Protein kinase A--induced myofilament desensitization to [Ca.sup.2+] as a result of phosphorylation of cardiac myosin-binding protein C

In skinned myocardium, cyclic AMP--dependent protein kinase A (PKA)-catalyzed phosphorylation of cardiac myosin--binding protein C (cMyBP-C) and cardiac troponin I (cTnI) is associated with a reduction in the [Ca.sup.2+] responsiveness of myofilaments and an acceleration in the kinetics of cross-bridge cycling, although the respective contribution of these two proteins remains controversial. To further examine the relative roles that cTnI and cMyBP-C phosphorylation play in altering myocardial function, we determined the [Ca.sup.2+] sensitivity of force ([pCa.sub.50]) and the activation dependence of the rate of force redevelopment ([k.sub.tr]) in control and PKA-treated mouse myocardium (isolated in the presence of 2,3-butanedione monoxime) expressing: (a) phosphorylatable cTnI and cMyBP-C (wild type [WT]), (b) phosphorylatable cTnI on a cMyBP-C--null background ([cMyBP-C.sup.-/-]), (c) nonphosphorylatable cTnI with [serines.sup.23/24/43/45] and [threonine.sup.144] mutated to alanines ([cTnI.sub.Ala5]), and (d) nonphosphorylatable cTnI on a cMyBP-C-null background ([cTnI.sub.Ala5]/[cMyBP-C.sup.-/-]). Here, PKA treatment decreased [pCa.sub.50] in WT, [cTnI.sub.Ala5], and [cMyBP-C.sup.-/-] myocardium by 0.13, 0.08, and 0.09 pCa units, respectively, but had no effect in [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium. In WT and [cTnI.sub.Ala5] myocardium, PKA treatment also increased [k.sub.tr] at submaximal levels of activation; however, PKA treatment did not have an effect on [k.sub.tr] in [cMyBP-C.sup.-/-] or [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium. In addition, reconstitution of [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium with recombinant cMyBP-C restored the effects of PKA treatment on [pCa.sub.50] and [k.sub.tr] reported in [cTnI.sub.Ala5] myocardium. Collectively, these results indicate that the attenuation in myofilament force response to PKA occurs as a result of both cTnI and cMyBP-C phosphorylation, and that the reduction in [pCa.sub.50] mediated by cMyBP-C phosphorylation most likely arises from an accelerated cross-bridge cycling kinetics partly as a result of an increased rate constant of cross-bridge detachment. 10.1085/jgp.201010448