Mitochondrial Polymerase Gamma Dysfunction and Aging Cause Cardiac Nuclear DNA Methylation Changes [100718_MM9_EXP]

Cardiomyopathy (CM) is an intrinsic weakening of the myocardium with contractile dysfunction and congestive heart failure (CHF). CHF has been postulated to result from decreased mitochondrial energy production and oxidative stress. The effects of decreased mitochondrial oxygen consumption can also accelerate with aging, with the mitochondrial theory of aging forming the basis of this knowledge. We previously showed DNA methylation changes in human hearts with CM. This was associated with mitochondrial DNA depletion, being another molecular marker of CM. We examined the relationship between mitochondrial dysfunction and cardiac epigenetic DNA methylation changes in both young and old mice. We used genetically engineered C57Bl/6 mice transgenic for a cardiac-specific mutant of the mitochondrial polymerase ï§ï€ (termed Y955C). Y955C mice undergo left ventricular hypertrophy (LVH) at a young age (~94 days old), and LVH decompensated to CHF at old age (~255 days old). In Y955C hearts, 95 differentially expressed genes were found, while 4,452 genes were differentially expressed in aged hearts. Moreover, cardiac DNA methylation patterns differed between Y955C (4,506 peaks with 68.5% hypomethylation) and aged hearts (73,286 peaks with 80.2% hypomethylated). Correlatively, of the 95 Y955C-dependent differentially expressed genes, 30 genes (31.6%) also displayed differential DNA methylation; in the 4,452 age dependent differentially expressed genes, 342 gene (7.7%) displayed associated DNA methylation changes. Both Y955C and aging demonstrated significant enrichment of CACGTG-associated E-box motifs in differentially methylated regions. Cardiac mitochondrial polymerase dysfunction alters nuclear DNA methylation. Furthermore, aging causes a robust change in cardiac DNA methylation that is partially associated with mitochondrial polymerase dysfunction. This study addresses how Y955C-mutated mitochondrial DNA polymerase g and aging affect cardiac (left ventricle) gene expression and epigenetic nuclear DNA methylation. Each sample was fluorescently labeled and hybridized to Roche Nimblegen 12X135kb MM9 Gene Expression Arrays.心肌病（Cardiomyopathy, CM）是一类以心肌内在性弱化、收缩功能障碍及充血性心力衰竭（Congestive Heart Failure, CHF）为特征的疾病。现有假说认为，充血性心力衰竭的发生与线粒体能量产生减少及氧化应激密切相关。线粒体耗氧量降低的负面影响可随衰老进程进一步加剧，而衰老的线粒体理论正是这一认知的核心理论基础。我们此前的研究已证实，人类心肌病患者的心脏组织中存在DNA甲基化异常改变，该改变与线粒体DNA缺失存在关联，而线粒体DNA缺失亦是心肌病的另一分子标志物。本研究以年轻及衰老小鼠为模型，探究了线粒体功能障碍与心脏表观遗传DNA甲基化改变之间的调控关系。我们使用了经基因工程改造的C57Bl/6小鼠，该小鼠心肌特异性表达线粒体聚合酶γ（mitochondrial polymerase γ）突变体Y955C。Y955C小鼠在幼年阶段（约94日龄）即可出现左心室肥厚（Left Ventricular Hypertrophy, LVH），并在衰老阶段（约255日龄）时左心室肥厚失代偿，进展为充血性心力衰竭。在Y955C小鼠的心脏组织中，共鉴定得到95个差异表达基因；而衰老小鼠的心脏组织中则存在4452个差异表达基因。此外，Y955C小鼠与衰老小鼠的心脏DNA甲基化模式存在显著差异：Y955C小鼠心脏中共检测到4506个甲基化峰，其中68.5%为低甲基化区域；衰老小鼠心脏中共检测到73286个甲基化峰，其中80.2%为低甲基化区域。相关性分析结果显示，在95个Y955C依赖性差异表达基因中，有30个（占比31.6%）同时存在DNA甲基化改变；而在4452个衰老依赖性差异表达基因中，有342个（占比7.7%）伴随DNA甲基化变化。在两组模型的差异甲基化区域中，均显著富集了与CACGTG序列相关的E-box基序。本研究证实，心脏线粒体聚合酶功能异常可改变细胞核DNA甲基化谱；此外，衰老会引发心脏DNA甲基化的大范围重塑，且该重塑过程部分与线粒体聚合酶功能异常相关。本研究旨在阐明Y955C突变型线粒体DNA聚合酶γ与衰老如何调控心脏（左心室）的基因表达及细胞核DNA表观遗传甲基化修饰。所有实验样本均经荧光标记后，与罗氏NimbleGen 12X135kb MM9基因表达芯片进行杂交。