Role of phosphorylation in cardiac muscle myosin

肌球蛋白调节轻链(RLC)的磷酸化在许多形式的心脏疾病中都会改变，既有遗传性的(HCM和DCM)，也有获得性的(衰竭、梗塞和糖尿病)。RLC的磷酸化通过影响钙敏感性、力量、力量和缩短速度来改变肌肉力学。目前，我们还不清楚这些对肌肉的影响是如何在跨桥的分子水平上产生的。为此，我们从猪心内膜分离出全长心肌肌球蛋白，并在体外改变了它的磷酸化水平。我们比较了完全(100%)RLC磷酸化、中间磷酸化(40%&50%)和消除磷酸化(0%)的肌球蛋白群体。因此，我们可以将RLC磷酸化所产生的酶变化和运动性变化联系起来。我们发现，当肌球蛋白拥有100%磷酸化的RLC时，ATPase的速度加快，在假定的卸载和加载(在-Actinin存在的情况下)条件下，肌动蛋白的滑动速度加快。这些结果与RLC磷酸化对过桥马达动力学的影响是一致的。RLC的磷酸化缩短了肌动球蛋白的连接寿命，这是在光学捕获三珠实验中测得的。这些结果提示RLC的磷酸化在调节心肌肌球蛋白的功能中起着重要的作用。-结果与RLC磷酸化正常水平降低可能导致心血管疾病和心力衰竭的假设一致。

Phosphorylation of the regulatory light chain of myosin (RLC) is altered in many forms of cardiac diseases, both inherited (HCM and DCM) and acquired (failure, infarction and diabetes). Phosphorylation of the RLC alters muscle mechanics by influencing calcium sensitivity, force, power and shortening velocity. Currently we do not understand how these effects on muscle are created at the molecular level of the cross-bridge. To this end, we isolated full-length cardiac myosin from porcine endocardium and altered its phosphorylation level in-vitro. We compare populations of myosins with complete (100%) RLC phosphorylation, intermediate phosphorylation (40% & 50%), and eradicated phosphorylation (0%). We could thereby correlate enzymatic and motility changes produced with RLC phosphorylation. We find that when myosin possesses 100% phosphorylated RLCs, the ATPase rate is accelerated and actin gliding velocities in putative unloaded and loaded (in presence of -actinin) conditions are accelerated. These results are consistent with the influence of RLC phosphorylation on the kinetics of the cross-bridge motor kinetics. RLC phosphorylation reduced the lifetime of actomyosin attachment as measured in the optical trap three-bead assay. These results suggest an important role for RLC phosphorylation in regulating the function of cardiac myosin. - The results are consistent with the hypothesis that reductions in normal levels of RLC phosphorylation may precipitate cardiovascular disorders and heart failure.