CALCIUM REGULATION IN THE DIABETIC HEART

心脏病是糖尿病患者的主要死亡原因， 现在有相当多的证据支持 独立于冠状动脉的特定的糖尿病心肌病 疾病和高血压。获取的功能和生化数据来自 糖尿病动物的多细胞心脏制剂支持这一观点 一次又一次控制细胞内钙离子的细胞机制 基础是不正常的，并导致松弛受损。的目标是 这个项目是为了描述糖尿病引起的 钙调节蛋白在离体心组织中的表达及功能 并确定高血糖在心肌梗死中的作用。 糖尿病心肌病的发病机制和病理生理。为了测试 单细胞出现异常钙处理的假说 水平，兴奋-收缩耦合的生物物理评估将 在糖尿病大鼠分离的心室肌细胞中进行。 电压钳技术将用于测定全细胞钙和 钠/钙交换电流作为肌膜L型钙通道的量度 和Na/Ca交换功能。视频边缘检测与钙离子交换 荧光测量(Fura-2)将作为额外手段用于 评价Na/Ca交换，分析肌浆网钙-三磷酸腺苷酶和兰尼定 受体功能。这些钙调节蛋白的mRNA丰度 将通过Northern印迹分析和蛋白质含量来确定 将通过西方印迹分析进行评估。来检验这一假设 高血糖影响这些钙离子的表达和功能 糖尿病患者分离的心室肌细胞的调节蛋白 而非糖尿病动物将被维持在类似糖尿病的介质中 (高糖)短期原代培养。我们将确定 高血糖在表达、修饰和功能中的特殊作用 钙调节蛋白。初步数据显示，在几天内， 在“类糖尿病”培养液中培养的正常心肌细胞 松弛时间延长，动作电位持续时间延长，速度减慢 胞浆内钙的清除，类似于糖尿病心肌细胞。这些 实验将为发病机制提供重要的新见解。 以及糖尿病心肌病的早期事件。这些加在一起， 实验将解决钙调节蛋白在 通过研究它们在糖尿病心肌病中的功能和表达 单细胞水平。成人心肌细胞培养系统提供一种 从阐明HIGH的基本机制入手的明确方法 葡萄糖可能参与心肌细胞功能障碍的发生 糖尿病患者。

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.