Shh信号通路受损致糖尿病心肌DNA双链断裂异常积累的作用及机制研究

The diabetes is the number one health challenge in China. The people with diabetes are 2 to 4 times more likely to have heart disease than people without diabetes. However, the mechanisms by which diabetes results in cardiac dysfunction still remains to be determined. The DNA double strand breaks (DSBs), as the most serious genomic lesions, their persistent exist and inappropriate repair can cause the overload of cellular DSB and subsequently induce cell apoptosis or senescence. Emerging evidence has shown the accumulation of DSBs damaged by metabolic disorder or oxidative stress may drive cardiac dysfunction in patients with diabetes. But how DSBs and their repair are regulated in diabetes are still unclear. Our recently study showed that impaired Shh pathway can augument cardiac dysfunction in type 1 diabetic mice with myocardial infarction. Based on the findings of Shh protection on human tumor cells by inducing DSBs repair, the Shh pathway has been implicated in the DSBs and their repairs pathway. So we hypothesize that Shh pathway is involved in the regulation of DSB and DSB repair in heart, its inactivation induced by diabetes may result in DSB too severe to overload in cardiomyocytes. .Brahma-related gene 1 (Brg1), a catalytic subunit of an important chromatin remodeling complex, regulates the nuclear access of DNA interacting proteins and recent investigations have revealed that it is involved in DDR and cell survival after DNA damage. Our preliminary data showed that γ-H2AX foci were dramatically increased in cardiomyocytes at the ischemic zone of acute myocardial infarction mice heart. Brg1 was also upregulated in this area and translocated to the nucleus, indicating that Brg1 is likely involved in DSB repair in cardiomyocytes. The overexpression of Brg1 in cultured neonatal rat cardiomyocytes attenuated γ-H2AX after oxidative stress suggesting that higher Brg1 levels may promote DSB repair. Furthermore, Brg1 has dual roles in the regulation of Shh signaling pathway. Our preliminary data also found Shh agonist upregulated the expression of Brg1 in heart; and Brg 1 overexpression in cardiomyocytes increased the Gli1、Ptch1 protein levels as well. These data indicated that Shh may regulate cardiac DSB and DSBs repair through the activation of Brg1..Based on these novel and exciting findings, we hypothesize that Shh pathway is directly involved in DSB and DNA repair; impaired Shh pathway induced by diabetes accelerates the DSBs and their dysregulation repair through inactivation of Brg1. We propose to create in vivo and in vitro models of DSBs in diabetic heart. With these models, we will test whether Shh pathway is required for or promotes DNA DSB repair. The completion of the proposed study will provide new effective ways to enhance DNA repair and thus prevent as well as treat cardiac dysfunction in patient with diabetes.

我国糖尿病和准糖尿病人已超过2亿。糖尿病心脏并发症导致的心功能障碍、心力衰竭是糖尿病常见和重要的死亡原因，但机制不完全清楚。DNA双链断裂（DSB）作为最严重的DNA损伤形式，在心肌中的异常堆积能诱导细胞凋亡导致心功能障碍。我们的研究已表明Shh通路受损是糖尿病心功能障碍的重要原因；Shh通路在肿瘤细胞能调控细胞DSB修复减少DNA损伤。染色质重塑复合物Brg1作为调控肿瘤细胞DSB修复的重要因子，在毛囊和端脑发育中介导Shh的作用。因此我们的研究设想是糖尿病受损的Shh通路通过抑制心脏Brg1造成DSB异常堆积，诱发细胞凋亡，导致心功能障碍。本项目主要研究糖尿病Shh通路受损与心脏DSB及修复的关系；探讨Shh通路通过Brg1调控心脏DSB修复的作用；研究Brg1对Shh通路的转录激活作用及机制。该研究将提出改善Shh通路治疗糖尿病心功能障碍的新策略和新观点

急性心肌梗死是威胁人类健康的常见心血管疾病之一。在心脏中，染色质重塑复合物Brg1通过调控基因表达促进心脏胚胎发育进程和心肌肥厚等病理生理过程。本项目构建腺病毒载体Ad-Brg1 WT、慢病毒载体Lenti-Brg1 shRNA 调控Brg1表达，在体内外缺血缺氧模型中研究Brg1对缺血心肌的保护作用。结果显示AMI时，激活Brg1使AMI小鼠心重比、心肌梗死面积、心肌纤维化显著降低，左室收缩末期内径和左室舒张末期内径显著降低，短轴缩短率和射血分数显著增加，梗死周边区凋亡细胞数目减少；沉默Brg1则使AMI小鼠心重比升高、心肌梗死面积升高、心肌纤维化增加，左室收缩末期内径和左室舒张末期内径显著增加，短轴缩短率和射血分数显著降低，梗死周边区凋亡细胞数目增多。激活Brg1使AMI小鼠心脏梗死周边区Shh信号通路Ptc1、Gli1、Gli2、Shh蛋白表达升高，沉默Brg1则使上述蛋白表达降低。利用ChIP方法发现心肌梗死周边区Shh信号通路靶基因Ptc1、Gli1/2和Shh启动子区域与Brg1结合增强，说明过表达Brg1后，能够促进Shh信号通路关键基因的转录，激活该通路。利用心肌细胞氧葡萄糖剥夺（oxygen and glucose deprivation, OGD）模拟体外心肌缺血的研究结果与体内实验一致。上述结果提示，Brg1是Shh信号通路的上游调控因子，心肌梗死时Brg1通过激活Shh信号通路发挥心脏保护作用。随后，项目组研究Shh信号通路在缺血缺氧诱导的心肌细胞DNA损伤中的保护作用。结果显示，与OGD 6 h、OGD 12 h组相比，激动Shh信号通路，OGD细胞γH2AX、p-ATM表达明显下降，细胞存活率上升，p-p53、cleaved-caspase-3表达下降， Bcl-2/Bax比例上升，细胞存活率上升；抑制Shh通路的心肌细胞较OGD组γH2AX、p-ATM表达明显上升，p-p53、cleaved-caspase-3表达上升， Bcl-2/Bax比例下降，细胞存活率下降。由此提示，激活Sonic hedgehog通路可以通过抑制ATM磷酸化，减少γH2AX表达，改善缺血缺氧诱导的心肌细胞DNA损伤，抑制细胞凋亡。本研究为心肌梗死的防治提供了新线索和思路。