Dystrophin-glycoprotein complex in viral cardiomyopathy

扩张型心肌病是一种多因素疾病，包括遗传性和获得性心肌病。最近的实验表明，人类遗传性心肌病可能与营养不良蛋白-糖蛋白复合体成分的遗传缺陷有关。此外，我们还发现，由肠道病毒感染引起的获得性心肌病与病毒蛋白2A介导的抗肌营养不良蛋白的裂解和抗肌营养不良蛋白-糖蛋白复合体的破坏有关。这对病毒介导的扩张型心肌病的发病机制提出了两个重要的问题。第一，dystrophin的裂解如何影响病毒复制和病毒繁殖的动力学；第二，dystrophin的裂解如何促进肠道病毒感染引起的心肌病？因此，本研究的总体目标是准确地确定dystrophin中的蛋白水解酶2A的裂解位点，并确定在培养细胞和完整心脏中，蛋白酶2A对dystrophin的裂解作用。为了实现这一目标，我们提出了以下具体目标：1.确定在体外和细胞培养中，是否需要在dystrophin的铰链3区域进行切割，以使人和小鼠的dystrophin在体外和细胞培养中被蛋白水解性切割；2.在细胞培养中，确定野生型和抗切割的dystrophin表达对病毒复制动力学和细胞死亡的影响；在小鼠中，确定CVB3感染对dystrophin相关细胞骨架蛋白和其他不属于dystrophin-糖蛋白复合体一部分的细胞骨架蛋白的组织的影响；以及在小鼠中，确定CVB3感染的缺乏dystrophin或dystrophin相关肌糖蛋白的小鼠中病毒复制的影响。3.确定酶2A对dystrophin的切割如何影响病毒复制的动力学和与CVB3感染相关的完全心肌炎/心肌病表型的诱导。4.确定柯萨奇病毒蛋白酶2A在心肌细胞中的条件性表达是否足以引起心肌病和通过切割dystrophin而导致肌膜破裂。

Dilated cardiomyopathy is a multifactorial disease that includes both hereditary and acquired forms of cardiomyopathy. Recent experiments have shown that hereditary cardiomyopathy in humans can be associated with genetic defects in components of the dystrophin-glycoprotein complex. In addition, we have shown that an acquired form of cardiomyopathy caused by enteroviral infection is associated with viral protease 2A mediated cleavage of dystrophin and disruption of the dystrophin-glycoprotein complex. This raises two important questions regarding the pathogenesis of viral mediated dilated cardiomyopathy. First, how does cleavage of dystrophin affect the kinetics of viral replication and viral propagation; and second, how does cleavage of dystrophin contribute to the cardiomyopathy caused by enteroviral infection? Therefore, the overall goal of this proposal is to precisely identify the protease 2A cleavage site in dystrophin and to determine the effect of protease 2A mediated cleavage of dystrophin in cultured cells and in the intact heart. In order to accomplish this goal, we propose the following Specific Aims: 1. Determine whether cleavage in the hinge 3 region of dystrophin is required for proteolytic cleavage of human and mouse dystrophin by protease 2A in vitro and in cell culture, 2. In cell culture determine the effect of wild type and cleavage resistant dystrophin expression on viral replication kinetics and cell death; in mice determine the impact of CVB3 infection on organization of the dystrophin associated cytoskeletal proteins and other cytoskeletal proteins that are not part of the dystrophin-glycoprotein complex; and in mice, determine the effect of viral replication in CVB3 infected mice that lack either dystrophin or dystrophin-associated sarcoglycans. 3. Determine how cleavage of dystrophin by protease 2A affects the kinetics of viral replication and induction of the full myocarditic/cardiomyopathic phenotype associated with CVB3 infection. 4. Determine whether conditional expression of Coxsackieviral protease 2A in the cardiac myocyte is sufficient to induce cardiomyopathy and disruption of the sarcolemma via cleavage of dystrophin.