PDF(Pubmed)
Abstract：
Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that influences sarcomere stiffness and modulates cardiac contraction-relaxation through its phosphorylation. Phosphorylation of cMyBP-C and ablation of cMyBP-C have been shown to increase the rate of MgADP release in the acto-myosin cross-bridge cycle in the intact sarcomere. The influence of cMyBP-C on Pi-dependent myosin kinetics has not yet been examined. We investigated the effect of cMyBP-C, and its phosphorylation, on myosin kinetics in demembranated papillary muscle strips bearing the β-cardiac myosin isoform from nontransgenic and homozygous transgenic mice lacking cMyBP-C. We used quick stretch and stochastic length-perturbation analysis to characterize rates of myosin detachment and force development over 0-12 mM Pi and at maximal (pCa 4.8) and near-half maximal (pCa 5.75) Ca2+ activation. Protein kinase A (PKA) treatment was applied to half the strips to probe the effect of cMyBP-C phosphorylation on Pi sensitivity of myosin kinetics. Increasing Pi increased myosin cross-bridge detachment rate similarly for muscles with and without cMyBP-C, although these rates were higher in muscle without cMyBP-C. Treating myocardial strips with PKA accelerated detachment rate when cMyBP-C was present over all Pi, but not when cMyBP-C was absent. The rate of force development increased with Pi in all muscles. However, Pi sensitivity of the rate force development was reduced when cMyBP-C was present versus absent, suggesting that cMyBP-C inhibits Pi-dependent reversal of the power stroke or stabilizes cross-bridge attachment to enhance the probability of completing the power stroke. These results support a functional role for cMyBP-C in slowing myosin detachment rate, possibly through a direct interaction with myosin or by altering strain-dependent myosin detachment via cMyBP-C-dependent stiffness of the thick filament and myofilament lattice. PKA treatment reduces the role for cMyBP-C to slow myosin detachment and thus effectively accelerates β-myosin detachment in the intact myofilament lattice.NEW & NOTEWORTHY Length perturbation analysis was used to demonstrate that β-cardiac myosin characteristic rates of detachment and recruitment in the intact myofilament lattice are accelerated by Pi, phosphorylation of cMyBP-C, and the absence of cMyBP-C. The results suggest that cMyBP-C normally slows myosin detachment, including Pi-dependent detachment, and that this inhibition is released with phosphorylation or absence of cMyBP-C.
摘要：
心肌肌球蛋白结合蛋白C（cMyBP-C）是一种粗丝蛋白，可影响肌节硬度并通过其磷酸化调节心脏收缩-舒张。已显示cMyBP-C的磷酸化和cMyBP-C的消融可增加完整肌节肌球蛋白跨桥循环中MgADP的释放速率。尚未研究cMyBP-C对Pi依赖性肌球蛋白动力学的影响。我们调查了cMyBP-C的效果，以及它的磷酸化，关于缺乏cMyBP-C的非转基因和纯合转基因小鼠的带有β-心脏肌球蛋白同工型的脱膜乳头状肌条中的肌球蛋白动力学。我们使用快速拉伸和随机长度扰动分析来表征0-12mMPi以及最大（pCa4.8）和近半最大（pCa5.75）Ca2激活时的肌球蛋白脱离和力发展速率。将蛋白激酶A（PKA）处理应用于一半的条带，以探测cMyBP-C磷酸化对肌球蛋白动力学Pi敏感性的影响。对于有和没有cMyBP-C的肌肉，Pi的增加也会增加肌球蛋白跨桥脱离率，尽管没有cMyBP-C的肌肉中这些比率更高。当cMyBP-C存在于所有Pi上时，用PKA加速脱离率治疗心肌条，但当cMyBP-C缺失时并非如此。在所有肌肉中，力的发展速度随Pi的增加而增加。然而,当cMyBP-C存在与不存在时，速率力发展的Pi敏感性降低，提示cMyBP-C抑制功率冲程的Pi依赖性逆转或稳定跨桥连接以提高完成功率冲程的概率。这些结果支持cMyBP-C在减缓肌球蛋白脱离率方面的功能作用，可能是通过与肌球蛋白直接相互作用或通过粗丝和肌丝晶格的cMyBP-C依赖性刚度改变应变依赖性肌球蛋白脱离。PKA治疗降低了cMyBP-C减缓肌球蛋白脱离的作用,从而有效地加速完整肌丝晶格中的β-肌球蛋白脱离。NEW＆NOTEWORTHY长度扰动分析用于证明Pi加速了完整肌丝晶格中β-心肌肌球蛋白特征的脱离和募集速率，cMyBP-C的磷酸化，和cMyBP-C的缺失结果表明，cMyBP-C通常可以减缓肌球蛋白的脱离，包括依赖Pi的分离，并且这种抑制作用随着cMyBP-C的磷酸化或不存在而释放。
