Protein kinase A–induced myofilament desensitization to Ca2+ 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 Ca2+ 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 Ca2+ sensitivity of force (pCa50) and the activation dependence of the rate of force redevelopment (ktr) 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−/−), (c) nonphosphorylatable cTnI with serines23/24/43/45 and threonine144 mutated to alanines (cTnIAla5), and (d) nonphosphorylatable cTnI on a cMyBP-C–null background (cTnIAla5/cMyBP-C−/−). Here, PKA treatment decreased pCa50 in WT, cTnIAla5, and cMyBP-C−/− myocardium by 0.13, 0.08, and 0.09 pCa units, respectively, but had no effect in cTnIAla5/cMyBP-C−/− myocardium. In WT and cTnIAla5 myocardium, PKA treatment also increased ktr at submaximal levels of activation; however, PKA treatment did not have an effect on ktr in cMyBP-C−/− or cTnIAla5/cMyBP-C−/− myocardium. In addition, reconstitution of cTnIAla5/cMyBP-C−/− myocardium with recombinant cMyBP-C restored the effects of PKA treatment on pCa50 and ktr reported in cTnIAla5 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 pCa50 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

Cross-bridge interaction kinetics in rat myocardium are accelerated by strong binding of myosin to the thin filament

To determine the ability of strong-binding myosin cross-bridges to activate the myocardial thin filament, we examined the Ca2+ dependence of force and cross-bridge interaction kinetics at 15°C in the absence and presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1) in chemically skinned myocardium from adult rats.Relative to control conditions, application of 6 μM NEM-S1 significantly increased Ca2+-independent tension, measured at pCa 9.0, from 0.8 ± 0.3 to 3.7 ± 0.8 mN mm−2. Furthermore, NEM-S1 potentiated submaximal Ca2+-activated forces and thereby increased the Ca2+ sensitivity of force, i.e. the [Ca2+] required for half-maximal activation (pCa50) increased from pCa 5.85 ± 0.05 to 5.95 ± 0.04 (change in pCa50 (ΔpCa50) = 0.11 ± 0.02). The augmentation of submaximal force by NEM-S1 was accompanied by a marked reduction in the steepness of the force-pCa relationship for forces less than 0.50 Po (maximum Ca2+-activated force), i.e. the Hill coefficient (n2) decreased from 4.72 ± 0.38 to 1.54 ± 0.07.In the absence of NEM-S1, the rate of force redevelopment (ktr) was found to increase from 1.11 ± 0.21 s−1 at submaximal [Ca2+] (pCa 6.0) to 9.28 ± 0.41 s−1 during maximal Ca2+ activation (pCa 4.5). Addition of NEM-S1 reduced the Ca2+ dependence of ktr by eliciting maximal values at low levels of Ca2+, i.e. ktr was 9.38 ± 0.30 s−1 at pCa 6.6 compared to 9.23 ± 0.27 s−1 at pCa 4.5. At intermediate levels of Ca2+, ktr was less than maximal but was still greater than values obtained at the same pCa in the absence of NEM-S1.NEM-S1 dramatically reduced both the extent and rate of relaxation from steady-state submaximal force following flash photolysis of the caged Ca2+ chelator diazo-2.These data demonstrate that strongly bound myosin cross-bridges increase the level of thin filament activation in myocardium, which is manifested by an increase in the rate of cross-bridge attachment, potentiation of force at low levels of free Ca2+, and slowed rates of relaxation

Determination of rate constants for turnover of myosin isoforms in rat myocardium: implications for in vivo contractile kinetics

The ventricles of small mammals express mostly α-myosin heavy chain (α-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express ∼10% α-MHC on a predominately β-MHC (slow isoform) background. In failing human ventricles, the amount of α-MHC is dramatically reduced, leading to the hypothesis that even small amounts of α-MHC on a predominately β-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment (fapp) and detachment (gapp) for myosins composed of 100% α-MHC or β-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% α-MHC or 100% β-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (ktr) in solutions of varying Ca2+ concentrations. The rate of ATP utilization was ∼2.5-fold higher in preparations expressing 100% α-MHC compared with those expressing only β-MHC, whereas ktr was 2-fold faster in the α-MHC myocardium. From these variables, we calculated fapp to be approximately threefold higher for α-MHC than β-MHC and gapp to be twofold higher in α-MHC. Mathematical modeling of isometric twitches predicted that small increases in α-MHC significantly increased the rate of force development. These results suggest that low-level expression of α-MHC has significant effects on contraction kinetics