Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK)and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca 2+ sensitivity of force (pCa 50 ), PKA treatment has been shown to decrease pCa 50 , presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca 2+ -independent force and maximum Ca 2+ -activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I 1,1 /I 1,0 ) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d 1,0 ) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I 1,1 /I 1,0 and, as hypothesized, treatment with MLCK also increased I 1,1 /I 1,0 , which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by ∼2 nm ( 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca 2+ sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

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 Ϫ/Ϫ ) 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 (0.22Ϯ0.03 versus 0.33Ϯ0.03). 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 Ϫ/Ϫ myocardium (0.40Ϯ0.06 versus 0.42Ϯ0.05). Although lattice spacing did not change after treatment in wild-type (45.68Ϯ0.84 nm versus 45.64Ϯ0.64 nm), treatment of cMyBP-C Ϫ/Ϫ myocardium increased lattice spacing (46.80Ϯ0.92 nm versus 49.61Ϯ0.59 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. (Circ Res. 2008;103:244-251.)Key Words: contractile protein structure Ⅲ cross-bridge kinetics Ⅲ cMyBP-C Ⅲ protein kinase A phosphorylation Ⅲ x-ray I n myocardium, the phosphorylation status of myofibrillar proteins affects protein function, which leads to changes in Ca 2ϩ -activated force and the rate at which force is developed, presumably by changing myofilament structure. In response to ␤-adrenergic stimulation of the heart, phosphorylation by protein kinase A (PKA) is a short-term modulator of myocardial work capacity. Cardiac myosin binding protein C (cMyBP-C), which binds tightly to myosin, is a substrate for PKA, and its phosphorylation is likely to play an important role in the regulation of cardiac contractility, 1,2 possibly by accelerating the rates of force development in systole and the rates of relaxation in diastole. 3,4 Conversely, the lack of cMyBP-C 3-8 or decreased levels of cMyBP-C phosphorylation 9 lead to cardiac dysfunction.Although cAMP activation of PKA targets cMyBP-C in the thick filament, PKA targets primarily troponin I (cTnI) in the thin filament. In skinned myocardium, phosphorylation of cTnI regulates the Ca 2ϩ -sensitivity of force, and phosphorylation of cMyBP-C regulates the rates of cross-bridge cycling. 3,4 With regard to the role of cMyBP-C in the regulation of contraction kin...

The speed and force of myocardial contraction during systolic ejection is largely dependent on the intrinsic contractile properties of cardiac myocytes. As the myosin heavy chain (MHC) isoform of cardiac muscle is an important determinant of the contractile properties of individual myocytes, we studied the effects of altered MHC isoform expression in rat myocardium on the mechanical properties of skinned ventricular preparations. Skinned myocardium from thyroidectomized rats expressing only the β MHC isoform displayed rates of force redevelopment that were about 2.5-fold slower than in myocardium from hyperthyroid rats expressing only the α MHC isoform, but the amount of force generated at a given level of Ca 2+ activation was not different. Because recent studies suggest that the stretch activation response in myocardium has an important role in systolic function, we also examined the effect of MHC isoform expression on the stretch activation response by applying a rapid stretch (1% of muscle length) to an otherwise isometrically contracting muscle fibre. Sudden stretch of myocardium resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force (i.e. stretch activation) to levels greater than prestretch force. β MHC expression dramatically slowed the overall rate of the stretch activation response compared to expression of α MHC isoform; specifically, the rate of force decay was ∼2-fold slower and the rate of delayed force development was ∼2.5-fold slower. In contrast, MHC isoform had no effect on the amplitude of the stretch activation response. Collectively, these data show that expression of β MHC in myocardium dramatically slows rates of cross-bridge recruitment and detachment which would be expected to decrease power output and contribute to depressed systolic function in end-stage heart failure.

