of PKA sites in cMyBP-C, thereby increasing the proximity of cross- bridges to actin and increasing the probability of interaction with actin on contraction.