甲基丙二酸对心肌缺血/再灌注损伤的影响及机制研究  

Effects and mechanisms of methylmalonic acid on myocardial ischemia/reperfusion injury

作　　者：刘志远 王心怡 孙芳芳 陶凌 陈希瑶 张富洋 LIU Zhiyuan;WANG Xinyi;SUN Fangfang;TAO Ling;CHEN Xiyao;ZHANG Fuyang(Department of Cardiology,Air Force Medical University,Xi'an 710032,China;Department of Geriatrics,Xijing Hospital,Air Force Medical University,Xi'an 710032,China)

机构地区：[1]空军军医大学西京医院心血管内科,陕西西安710032 [2]空军军医大学西京医院老年病科,陕西西安710032

出　　处：《空军军医大学学报》2025年第2期160-166,共7页Journal of Air Force Medical University

基　　金：国家自然科学基金(82270389,82422004,82170337)。

摘　　要：目的明确甲基丙二酸(MMA)对心肌缺血/再灌注(MI/R)损伤的影响,并探索其下游分子机制。方法通过皮下植入微量渗透泵向C57BL/6J小鼠持续泵注溶媒(Vehicle)或MMA后,小鼠随机接受冠状动脉左前降支结扎所建立的MI/R手术或假手术。术后3 h取左心室组织并利用RNA测序(RNA-Seq)分析左心室的基因表达变化。术后24 h利用M型超声心动图评估左心室收缩功能,利用TUNEL染色评估心肌细胞凋亡比例。术后14 d利用Masson三色法染色评估左心室的间质纤维化程度。分离C57BL/6J乳鼠原代心肌细胞并给予具有细胞膜通透性的甲基丙二酸二乙酯(D-MMA)或Vehicle孵育12 h,利用细胞活力检测试剂盒检测心肌细胞活力,利用透射电镜检测心肌细胞线粒体超微结构变化,利用Seahorse能量代谢分析仪检测心肌细胞线粒体功能变化。结果MI/R+MMA组与MI/R+Vehicle组相比,MMA显著加重MI/R导致的左心室收缩功能障碍(P<0.01),增加心肌细胞凋亡比例(P<0.01),并加重术后14 d心肌间质纤维化程度(P<0.01)。RNA-Seq结果提示,MMA显著抑制心肌细胞一系列线粒体相关通路的基因表达。与在体实验结果一致,D-MMA孵育直接导致乳鼠原代心肌细胞活力降低(P<0.05,P<0.01),并破坏线粒体超微结构。Seahorse能量代谢分析检测显示D-MMA孵育显著抑制乳鼠原代心肌细胞线粒体呼吸功能(P<0.01)。结论MMA导致心肌细胞线粒体功能障碍并加重MI/R损伤。Objective To elucidate the impact of methylmalonic acid(MMA) on myocardial ischemia/reperfusion(MI/R) injury and to explore its downstream molecular mechanisms.Methods C57BL/6J mice were subcutaneously implanted with micro-osmotic pumps to continuously infuse either Vehicle or MMA.The mice were then randomly subjected to MI/R via left anterior descending coronary artery ligation or a Sham operation.Left ventricular tissues were collected 3 h post-surgery,and the changes of gene expression in left ventricle were analyzed by RNA-sequencing(RNA-Seq).Left ventricular systolic function was assessed using M-mode echocardiography 24 h post-surgery.The percentage of apoptosis in cardiomyocytes was evaluated using TUNEL staining.The degree of left ventricular interstitial fibrosis was assessed using Masson's trichrome staining 14 d post-surgery.Primary cardiomyocytes isolated from neonatal C57BL/6J mice were incubated with cell membrane-permeable diethyl methylmalonic acid(D-MMA) or Vehicle for 12 h.Cardiomyocyte viability was determined using a cell viability assay kit.Mitochondrial ultrastructural changes were observed via transmission electron microscopy,and mitochondrial function changes of cardiomyocytes were detected using the Seahorse analyzer.Results Compared with MI/R + Vehicle group,MMA significantly exacerbated MI/R-induced left ventricular systolic dysfunction(P<0.01),increased the percentage of apoptosis in cardiomyocytes(P<0.01),and worsened myocardial interstitial fibrosis 14 d post-surgery(P<0.01).RNA-Seq analysis indicated that MMA significantly inhibited gene expression of a series of mitochondrial-related pathways in cardiomyocytes.Consistent with the results of in vivo experiments,D-MMA incubation directly reduced neonatal mouse cardiomyocyte viability(P<0.05,P<0.01),and damaged the ultrastructure of mitochondria.Seahorse analysis showed that D-MMA incubation significantly inhibited mitochondrial respiration of primary neonatal rat cardiomyocytes(P<0.01).Conclusion MMA induces mitochondrial dysfunction

关 键 词：甲基丙二酸 缺血/再灌注损伤 线粒体 小鼠 

分 类 号：R542.22[医药卫生—心血管疾病]