糖尿病导致冠脉舒张功能受损的一项重要机制：ROS促进MuRF1介导的BK-β1亚基降解

Cardiovascular diseases are the leading cause of death in patients with diabetes mellitus. The large conductance Ca2+-activated K+ (BK) channels are abundantly expressed in coronary artery smooth muscle cells (SMCs). It has been shown that BK channel plays a pivotal role in the regulation of coronary blood flow. However, vascular BK channels are dysfunction in both type 1 and type 2 diabetes. Thus, a better understanding of the mechanism leading to BK channel dysfunction in diabetic vessels is of therapeutic significance for diabetic vascular complications. We and other investigators have reported that diminished BK channel function is mainly due to the downregulation of BK-β1protein level in diabetic vessels. Most importantly, overexpression of BK-β1 restored BK channel activity and preserved coronary function in diabetic mice, indicating that BK-β1 is a bona fide target for the treatment of diabetic vascular disorders. We are the first to demonstrate that reduced BK-β1 expression is associated with increase in muscle RING finger-1 (MuRF1) protein, a muscle specific E3 ligase, in diabetic vessels. Moreover, knockdown of MuRF1 gene rescues BK-β1 expression and protects BK channel function against diabetes. However, the molecular mechanism and signaling pathway underlying MuRF1-dependent BK-β1 proteolysis have not been delineated. In this proposal, we hypothesize that reactive oxygen species (ROS) promote MuRF1 expression and BK-β1 protein tyrosine nitration in coronary SMCs, which facilitates BK-β1 proteolysis and diminishes BK channel-mediated coronary vasodilation in diabetes. MuRF1 may serve as a molecular target for the treatment of diabetic vascular complications. Three specific aims are proposed: Aim 1: To determine the role of MuRF1 in the regulation of BK-β1 expression and BK channel-mediated coronary vasodilation in streptozotocin (STZ)-induced type 1 diabetic mice. Aim 2: To determine the roles of ROS signaling and NF-κB activity in MuRF1-dependent BK-β1degradation and coronary dysfunction in STZ-induced diabetic mice. Aim 3: To determine whether enhanced BK-β1 protein tyrosine nitration facilitates MuRF1 recognition and accelerates BK-β1 protein degradation in oxidative stress. Results from this proposed studyare clinically relevant. It will help us better understand the molecular mechanism leading to BK channelopathy and vasculopathy in diabetes, and help us identify a molecular target to develop a novel strategy for the treatment of diabetic vascular complications.

糖尿病导致冠脉舒缩功能下降是诱发冠心病的机制之一。大电导钙激活钾通道(BK)在冠脉平滑肌内广泛表达，糖尿病时BK通道介导的血管扩张功能受损，这与BK-β1亚基表达降低有关。我们首次发现在糖尿病冠脉平滑肌中MuRF1作为E3连接酶参与到BK-β1蛋白水解过程，但其分子机制及信号调节通路却不清楚。活性氧簇(ROS)是糖尿病血管疾病发生的关键因子，但ROS对离子通道的作用却所知甚少，我们首次发现过氧化氢(H2O2)对BK通道的影响与高糖环境作用相似，加速BK-β1亚基降解，并增加了BK-β1亚基的硝基化水平。据此，我们假设ROS促进冠脉平滑肌内MuRF1的表达及BK-β1蛋白的硝基化，两者共同加速了BK-β1亚基的蛋白水解，导致冠脉BK通道活性降低，舒缩功能受损。我们拟通过膜片钳及其他分子生物学手段，证实该假设，为糖尿病血管并发症的治疗提供一个新的方向。

背景：糖尿病导致冠脉舒缩功能下降是诱发冠心病的机制之一。大电导钙激活钾通道(BK)在冠脉平滑肌内广泛表达，糖尿病时BK通道介导的血管扩张功能受损，这与BK-β1亚基表达降低有关。MuRF1作为E3连接酶参与到BK-β1蛋白水解过程，但其分子机制及信号调节通路却不清楚。方法：应用糖尿病小鼠及miR技术、药物给予，以及膜片钳、其他生物学手段。结果：证实糖尿病血管平滑肌内BK-β1亚基表达降低，从而导致血管平滑肌内的BK通道功能下调，冠脉内MuRF1蛋白表达增加，且MuRF1作为E3介导冠脉BK-β1亚基的蛋白水解。糖尿病血管平滑肌内ROS生成增加、NFκB激活增加，更为重要的是，H2O2对BK通道动力学的影响与高糖环境作用相似，同时糖尿病冠脉平滑肌内BK-β1亚基蛋白的硝基化水平升高，证实糖尿病血管中ROS过表达在BK-β1亚基降解中扮演着重要角色，不仅通过NFκB信号途径导致MuRF1转录增加，而且同时增加了BK-β1亚基的转录后修饰，促使了MuRF1与BK-β1亚基的结合。