Locher MR, Razumova MV, Stelzer JE, Norman HS, Patel JR, Moss RL. Determination of rate constants for turnover of myosin isoforms in rat myocardium: implications for in vivo contractile kinetics. Am J Physiol Heart Circ Physiol 297: H247-H256, 2009. First published April 24, 2009 doi:10.1152/ajpheart.00922.2008The ventricles of small mammals express mostly ␣-myosin heavy chain (␣-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express ϳ10% ␣-MHC on a predominately ␤-MHC (slow isoform) background. In failing human ventricles, the amount of ␣-MHC is dramatically reduced, leading to the hypothesis that even small amounts of ␣-MHC on a predominately ␤-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% ␣-MHC or ␤-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% ␣-MHC or 100% ␤-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (ktr) in solutions of varying Ca 2ϩ concentrations. The rate of ATP utilization was ϳ2.5-fold higher in preparations expressing 100% ␣-MHC compared with those expressing only ␤-MHC, whereas ktr was 2-fold faster in the ␣-MHC myocardium. From these variables, we calculated fapp to be approximately threefold higher for ␣-MHC than ␤-MHC and gapp to be twofold higher in ␣-MHC. Mathematical modeling of isometric twitches predicted that small increases in ␣-MHC significantly increased the rate of force development. These results suggest that low-level expression of ␣-MHC has significant effects on contraction kinetics.␣-myosin heavy chain; rate constants of cross-bridge attachment and detachment; rate of rise of force THE MYOSIN MOLECULE, a hexamer composed of two heavy chains (ϳ220 kDa) and four light chains (16 -25 kDa), is the principal component of the thick filament and is responsible for the generation of force in mammalian striated muscle. The rod-shaped COOH-terminal domain of the myosin molecule (light meromyosin) forms the backbone of the thick filament, whereas the globular NH 2 -terminus (subfragment 1) contains the actin-binding and catalytic domains (13). Thus, S1 is an important determinant of contractile speed and power developed in the mammalian myocardium.Two isoforms of myosin heavy chain (MHC), ␣ and ␤, have been identified in cardiac muscle (23). These MHC isoforms share 93% amino acid sequence homology (32), and yet they confer distinct functional properties to the myocardium: ␣-MHC has been shown to have markedly higher ATPase activity, faster shortening velocity (V max ), and faster rates of force development (42), whereas ␤-MHC exhibits a lower tension cost and is thus more efficient (1,24,40).Expression levels of the ventricular MHC isoforms are sp...

Locher MR, Razumova MV, Stelzer JE, Norman HS, Moss RL. Effects of low-level ␣-myosin heavy chain expression on contractile kinetics in porcine myocardium. Am J Physiol Heart Circ Physiol 300: H869 -H878, 2011. First published January 7, 2011 doi:10.1152/ajpheart.00452.2010.-Myosin heavy chain (MHC) isoforms are principal determinants of work capacity in mammalian ventricular myocardium. The ventricles of large mammals including humans normally express ϳ10% ␣-MHC on a predominantly ␤-MHC background, while in failing human ventricles ␣-MHC is virtually eliminated, suggesting that low-level ␣-MHC expression in normal myocardium can accelerate the kinetics of contraction and augment systolic function. To test this hypothesis in a model similar to human myocardium we determined composite rate constants of cross-bridge attachment (fapp) and detachment (gapp) in porcine myocardium expressing either 100% ␣-MHC or 100% ␤-MHC in order to predict the MHC isoform-specific effect on twitch kinetics. Right atrial (ϳ100% ␣-MHC) and left ventricular (ϳ100% ␤-MHC) tissue was used to measure myosin ATPase activity, isometric force, and the rate constant of force redevelopment (ktr) in solutions of varying Ca 2ϩ concentration. The rate of ATP utilization and ktr were approximately ninefold higher in atrial compared with ventricular myocardium, while tension cost was approximately eightfold greater in atrial myocardium. From these values, we calculated fapp to be ϳ10-fold higher in ␣-compared with ␤-MHC, while gapp was 8-fold higher in ␣-MHC. Mathematical modeling of an isometric twitch using these rate constants predicts that the expression of 10% ␣-MHC increases the maximal rate of rise of force (dF/dt max) by 92% compared with 0% ␣-MHC. These results suggest that low-level expression of ␣-MHC significantly accelerates myocardial twitch kinetics, thereby enhancing systolic function in large mammalian myocardium. myosin heavy chain isoforms; cross-bridge cycling kinetics; ventricular myocardium THE ABILITY OF MAMMALIAN MYOCARDIUM to perform work depends on a multitude of factors that modulate contractility both acutely and chronically. Beat-to-beat variations in work capacity can be mediated by several mechanisms, including the Frank-Starling response to variations in ventricular end-diastolic volume, as well as phosphorylation of myofibrillar and Ca 2ϩ handling proteins. In contrast, chronic alterations in myocardial work capacity are determined in large part by the level of gene expression of myosin heavy chain (MHC) isoforms. The expression and distribution of the MHC isoforms in myocardium play a crucial role in determining the intrinsic rate of ATP turnover and thus are important determinants of myocyte shortening velocity (V max ) (44) and power output (21,22,28).Cardiac myosin is a hexamer consisting of two heavy chain isoforms, ␣ and ␤ (24), and four associated light chains. The MHC isoforms are distinguished primarily by the nucleotide turnover rate of the globular head (S1) region of the thick filament, which converts ...