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.

Recent evidence suggests that ventricular ejection is partly powered by a delayed development of force, i.e., stretch activation, in regions of the ventricular wall due to stretch resulting from torsional twist of the ventricle around the apex-to-base axis. Given the potential importance of stretch activation in cardiac function, we characterized the stretch activation response and its Ca2+ dependence in murine skinned myocardium at 22°C in solutions of varying Ca2+ concentrations. Stretch activation was induced by suddenly imposing a stretch of 0.5–2.5% of initial length to the isometrically contracting muscle and then holding the muscle at the new length. The force response to stretch was multiphasic: force initially increased in proportion to the amount of stretch, reached a peak, and then declined to a minimum before redeveloping to a new steady level. This last phase of the response is the delayed force characteristic of myocardial stretch activation and is presumably due to increased attachment of cross-bridges as a consequence of stretch. The amplitude and rate of stretch activation varied with Ca2+ concentration and more specifically with the level of isometric force prior to the stretch. Since myocardial force is regulated both by Ca2+ binding to troponin-C and cross-bridge binding to thin filaments, we explored the role of cross-bridge binding in the stretch activation response using NEM-S1, a strong-binding, non-force–generating derivative of myosin subfragment 1. NEM-S1 treatment at submaximal Ca2+-activated isometric forces significantly accelerated the rate of the stretch activation response and reduced its amplitude. These data show that the rate and amplitude of myocardial stretch activation vary with the level of activation and that stretch activation involves cooperative binding of cross-bridges to the thin filament. Such a mechanism would contribute to increased systolic ejection in response to increased delivery of activator Ca2+ during excitation–contraction coupling.

Nonmuscle gamma(cyto)-actin is expressed at very low levels in skeletal muscle but uniquely localizes to costameres, the cytoskeletal networks that couple peripheral myofibrils to the sarcolemma. We generated and analyzed skeletal muscle-specific gamma(cyto)-actin knockout (Actg1-msKO) mice. Although muscle development proceeded normally, Actg1-msKO mice presented with overt muscle weakness accompanied by a progressive pattern of muscle fiber necrosis/regeneration. Functional deficits in whole-body tension and isometric twitch force were observed, consistent with defects in the connectivity between muscle fibers and/or myofibrils or at the myotendinous junctions. Surprisingly, gamma(cyto)-actin-deficient muscle did not demonstrate the fibrosis, inflammation, and membrane damage typical of several muscular dystrophies but rather presented with a novel progressive myopathy. Together, our data demonstrate an important role for minimally abundant but strategically localized gamma(cyto)-actin in adult skeletal muscle and describe a new mouse model to study the in vivo relevance of subcellular actin isoform sorting.

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...

Myosin-binding protein-C (MyBP-C) is a thick filament-associated protein that binds tightly to myosin. Given that cMyBP-C may act to modulate cooperative activation of the thin filament by constraining the availability of myosin cross-bridges for binding to actin, we investigated the role of MyBP-C in the regulation of cardiac muscle contraction. We assessed the Ca(2+) sensitivity of force (pCa(50)) and the activation dependence of the rate of force redevelopment (k(tr)) in skinned myocardium isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) mice. Mechanical measurements were performed at 22 degrees C in the absence and presence of a strong-binding, nonforce-generating analog of myosin subfragment-1 (NEM-S1). In the absence of NEM-S1, maximal force and k(tr) and the pCa(50) of isometric force did not differ between WT and cMyBP-C(-/-) myocardium; however, ablation of cMyBP-C-accelerated k(tr) at each submaximal force. Treatment of WT and cMyBP-C(-/-) myocardium with 3 muM NEM-S1 elicited similar increases in pCa(50,) but the effects of NEM-S1 to increase k(tr) at submaximal forces and thereby markedly reduce the activation dependence of k(tr) occurred to a greater degree in cMyBP-C(-/-) myocardium. Together, these results support the idea that cMyBP-C normally acts to constrain the interaction between myosin and actin, which in turn limits steady-state force development and the kinetics of cross-bridge interaction.