线粒体A激酶锚定蛋白121表达下调致糖尿病心肌缺血易损性增加及机制

Morbidity and mortality from ischemic heart disease is significantly increased in type 2 diabetic patients compared with non-diabetic control subjects. We previously demonstrated that hyperglycemia aggravated myocardial oxidative stress and subsequently, amplification of cardiac injury following ischemia/repefusion (MI/R) (J Cell Mol Med 2013). Moreover, augmenting insulin signaling inhibited oxidative stress in ischemic/reperfused hearts and thus reduced myocardial injury (Am J Physiol Endocrinol Metab 2010). These results indicated that oxidative stress was closely associated with cardiac ischemic intolerence. The scaffold protein A-kinase anchor protein 121 (AKAP121) assembles a multivalent signaling complex on the outer mitochondrial membrane, playing a pivotal role in mtochondrial functions and cell survival. Our preliminary experiments showed that AKAP121 expression was reduced in ob/ob mouse hearts. Meanwhile, increased production of reactive oxygen species (ROS) and aggravated MI/R injury were observed in ob/ob mice. Furthermore, overexpression of AKAP121 in cardiomyocytes abolished high glucose/high fat-induced mitochondrial ROS production. Based on these data, we hypothesized that myocardial AKAP121 downregulation and its induced mitochondrial oxidative stress contributes to the increased vulnerability of diabetic heart to ischemia/reperfusion injury. The present study was designed to determine the exact relationship among myocardial AKAP121 expression, mitochondrial oxidative stress and cardiac ischemic intolerance in diabetes and also determine whether AKAP121 downregulation modulates mitochondrial function during MI/R and if so, to investigate its molecular basis. All of these would be tested both in AKAP121 knockout and ob/ob mice. Clarifying this hypothesis might gain further insight into the pathogenesis of myocardial ischemic intolerance in diabetic hearts and provide novel possible targets for cardioprotective interventions.

糖尿病患者缺血性心脏病的发病率和死亡率均远高于正常人群，亟需探寻有效防治措施。我们前期发现，高血糖可加剧缺血/再灌注（I/R）心肌氧化应激，使损伤加重（JCMM 2013），而强化胰岛素治疗可抑制I/R心肌氧化/硝化应激，减轻心肌损伤（AJP-E&M 2010），提示氧化应激加重与糖尿病缺血心肌易损性增加相关。我们新近预实验表明，糖尿病ob/ob小鼠心肌中A激酶锚定蛋白121（AKAP121）表达减少，且伴随心肌ROS生成增多，上调AKAP121表达可抑制线粒体ROS生成。据此我们提出科学假设：AKAP121表达缺失导致的线粒体氧化应激加重可能是糖尿病心肌缺血易损性增加的机制之一。本项目拟在前期基础上，应用药理学与基因干预方法，研究糖尿病状态下AKAP121表达变化及机制，及其调控的线粒体氧化应激对心肌缺血易损性的影响，期望为探索糖尿病性缺血性心脏病的发病机制及防治策略提供新思路和新靶点。

糖尿病患者缺血性心脏病的发病率和死亡率均远高于正常人群，急需探寻有效防治措施。我们前期实验发现，高血糖可加剧缺血/再灌注心肌氧化应激，使损伤加重，而强化胰岛素治疗可抑制心肌氧化应激，减轻心肌损伤，提示氧化应激加重与糖尿病缺血心肌易损性增加相关。进一步实验结果表明，糖尿病小鼠心肌中A激酶锚定蛋白121（AKAP121，由AKAP1基因编码）表达减少，且伴随心肌ROS生成增多，上调AKAP121表达可抑制线粒体ROS生成。据此我们提出科学假设：AKAP121表达缺失导致的线粒体氧化应激加重可能是糖尿病心肌缺血易损的机制之一。.本项目在前期基础上，上调糖尿病心肌AKAP121的表达，发现过表达AKAP121的糖尿病小鼠心脏功能明显高于对照组。同时，上调AKAP121可以改善糖尿病小鼠慢性心肌缺血所致的心肌纤维化。此外，我们发现缺血本身即可抑制AKAP121的表达，随着心梗时间延长，存活心肌的AKAP121表达显著减少。上调AKAP121可抑制心肌细胞ROS生成并减少心肌细胞凋亡。随即，我们利用CRISPR/Cas9技术成功建立了AKAP1基因敲除小鼠。我们发现与野生型相比，AKAP1敲除鼠的左室内径显著增大，左室重量明显增加，心功能有下降趋势，提示长期AKAP1敲除可促进心肌肥大，降低心功能。此外在AKAP1基因敲除小鼠的心脏，肝脏，骨骼肌和白色脂肪组织中，线粒体钙摄取的关键分子MICU1的表达均显著降低，提示AKAP1调控线粒体钙摄取。进一步研究发现糖尿病小鼠心肌线粒体钙摄取降低，上调糖尿病鼠线粒体钙摄取可通过增强线粒体抗氧化系统活性抑制线粒体ROS生成，减少心肌细胞凋亡，最终改善糖尿病心脏功能，抑制心肌损伤。.本项目的开展明确了AKAP121可有效保护糖尿病缺血心肌，减轻心肌损伤，抑制ROS生成并减少心肌细胞凋亡；进一步研究发现AKAP1敲除后线粒体钙摄取关键分子MICU1表达降低，而糖尿病心肌MICU1及其介导的线粒体钙摄取显著减少，上调心肌线粒体钙摄取可抑制心肌线粒体ROS生成并改善糖尿病心脏功能，抑制心肌损伤。本研究提示AKAP121在糖尿病心肌保护中发挥重要作用，有望为糖尿病心肌保护提供新思路和新靶点。