ASMase调控线粒体损伤依赖性铁死亡诱发糖尿病心肌病的机制研究

Myocardial mitochondrial damage is the leading cause of diabetic cardiomyopathy (DCM). However, the underlying mechanisms were remained obscure. Our previous studies had indicated that acid sphingomyelinase (ASMase) is highly expressed in diabetic hearts, whereas knockout of ASMase prevented myocardial mitochondrial damage and rescued DCM in diabetic mice. Together with the evidences that mitochondrial damage is known to induce ferroptosis, we propose speculation that ASMase may regulate mitochondrial injury-dependent ferroptosis which results in diabetic cardiomyopathy. To test this hypothesis, firstly, a model of high-glucose (HG, 30 mM) and palmitic acid (PA, 300 μM) cultured neonatal rat cardiomyocytes (NRCMs) is established to illuminate the underlying molecular mechanisms of myocardial mitochondrial damage. Secondly, we will observe the relationship between ferroptosis and DCM by investigation of activation of ferroptosis or myocardial mitochondrial damage in the development of DCM in multiple diabetic animal models with duration of diabetes, and then to reveal the important role of ferroptosis in development of DCM by observation the impact of ferroptosis on cardiomyocyte hypertrophy in HG+PA or C16:0 CER incubated NRCMs with different courses. Finally, the gene therapy (ASMase-/-) and drug treatment (ferroptosis inhibitor) were performed in diabetic rat models in order to explore new target for clinical diagnosis, prevention and treatment of DCM as well as the development of new drug.

心肌线粒体损伤是导致糖尿病心肌病（DCM）的重要原因，但其中的机制尚未完全阐明。我们前期研究发现，酸性鞘磷脂酶（ASMase）在糖尿病心肌中高表达，敲除ASMase可抑制糖尿病小鼠心肌线粒体损伤，改善DCM。已知线粒体损伤可诱发铁死亡。据此我们提出：ASMase通过调控心肌线粒体损伤介导的铁死亡，进一步诱导DCM的发生发展这一假说。为验证此假说，我们将：①构建高糖/高脂孵育诱导的心肌细胞肥大模型，阐明ASMase影响心肌线粒体损伤的分子机制；②构建不同病程糖尿病小鼠模型，查明在DCM发生发展过程中心肌线粒体损伤与铁死亡的激活情况；③给予糖尿病小鼠铁死亡抑制剂或敲除ASMase，揭示抑制铁死亡对DCM的治疗作用。本项目将从铁死亡这个新视角拓展对DCM发病机制的认识并为DCM的防治提供新思路。

糖尿病心肌病（DCM）是导致糖尿病患者死亡的重要原因，但发病机制不明且缺乏有效防治措施。本项目研究发现使用酸性鞘磷脂酶ASMase抑制剂丙咪嗪，siRNA-ASMase或心脏特异性敲除ASMase均可显著减轻高脂饮食诱导的小鼠心功能障碍、心脏重构和细胞凋亡，以及高糖+高脂、C16神经酰胺孵育诱导的心肌细胞肥大、ROS产生和细胞凋亡，这一作用可被NOX4抑制剂阻断，表明ASMase促进DCM的发生发展且其介导的心肌损伤依赖于NOX4活化。本项目还发现铁死亡和线粒体功能障碍与糖尿病心肌病密切相关，通过腹腔注射或体外预孵育铁死亡抑制剂ferostatin-1在体内外显著抑制心肌细胞铁死亡，逆转心功能障碍和心脏重构。二甲双胍作为治疗糖尿病的一线药物，通过抑制铁死亡、改善心肌线粒体功能发挥对糖尿病心肌细胞的保护作用，它降低了棕榈酸和铁死亡诱导剂erastin增加的LPO、MDA水平和ROS生成，并上调了线粒体膜电位，提示二甲双胍直接抑制铁死亡，但加入AMPK抑制剂Compound C阻断了二甲双胍或铁死亡抑制剂对LPO、MDA、GSH、ROS生成、膜电位、GPX和AMPK蛋白表达等的影响，提示二甲双胍激活AMPK构成重要分子机制。本项目从鞘磷脂代谢和细胞死亡这个新视角拓展对DCM发病机制的认识并为DCM的防治提供了新思路。