Novel function of a mitochondria phosphatase in cardiac development

项目摘要 线粒体是心肌细胞分化和心脏形态发生所必需的。突变 编码线粒体蛋白质的基因经常导致先天性心脏病，这突出了 阐明心脏发育过程中线粒体稳态的关键分子途径。蛋白酪氨酸 线粒体磷酸酶1（PTPMT 1）是一种双特异性线粒体磷酸酶 由核DNA编码。PTPMT 1仅定位于线粒体，锚定在线粒体的内部。 线粒体膜PTPMT 1在几个发育阶段的CM中表达。到 为了确定PTPMT 1在CM中的作用，我们建立了Ptpmt 1组成型CM特异性敲除（cKO）小鼠 模型我们的初步数据显示，Ptpmt 1 cKO小鼠显示胚胎致死性。Ptpmt 1 cKO小鼠 显示致密带心肌变薄，CM增殖减少。我们还观察到， Ptpmt 1 cKO心脏中线粒体呼吸速率降低和线粒体形态异常， 证明PTPMT 1在CM的发展和维持正常线粒体中起着关键作用， 体内平衡我们还检查了先前描述的Ptpmt 1 cKO心脏中的PTPMT 1底物， 对照，但没有发现证据支持它们是CMs中PTPMT 1的直接底物。为了获得进一步 为了深入了解受PTPMT 1丢失影响的途径，我们对Ptpmt 1 cKO心脏进行了RNA-seq分析。 生物信息学分析显示，PTPMT 1的缺失显著激活了转录激活因子 4（ATF 4）途径。ATF 4控制多种适应性基因的表达，使细胞得以存活 线粒体应激时期。然而，在持续的应激条件下，ATF 4促进细胞凋亡的诱导。 细胞周期阻滞、凋亡和衰老。值得注意的是，在整体Atf 4单倍缺陷和 平滑肌特异性Atf 4基因敲除小鼠可减轻ER应激并降低中膜和动脉粥样硬化 钙化，突出强调了有利于缓解压力的适应性效果而不是 通过调节ATF 4激活产生适应不良效应。上述证据使我们得出这样的假设， PTPMT 1通过调节特定底物在心脏发育中起重要作用， 在Ptpmt 1敲除的CM中，响应于线粒体应激而激活的ATF 4的部分缺失，可能会改善， 但ATF 4的完全丧失可能加重Ptpmt 1 cKO表型。因此，我们的具体目标是：1。 阐明PTPMT 1在CM线粒体稳态和心脏发育和功能中的作用， 分析Ptpmt 1 cKO小鼠，并通过进行以下操作鉴定CM中PTPMT 1的内源性底物： 无偏脂质组学和磷酸蛋白质组学分析;以及2.确定部分或 通过分析CM特异性Ptpmt 1，在Ptpmt 1 cKO小鼠表型上CM中的ATF 4完全丢失 敲除/Atf 4杂合（hcKO）敲除小鼠和CM特异性Ptpmt 1/Atf 4双敲除（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.