Genetic Editing of Ca Cycling in Diabetic Cardiomyopathy

描述（由申请人提供）：糖尿病损害左心室收缩和舒张功能，并与兴奋-收缩偶联和信号传导途径的各个步骤中细胞水平的许多异常相关。实验性糖尿病性心肌病的两个关键异常是肌浆网（SR）功能缺陷（与细胞内钙处理异常相关）和磷脂酰肌醇3-OH（PI3-）激酶活性和GLUT-4表达降低。在糖尿病心脏中已经确定了舒张期间SR Ca 2+摄取不足，并且与SR Ca 2 +-ATP酶（SERCA2a）的表达和活性降低相关。此外，PI3激酶活性降低导致Akt活化降低，随后GLUT-4表达降低导致葡萄糖摄取受损和肥大。利用基因转移靶向心肌细胞中的特定通路，我们将试图了解糖尿病心脏中的兴奋-收缩偶联变化是否发生在明显心力衰竭发展之前和/或参与变化的开始或恶化。我们将检验以下假设：1）PI3-激酶活性的降低与EC偶联途径有关，因此导致糖尿病心脏的收缩功能障碍：2）代谢干预将防止糖尿病心脏的心脏收缩功能障碍：和3）胰岛素的调节-在糖尿病大鼠中，体内的EC介导的信号转导通路可以有利地调节EC偶联通路的改变，并改善心脏能量学和存活率。为了验证这些假设，我们将使用病毒载体在体外和体内心肌细胞中表达野生型和突变形式的特异性信号分子。我们将研究PI3激酶、Akt和GLUT-4对糖尿病心肌病心脏收缩功能、能量学、存活和重塑的影响。除了人和成年大鼠心室肌细胞的原代培养物外，还将使用1型和2型糖尿病的动物模型。

DESCRIPTION (provided by applicant): Diabetes impairs left ventricular systolic and diastolic function and is associated with a number of abnormalities at the cellular level in the various steps of excitation-contraction coupling and signaling pathways. Two key abnormalities in experimental diabetic cardiomyopathy are a defect in sarcoplasmic reticulum (SR) function, which is associated with abnormal intracellular calcium handling, and a reduction in phosphatidyl-inositol 3-OH (PI3-) kinase activity and GLUT-4 expression. Deficient SR Ca2+ uptake during relaxation has been identified in diabetic hearts and has been associated with a decrease in the expression and activity of SR Ca2+-ATPase (SERCA2a). Furthermore, a decrease in PI3 kinase activity leading to a decrease in Akt activation and subsequently a decrease in GLUT-4 expression leads to impaired glucose uptake and hypertrophy. Using gene transfer to target specific pathways in cardio-myocytes, we will try to understand whether the excitation-contraction coupling changes in diabetic hearts occur before overt heart failure develops and/or participate in the initiation or the worsening of the changes. We will test the following hypotheses: 1) that the decrease in PI3-Kinase activity is linked to the EC coupling pathways and so contributes to the contractile dysfunction in diabetic hearts: 2) that metabolic interventions will protect against cardiac contractile dysfunction in diabetic hearts: and 3) that modulation of insulin-mediated signal transduction pathways in vivo can favorably modulate alterations of the EC coupling pathways and improve cardiac energetics and survival in diabetic rats. To test these hypotheses, we will use viral vectors to express wild-type and mutant forms of specific signaling molecules in cardiomyocytes in vitro and in vivo. We will examine the effects of PI3-Kinase, Akt, and GLUT-4 on contractile function, energetics and survival and remodeling in diabetic cardiomyopathic hearts. Animal models of type 1 and type 2 diabetes will be utilized in addition to primary cultures of human and adult rats ventricular myocytes.