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肌营养不良蛋白-糖蛋白复合物成分的遗传缺陷有关。此外，我们已经证明，由肠病毒感染引起的获得性心肌病与病毒蛋白酶2A介导的肌营养不良蛋白的裂解和肌营养不良蛋白-糖蛋白复合物的破坏有关。这就提出了关于病毒介导的扩张型心肌病发病机制的两个重要问题。首先，肌营养不良蛋白的分裂如何影响病毒复制和病毒繁殖的动力学；第二，肌营养不良蛋白的分裂是如何导致肠病毒感染引起的心肌病的？因此，本课题的总体目标是精确识别肌营养不良蛋白中蛋白酶2A的裂解位点，并确定蛋白酶2A介导的肌营养不良蛋白在培养细胞和完整心脏中的裂解作用。为实现这一目标，我们提出以下具体目标：确定在体外和细胞培养中，蛋白酶2A对人和小鼠肌营养不良蛋白的蛋白水解裂解是否需要在肌营养不良蛋白的铰链3区进行切割。在细胞培养中测定野生型和抗裂型抗肌营养不良蛋白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感染对肌营养不良蛋白相关的细胞骨架蛋白和其他不属于肌营养不良蛋白-糖蛋白复合物的细胞骨架蛋白组织的影响；在小鼠中，确定病毒复制对缺乏肌营养不良蛋白或肌营养不良蛋白相关肌聚糖的CVB3感染小鼠的影响。3. 确定蛋白酶2A切割肌营养不良蛋白如何影响病毒复制动力学和诱导与CVB3感染相关的全心肌/心肌病表型。4. 确定心肌细胞中柯萨奇病毒蛋白酶2A的条件表达是否足以诱导心肌病和通过肌营养不良蛋白的裂解破坏肌膜。

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.