Cardiac Dystrophy: Cellular Mechanisms

描述（由申请人提供）：Duchenne和Becker肌营养不良症（分别为DMD和BMD）是由肌营养不良蛋白基因突变引起的广泛和严重形式的横纹肌疾病。它们的特征是肌肉功能的进行性退化。大多数DMD和BMD男孩在20岁时出现心脏异常。心力衰竭是死亡的第二大原因。随着骨骼肌无力的治疗选择的改善，心脏病变得越来越限制年轻患者的生存。进一步延长其寿命取决于对心脏缺陷的机械理解。在营养不良的心脏中，已知力的产生受损。功能丧失的病理解剖学基础主要是结缔组织和脂肪（纤维化）替代心肌。我们的初步研究结果表明，增强的Ca 2+信号，Na+超载和氧化/亚硝化应激在收缩功能障碍和进行性损伤的营养不良的心脏组织的发展中发挥重要作用。我们的观察使我们得出三个假设，将在这个项目中进行测试：1）。过量的Ca 2+信号来自于RyR对Ca 2+的敏感性升高。2）。升高的RyR Ca 2+敏感性与疾病发作时的EC偶联相容，甚至确保其可靠性。然而，在疾病的进展过程中，这种最初有益的变化变得适应不良，并导致心肌功能恶化。3a）。升高的[Na+]i限制了肌膜Na+-Ca 2+交换器从营养不良的心肌细胞中排出Ca 2+的能力，从而促进了Ca 2+在胞质溶胶中的积累，从而导致细胞损伤。2b）。[Na+]i的增加增强了线粒体中Ca 2+的去除，减少了线粒体Ca 2+的积累，并改变了线粒体的代谢状态。在三个密切相关的特定目标中，我们将1）确定RyR敏感性变化的潜在机制，2）检查心脏营养不良发展过程中EC偶联的改变，3）评估细胞内Na+处理的变化，并确定它们如何影响胞质和线粒体Ca 2+信号传导和线粒体代谢状态。为了实现这些目标，将使用多种成像、电生理和生物化学技术。将对从不同年龄组动物中分离的心肌细胞进行实验，以确定细胞异常与心肌病发生之间的相关性和可能的因果关系。将采用两种营养不良的动物模型：mdx小鼠，缺乏肌营养不良蛋白，和mdx/utrophin-/-小鼠，其中utrophin也被敲除。我们的总体假设是，肌营养不良症中的心肌病是一种缓慢发展的病理学，这是由于Ca 2+信号传导和Na+处理的多重缺陷的累积效应。该提案将确定导致缺陷的关键细胞过程，并为开发治疗干预提供坚实的基础。

DESCRIPTION (provided by applicant): Duchenne and Becker muscular dystrophy (DMD and BMD, respectively) are widespread and severe forms of striated muscle diseases caused by dystrophin gene mutations. They are characterized by progressive degeneration of muscle function. Cardiac abnormality is present in the majority of boys with DMD and BMD by age 20. Heart failure is the second leading cause of fatalities. With improving therapeutic options for the skeletal muscle weakness, cardiac disease becomes increasingly limiting for the survival of the young patients. Further prolongation of their life depends on a mechanistic understanding of the cardiac defects. In dystrophic heart, force generation is known to be impaired. The pathoanatomic basis for the loss of function is mainly the replacement of myocardium by connective tissue and fat (fibrosis). Our preliminary findings indicate that augmented Ca2+ signaling, Na+ overload and oxidative/nitrosative stress play an important role in the development of contractile dysfunction and progressive damage of dystrophic cardiac tissue. Our observations led us to three hypotheses that will be tested in this project: 1). Excessive Ca2+ signals arise from an elevated RyR sensitivity to Ca2+. 2). Elevated RyR Ca2+ sensitivity is compatible with or even ensures reliability of EC-coupling at the onset of the disease. During the progression of the disease however, this initially beneficial change becomes maladaptive and contributes to the deterioration of cardiac muscle function. 3a). An elevated [Na+]i limits the ability of the sarcolemmal Na+-Ca2+ exchanger to extrude Ca2+ from dystrophic cardiac myocytes, thus promoting Ca2+ accumulation in the cytosol and consequently cellular damage. 2b). An increase in [Na+]i enhances Ca2+ removal from the mitochondria, decreases mitochondrial Ca2+ accumulation and changes mitochondrial metabolic state. In three intimately connected Specific Aims we will 1) determine the mechanisms underlying changes in the sensitivity of RyR, 2) examine alterations of EC-coupling during development of cardiac dystrophy and 3) evaluate changes in intracellular Na+ handling and establish how they affect cytosolic and mitochondrial Ca2+ signaling and mitochondrial metabolic state. To achieve these goals a multitude of imaging, electrophysiological, and biochemical techniques will be used. The experiments will be carried out on cardiomyocytes isolated from the animals of different age groups in order to establish a correlation and possibly causal relationship between cellular abnormalities and the development of cardiac myopathy. Two animal models of dystrophy will be employed: mdx mice, lacking dystrophin, and mdx/utrophin-/- mice, in which utrophin has also been knocked out. Our overall hypothesis is that cardiac myopathy in muscular dystrophy is a slowly developing pathology due to the cumulative effects of multiple defects in Ca2+ signaling and Na+ handling. This proposal will identify the key cellular processes contributing to the defects and provide a solid basis for developing therapeutic intervention.