CALCIUM REGULATION IN THE DIABETIC HEART

心脏病是糖尿病患者死亡的主要原因， 现在有大量证据支持存在一种 不依赖于冠状动脉的特异性糖尿病心肌病 疾病和高血压。功能和生化数据从 糖尿病动物的多细胞心脏标本支持这种观点 细胞机制控制细胞内Ca 2+的心跳 基础是异常的，并有助于受损的放松。的目标 该项目旨在描述糖尿病引起的 离体心肌细胞Ca ~（2+）调节蛋白的表达及功能 肌细胞，并确定高血糖症的作用， 糖尿病性心肌病的发病机制和病理生理。测试 假设异常Ca 2+处理发生在单个细胞 水平，兴奋-收缩偶联的生物物理评估将 在分离自糖尿病大鼠的心室肌细胞中进行。 电压钳技术将用于确定全细胞Ca 2+和 肌膜L型钙通道Na/Ca交换电流的测定 Na/Ca交换功能。视频边缘检测和Ca 2 + 荧光测量（fura-2）将用作额外手段， 评价Na/Ca交换并分析SR Ca 2 + ATP酶和ryanodine 受体功能这些Ca 2+调节蛋白的mRNA的缺失 将使用北方印迹分析和蛋白质的量来确定 将通过蛋白质印迹分析进行评估。为了验证这个假设， 高血糖影响这些Ca 2+的表达和功能， 调节蛋白，分离的心室肌细胞，从两个糖尿病 而非糖尿病动物将维持在“糖尿病样”培养基中 (high葡萄糖）在短期原代培养中。康贝特人将以 高血糖对表达、修饰和功能特异性作用 钙调节蛋白质。初步数据显示，几天之内， 在“糖尿病样”培养基中培养的正常肌细胞表现出 松弛时间延长，动作电位持续时间延长， 胞浆Ca 2+清除，类似于糖尿病心肌细胞。这些 实验将提供重要的新见解的发病机制 和糖尿病心肌病的早期事件。综上所述各项 实验将解决Ca 2+调节蛋白在 糖尿病心肌病，研究其功能和表达， 单细胞水平。成人肌细胞培养系统提供了一种 明确的方法，从阐明基本机制的高 可能导致肌细胞功能障碍的葡萄糖 在糖尿病中。

Heart disease is the leading cause of death in diabetic patients, and considerable evidence is now available to support the existence of a specific diabetic cardiomyopathy that is independent of coronary artery disease and hypertension. Functional and biochemical data acquired from multicellular cardiac preparations of diabetic animals support the view that cellular mechanisms controlling cytosolic Ca2+ on a beat-to-beat basis are abnormal and contribute to impaired relaxation. The goal of this project is to characterize diabetes-induced changes in the expression and function of Ca2+ regulating proteins in isolated cardiac myocytes, and to determine the role of hyperglycemia in the pathogenesis and pathophysiology of diabetic cardiomyopathy. To test the hypothesis that abnormal Ca2+ handling occurs at the single cell level, biophysical assessment of excitation-contraction coupling will be carried out in ventricular myocytes isolated from diabetic rats. Voltage clamp techniques will be used to determine whole-cell Ca2+ and Na/Ca exchange currents as a measure of sarcolemmal L-type Ca2+ channel and Na/Ca exchanger function. Video edge-detection and Ca2+ fluorescence measurements (fura-2) will be used as additional means to evaluate Na/Ca exchange and to analyze SR Ca2+ ATPase and ryanodine receptor function. Abundance of mRNA for these Ca2+ regulating proteins will be determined using northern blot analysis and amount of protein will be assessed by western blot analyses. To test the hypothesis that hyperglycemia influences the expression and function of these Ca2+ regulating proteins, isolated ventricular myocytes from both diabetic and nondiabetic animals will be maintained in a "diabetic-like" medium (high glucose) in short-term primary culture. We will determine the specific role of hyperglycemia on expression, modification and function of calcium regulating proteins. Preliminary data show that within days, normal myocytes cultured in the "diabetic-like" medium exhibit prolonged relaxation, prolonged action potential durations, and slowed cytosolic Ca2+ clearing, similar to that of diabetic myocytes. These experiments will provide important new insights into the pathogenesis and early events of diabetic cardiomyopathy. Taken together, these experiments will resolve the role of Ca2+ regulating proteins in diabetic cardiomyopathy by studying their function and expression at the single cell level. The adult myocyte culture system provides a well-defined method from elucidating fundamental mechanisms of high glucose that may contribute to the development of myocyte dysfunction in diabetes.