Novel function of a mitochondria phosphatase in cardiac development

项目总结 线粒体是心肌细胞分化和心脏形态发生所必需的。基因突变 编码线粒体蛋白的基因经常会导致先天性心脏病，这突显了 阐明心脏发育过程中线粒体动态平衡的关键分子途径(S)。蛋白质酪氨酸 定位于线粒体1的磷酸酶(PTPMT1)是一种双特异性线粒体磷酸酶 由核DNA编码。PTPMT1仅定位于线粒体，锚定在内部 线粒体膜。PTPMT1在不同发育阶段的细胞质雄性不育中均有表达。至 为了确定PTPMT1在CMS中的作用，我们产生了Ptpmt1结构性CM特异性基因敲除(CKO)小鼠 模特。我们的初步数据显示，Ptpmt1 CKO小鼠表现出胚胎致死性。Ptpmt1 CKO小鼠 表现为致密带心肌变薄，心肌细胞增殖减少。我们还显著地观察到 Ptpmt1 CKO心脏线粒体呼吸频率降低和线粒体形态异常， 证明PTPMT1在CMS的发育和维持正常线粒体中起关键作用 动态平衡。我们还检查了之前描述的Ptpmt1CKO心脏中的PTPMT1底物与 对照，但没有发现证据支持它们是CMS中PTPMT1的直接底物。为了获得更多 为了深入了解PTPMT1缺失对通路的影响，我们对Ptpmt1 CKO心脏进行了RNA-SEQ分析。 生物信息学分析表明，PTPMT1的缺失显著激活了激活的转录因子 4(ATF4)途径。ATF4调控一系列允许细胞存活的适应性基因的表达 线粒体应激期。然而，在持续的应激条件下，ATF4促进细胞- 细胞周期停滞、细胞凋亡和衰老。值得注意的是，ATF4在全球ATF4单倍体缺陷和 平滑肌特异性ATF4基因敲除小鼠减轻内质网应激，减轻内侧动脉和动脉粥样硬化 钙化，突出了通过偏爱缓解压力的适应效果而不是 通过调节ATF4的激活而产生的不适应效应。上述证据使我们得出这样的假设： PTPMT1通过调节特定底物在心脏发育中发挥重要作用。 在Ptpmt1基因敲除的CMS中，线粒体应激激活的ATF4的部分丢失可能会改善， 但ATF4的完全缺失可能会加剧Ptpmt1 CKO表型。据此，我们的具体目标是：1. PTPMT1在心肌线粒体动态平衡和心脏发育与功能中的作用 分析Ptpmt1 CKO小鼠，并通过执行以下步骤鉴定CMS中PTPMT1的内源性底物 无偏见的脂类组学和磷蛋白质组学分析；以及2.确定部分或 CMS中ATF4基因完全缺失对CKO小鼠Ptpmt1表型的影响 基因敲除/ATF4杂合(HcKO)小鼠和CM特异性Ptpmt1/ATF4双基因敲除(DcKO)小鼠。

PROJECT SUMMARY Mitochondria are essential for cardiomyocyte (CM) differentiation and cardiac morphogenesis. Mutations in genes encoding mitochondrial proteins frequently result in congenital heart disease, highlighting the need to elucidate key molecular pathway(s) in mitochondrial homeostasis during heart development. Protein Tyrosine Phosphatase localized to the Mitochondrion 1 (PTPMT1) is a dual-specificity mitochondrial phosphatase encoded by nuclear DNA. PTPMT1 is exclusively localized to mitochondria, being anchored to the inner mitochondrial membrane. PTPMT1 is expressed in CMs throughout several developmental stages. To determine the role of PTPMT1 in CMs, we generated a Ptpmt1 constitutive CM-specific knockout (cKO) mouse model. Our preliminary data revealed that Ptpmt1 cKO mice display embryonic lethality. Ptpmt1 cKO mice displayed thinner compact zone myocardium, with decreased CM proliferation. We also observed significantly decreased mitochondrial respiration rate and abnormal mitochondrial morphology in Ptpmt1 cKO hearts, demonstrating that PTPMT1 plays a critical role in developing CMs and in maintaining normal mitochondrial homeostasis. We also examined previously described PTPMT1 substrates in Ptpmt1 cKO hearts relative to controls, but could find no evidence to support them being direct substrates of PTPMT1 in CMs. To gain further insight into pathways affected by loss of PTPMT1, we performed RNA-seq analysis of Ptpmt1 cKO hearts. Bioinformatics analysis revealed that loss of PTPMT1 significantly activated the Activating Transcription Factor 4 (ATF4) pathway. ATF4 controls expression of a wide range of adaptive genes that allow cells to survive periods of mitochondrial stress. However, under persistent stress conditions, ATF4 promotes induction of cell- cycle arrest, apoptosis and senescence. Notably, reduced expression of ATF4 in global Atf4-haplodeficient and smooth muscle-specific Atf4 knockout mice attenuates ER stress and reduces medial and atherosclerotic calcification, highlighting new opportunities afforded by favoring a stress-relief adaptive effect over a maladaptive effect by modulating ATF4 activation. The foregoing evidence leads us to the hypothesis that PTPMT1 plays an essential role in cardiac development through modulation of specific substrates, and that partial loss of ATF4, activated in response to mitochondrial stress in Ptpmt1 knockout CMs, may ameliorate, but complete loss of ATF4 may exacerbate, Ptpmt1 cKO phenotypes. Accordingly, our specific aims are to: 1. Elucidate the role of PTPMT1 in CM mitochondrial homeostasis and cardiac development and function by analyzing Ptpmt1 cKO mice, and to identify endogenous substrates of PTPMT1 in CMs by performing unbiased lipidomics and phosphoproteomics analyses; and 2. Determine the consequences of partial or complete loss of ATF4 in CMs on phenotypes of Ptpmt1 cKO mice by analyzing CM-specific Ptpmt1 knockout/Atf4 heterozygous (hcKO) knockout mice and CM-specific Ptpmt1/Atf4 double knockout (dcKO) mice.