Regulation of Contractile Function by cMLC2v Phosphorylation in Heart Failure

 描述（由申请人提供）：心力衰竭（HF）是一种预后极差的主要临床问题。虽然已知细丝蛋白的磷酸化改变有助于HF进展，但对粗丝蛋白心肌肌球蛋白调节轻链（cMLC 2 v）的磷酸化知之甚少。cMLC 2 v通过改变肌球蛋白头部方向来调节力的产生。其磷酸化状态由肌球蛋白轻链激酶（cMLCK）和磷酸酶（MLCP）控制。在患者和动物模型中观察到HF期间cMLC 2 v磷酸化和cMLCK表达降低，尽管也报告了磷酸化增加，可能是一种适应性机制。在小鼠中，cMLCK过表达改善HF;相反，cMLCK破坏加重HF。然而，小鼠有两个cMLC 2 v磷酸化位点，每个位点可能具有单独的功能，而人cMLC 2 v只有一个磷酸化位点。因此，尽管cMLC 2 v磷酸化是收缩的关键调节因子，但其在人HF中的作用在很大程度上是未知的。确定cMLC 2 v磷酸化在人类健康和HF中的作用将开辟新的治疗途径。目前的HF治疗，如β受体阻滞剂和利尿剂，是非特异性的，主要是姑息性的;相反，靶向cMLC 2 v的治疗将是心脏特异性的，可以在收缩蛋白的基础水平上改善心脏功能。 我们假设cMLC 2 v磷酸化是人类心肌细胞功能的关键调节因子，操纵cMLC 2 v磷酸化可能是治疗HF的新疗法。在目的1中，我们将使用皮肤心脏制备物来研究衰竭和非衰竭人类心脏中cMLC 2 v磷酸化对收缩功能的调节。我们预测cMLC 2 v磷酸化在HF期间将减少，并且实验性增加cMLC 2 v磷酸化将改善收缩功能，特别是在衰竭与非衰竭心脏中。由于cMLC 2 v也可能影响Ca 2+瞬变，并可能优先改变动态和负荷收缩，因此Aim 2将研究cMLC 2 v磷酸化对衰竭和非衰竭兔心脏完整电刺激心肌细胞（与人具有高度cMLC 2 v同源性，磷酸化位点相同）动态收缩功能和Ca 2+处理的调节。我们预期通过cMLCK转染增加cMLC 2 v磷酸化将改善收缩功能，如通过模拟PV环评估的，并且将增加Ca 2+瞬变的幅度但减少持续时间;我们预期cMLCP过表达将具有相反的效果。总之，我们假设cMLC 2 v磷酸化减少是人HF收缩功能障碍的主要机制，cMLC 2 v磷酸化的恢复可能是一个新的治疗靶点。未来的研究将使用药理学和/或基因治疗来治疗动物模型中的HF，并最终在患者中。

 DESCRIPTION (provided by applicant): Heart failure (HF) is a major clinical problem with extremely poor prognosis. Though altered phosphorylation of thin filament proteins is known to contribute to HF progression, much less is known about phosphorylation of the thick filament protein cardiac myosin regulatory light chain (cMLC2v). cMLC2v modulates force production by altering myosin head orientation. Its phosphorylation status is controlled by myosin light chain kinase (cMLCK) and phosphatase (MLCP). Decreased cMLC2v phosphorylation and cMLCK expression are observed during HF in patients and animal models, though increased phosphorylation has also been reported and may be an adaptive mechanism. In mice, cMLCK overexpression ameliorates HF; conversely, cMLCK disruption exacerbates HF. However, mice have two cMLC2v phosphorylation sites, each of which may have a separate function, whereas human cMLC2v has only one phosphorylation site. Thus, though cMLC2v phosphorylation is a key regulator of contraction, its role in human HF is largely unknown. Defining the role of cMLC2v phosphorylation in human health and HF will open new therapeutic avenues. Current HF therapies, such as beta-blockers and diuretics, are non-specific and largely palliative; in contrast, therapies targeting cMLC2v would be cardiac-specific and could improve heart function at the fundamental level of the contractile proteins. We hypothesize that cMLC2v phosphorylation is a critical regulator of human cardiomyocyte function, and manipulation of cMLC2v phosphorylation may be a novel therapy for treatment of HF. In Aim 1, we will use skinned cardiac preparations to study regulation of contractile function by cMLC2v phosphorylation in failing and non-failing human heart. We predict that cMLC2v phosphorylation will be decreased during HF, and that experimentally increasing cMLC2v phosphorylation will improve contractile function, especially in failing versus non-failing hearts. Because cMLC2v may also impact Ca2+ transients and may preferentially alter dynamic and loaded contractions, Aim 2 will investigate regulation of dynamic contractile function and Ca2+ handling by cMLC2v phosphorylation in intact electrically stimulated cardiomyocytes from failing and non-failing rabbit hearts (which have high cMLC2v homology with human and identical phosphorylation site). We expect that increasing cMLC2v phosphorylation by cMLCK transfection will improve contractile function as assessed by simulated PV loops, and will increase amplitude but decrease duration of the Ca2+ transient; we expect that cMLCP overexpression will have the opposite effect. In summary, we hypothesize that reduced cMLC2v phosphorylation is a major mechanism underlying contractile dysfunction in human HF, and restoration of cMLC2v phosphorylation may be a novel therapeutic target. Future studies will use pharmacological and/or gene therapy to treat HF in animal models and eventually in patients.