心肌梗死后心肌纤维化小鼠心肌线粒体功能和能量代谢重塑相关性的转录组学分析

A transcriptomic analysis of correlation between mitochondrial function and energymetabolism remodeling in mice with myocardial fibrosis following myocardial infarction

目的探究心肌梗死小鼠心肌纤维化过程中线粒体呼吸功能的变化,阐述其与糖酵解通量增加的相关性。方法选取40只C57BL/6N小鼠,随机分为实验组(心肌梗死组)和对照组(假手术组),20只/组。实验组采用结扎冠状动脉左前降支的方法构建小鼠心肌梗死模型,对照组除不对左前降支进行结扎外,其他操作均与实验组相同。选取心肌梗死后28d和假手术组小鼠各5只进行安乐死取材,取左心室组织样本进行转录组学测序,采用FPKM法计算基因表达量,寻找差异表达基因。并通过GO和KEGG数据库对差异基因进行富集分析,寻找影响疾病进程的相关通路。通过绘制热图,展示富集分析中显示的通路和相关基因的表达差异,并对体外培养的小鼠原代CFs通过促纤维化激动剂TGF-β1或溶剂对照干预,通过Seahorse实验检测其线粒体呼吸和糖酵解水平。结果通过心脏大体图和心脏超声验证心梗模型构建成功,心肌梗死组舒张期左心室内径增加(P<0.05),收缩期左心室内径增加更为显著(P<0.001),左心室射血分数明显下降,差异有统计学意义(P<0.0001)。与假手术组相比,心肌梗死组上调基因124个,下调基因106个。GO和KEGG富集分析显示差异表达基因显著富集在脂肪酸代谢、细胞器等代谢通路和线粒体中。基因表达热图显示脂肪酸β氧化和线粒体功能障碍,相反糖酵解水平增加。Seahorse实验中,与溶剂对照组(Vehicle组)相比,TGF-β1干预组心肌成纤维细胞基础和最大呼吸水平明显降低,基础和最大糖酵解水平升高,差异均有统计学意义(P<0.0001)。结论在心肌纤维化过程中,心肌成纤维细胞能量代谢重塑,线粒体功能下降,相反更倾向于通过糖酵解产生能量。

Objective To investigate the changes of mitochondrial respiratory function during myocardial fibrosis in mice with myocardial infarction(MI)and its correlation with the increase of glycolytic flux.Methods Forty C57BL/6N mice were randomized into two equal groups to receive sham operation or ligation of the left anterior descending coronary artery to induce acute MI.At 28 days after the operation,5 mice from each group were euthanized and left ventricular tissue samples were collected for transcriptomic sequencing.FPKM method was used to calculate gene expression levels to identify the differentially expressed genes(DEGs)in MI mice,which were analyzed using GO and KEGG databases to determine the pathways affecting the disease process.Heat maps were drawn to show the differential expressions of the pathways and the related genes in the enrichment analysis.In primary cultures of neonatal mouse cardiac fibroblasts(CFs),the changes in mitochondrial respiration and glycolysis levels in response to treatment with the pro-fibrotic agonist TGF-β1 were analyzed using Seahorse experiment.Results The mouse models of MI showed significantly increased diastolic and systolic left ventricular diameter(P<0.05)and decreased left ventricular ejection fraction(P<0.0001).A total of 124 up-regulated and 106 down-regulated DEGs were identified in the myocardial tissues of MI mice,and GO and KEGG enrichment analysis showed that these DEGs were significantly enriched in fatty acid metabolism,organelles and other metabolic pathways and in the mitochondria.Heat maps revealed fatty acid beta oxidation,mitochondrial dysfunction and increased glycolysis levels in MI mice.In the primary culture of CFs,treatment with TGF-β1 significantly reduced the basal and maximum respiratory levels and increased the basal and maximum glycolysis levels(P<0.0001).Conclusion During myocardial fibrosis,energy metabolism remodeling occurs in the CFs,manifested by lowered mitochondrial function and increased energy generation through glycolysis.