Role of phosphorylation in cardiac muscle myosin

了解心肌肌球蛋白调节轻链（RLC）磷酸化如何改变心肌力学是很重要的，因为它经常在心脏疾病中改变。这种蛋白质磷酸化在缩短速度的生理范围内对肌肉力学的影响尚未得到解决，在此期间，心脏产生动力并做功。我们表达并磷酸化重组褐家鼠左心室RLC。在体外，我们磷酸化这些重组物种与心肌肌球蛋白轻链激酶和拉链相互作用蛋白激酶。我们比较大鼠透化心脏小梁，这已经经历了不同的磷酸化RLC物种交换。与对照组相比，我们能够富集小梁RLC磷酸化40%，并且在单独的系列中，将RLC磷酸化降低至对照值的60%。与低水平RLC磷酸化的小梁相比，RLC磷酸化富集使等长收缩力增加了3倍以上，峰值功率输出增加了7倍以上，最大缩短速度和产生峰值功率的缩短速度增加了约一倍。我们通过观察来自内皮细胞左心室匀浆的人和大鼠HF样品的RLC磷酸化增加来增强这些测量。这些结果表明，增加RLC磷酸化的心肌性能的上调的重要性，并表明，减少RLC磷酸化是在患病心肌收缩功能受损的一个关键方面。
 我们目前正在生产全长心肌肌球蛋白，并在体外改变相关的RLC磷酸化水平。我们能够使用Phos标签分子确定RLC磷酸化水平，以区分磷酸化的轻链与无法直接定量的凝胶电泳方法中的轻链。我们正在使用这些分子进行肌动蛋白滑动试验，以评估RLC磷酸化对滑动速度的影响。我们采用NADH偶联ATP酶法测定了低盐条件下不同RLC磷酸化水平的全长心肌肌球蛋白的ATP酶速率。我们将这些测量与三珠光阱配对，以测量具有不同RLC磷酸化水平的单个心脏全长肌球蛋白分子的附着持续时间、步长和力产生。 与Don A博士合作。Winkelmann（Robert Wood约翰逊医学院，罗格斯大学）和伊娃Forgacs-Lonart（东弗吉尼亚医学院），我们正在使用在C2 C12肌肉细胞系中重组表达的心肌肌球蛋白-2，通过双光束光镊装置测量单分子肌动球蛋白力学。这些重组表达的蛋白质中的一些编码有扩张型或肥厚型心肌病（DCM和HCM）的原因的点突变。

Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle mechanics is important because it is often altered in cardiac disease. The effect this protein phosphorylation has on muscle mechanics during a physiological range of shortening velocities, during which the heart generates power and performs work, has not been addressed. We have expressed and phosphorylated recombinant Rattus norvegicus left ventricular RLC. In vitro we have phosphorylated these recombinant species with cardiac myosin light chain kinase and zipper-interacting protein kinase. We compare rat permeabilized cardiac trabeculae, which have undergone exchange with differently phosphorylated RLC species. We were able to enrich trabecular RLC phosphorylation by 40% compared with controls and, in a separate series, lower RLC phosphorylation to 60% of control values. Compared with the trabeculae with a low level of RLC phosphorylation, RLC phosphorylation enrichment increased isometric force by more than 3-fold and peak power output by more than 7-fold and approximately doubled both maximum shortening speed and the shortening velocity that generated peak power. We augmented these measurements by observing increased RLC phosphorylation of human and rat HF samples from endocardial left ventricular homogenate. These results demonstrate the importance of increased RLC phosphorylation in the up-regulation of myocardial performance and suggest that reduced RLC phosphorylation is a key aspect of impaired contractile function in the diseased myocardium. 
 We are currently producing full-length cardiac myosin and altering the associated RLC phosphorylation level in-vitro. We are able to determine the RLC phosphorylation level using the Phos-tag molecule for differentiation light chains that are phosphorylated form those that are not in a directly quantifiable gel electrophoresis method. We are using these molecules to run actin-gliding assays to assess the effect of RLC phosphorylation on sliding velocity. We have adapted the NADH-coupled ATPase assay to measure the ATPase rate of full-length cardiac myosin in low salt with different RLC phosphorylation levels. We are twinning these measurements with the three bead optical trap to measure attachment duration, step length and force production by single cardiac full length myosin molecules with different RLC phosphorylation levels. Furthermore, in collaboration with Drs. Don A. Winkelmann (Robert Wood Johnson Medical School, Rutgers University) and Eva Forgacs-Lonart (Eastern Virginia Medical School), we are measuring the single molecule actomyosin mechanics via a dual beam optical tweezers apparatus using cardiac muscle myosin-2 which are recombinantly expressed in C2C12 muscle cell line. Some of these recombinantly expressed proteins are encoded with point mutations that are the cause of dilated or hypertrophic cardiomyopathy (DCM and HCM).