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

Abstract

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.

Figure 1

Intensity traces along the equator from X-ray patterns of untreated (- PKA) and treated (+ PKA) WT and cMyBP-C knockout (KO) skinned myocardium.

Figure 2

a) Ratio of intensities of the 1,1 and 1,0 equatorial X-ray reflections (I₁₁/I₁₀) from WT or cMyBP-C knockout (KO) skinned myocardium with and without PKA treatment. (*) Asterisks denote significant difference in I₁₁/I₁₀ with PKA treatment, p<0.05. b)d₁₀ lattice spacing from WT and KO with and without PKA treatment. (†) Crosses denote significant difference in d₁₀, p<0.02. NS indicates no significant difference following PKA treatment.

Figure 3

Phosphorylation status of myofibrillar proteins from WT and cMyBP-C knockout (KO) myocardial samples with (+) and without (−) PKA treatment was assessed following exposure to X-ray beam, as shown in this representative SDS-PAGE. SR indicates SYPRO Ruby-stained gel for relative abundance of total proteins. PQ indicates Pro-Q Diamond-stained gel specific for relative abundance of phosphorylated proteins.

Figure 4

Basal and PKA-induced phosphorylation levels of the myofibrillar proteins cMyBP-C and cTnI were determined in WT and cMyBP-C knockout (KO) myocardial samples with (+) and without (−) PKA treatment following X-ray diffraction experiments. Phosphorylation status was determined by dividing Pro-Q Diamond-stained intensity by SYPRO Ruby-stained intensity. Lanes containing KO samples (−) had no visible bands in the cMyBP-C region. (*) Asterisks denote significant difference in phosphorylation status of cMyBP-C or cTnI from WT and KO with and without PKA treatment. There was no difference between basal or PKA-induced phosphorylation levels of cTnI when comparing KO and WT samples.