Novel function of a mitochondria phosphatase in cardiac development

线粒体磷酸酶在心脏发育中的新功能

基本信息

批准号：
10687847
负责人：
Ju Chen
金额：
$ 53.91万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10687847
关键词：
Address Affect Apoptosis Attenuated Bioinformatics Biological Process Cardiac Cardiac Myocytes Cell Cycle Arrest Cells Crista ampullaris DNA Data Defect Development Embryo Fibroblasts Genes Genetic Transcription Heart Heart Abnormalities Heterozygote Homeostasis Inner mitochondrial membrane Knock-out Knockout Mice LoxP-flanked allele Medial Mitochondria Mitochondrial Proteins Modeling Molecular Morphogenesis Morphology Mus Mutation Myocardium Nuclear Oxygen Consumption Pathway interactions Phenotype Phosphatidylinositol Phosphates Phospholipids Phosphoric Monoester Hydrolases Phosphorylation Physiological Play Process Proliferating Protein Dephosphorylation Protein Tyrosine Phosphatase Respiration Role Signal Transduction Smooth Muscle Specificity Stress Succinate Dehydrogenase Thinness Zebrafish activating transcription factor 4 biological adaptation to stress calcification cardiogenesis congenital heart disorder embryonic stem cell endoplasmic reticulum stress heart function implantation in vivo inhibitor insight lipidomics loss of function mitochondrial dysfunction mouse model novel phosphatidylglycerophosphate phosphoproteomics response senescence transcriptome sequencing

项目摘要

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.

项目概要线粒体对于心肌细胞（CM）分化和心脏形态发生至关重要。突变在编码线粒体蛋白的基因经常导致先天性心脏病，强调需要阐明心脏发育过程中线粒体稳态的关键分子途径。蛋白质酪氨酸定位于线粒体 1 (PTPMT1) 的磷酸酶是一种双特异性线粒体磷酸酶由核DNA编码。 PTPMT1 专门定位于线粒体，锚定于线粒体内部线粒体膜。 PTPMT1 在多个发育阶段的 CM 中表达。到为了确定 PTPMT1 在 CM 中的作用，我们生成了 Ptpmt1 组成型 CM 特异性敲除 (cKO) 小鼠模型。我们的初步数据显示 Ptpmt1 cKO 小鼠表现出胚胎致死性。 Ptpmt1 cKO小鼠显示心肌致密区变薄，CM 增殖减少。我们还观察到显着 Ptpmt1 cKO 心脏中线粒体呼吸速率降低和线粒体形态异常，证明 PTPMT1 在 CM 的形成和维持正常线粒体方面发挥着关键作用体内平衡。我们还检查了之前描述的 Ptpmt1 cKO 心脏中的 PTPMT1 底物相对于对照，但找不到证据支持它们是 CM 中 PTPMT1 的直接底物。为了进一步获得为了深入了解 PTPMT1 缺失影响的通路，我们对 Ptpmt1 cKO 心脏进行了 RNA 测序分析。生物信息学分析表明，PTPMT1 的缺失显着激活了激活转录因子 4 (ATF4) 途径。 ATF4 控制多种适应性基因的表达，使细胞能够生存线粒体应激期。然而，在持续的应激条件下，ATF4 会促进细胞- 周期停滞、细胞凋亡和衰老。值得注意的是，在全球 Atf4 单倍体缺陷和平滑肌特异性 Atf4 基因敲除小鼠可减轻 ER 应激并减少内侧和动脉粥样硬化钙化，强调了通过有利于缓解压力的适应性效应而不是通过调节 ATF4 激活产生适应不良效应。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，并且 Ptpmt1 敲除 CM 中响应线粒体应激而激活的 ATF4 部分丢失可能会改善，但 ATF4 的完全丧失可能会加剧 Ptpmt1 cKO 表型。因此，我们的具体目标是： 1. 通过以下方式阐明 PTPMT1 在 CM 线粒体稳态以及心脏发育和功能中的作用分析 Ptpmt1 cKO 小鼠，并通过执行来鉴定 CM 中 PTPMT1 的内源性底物无偏见的脂质组学和磷酸化蛋白质组学分析； 2. 确定部分或部分的后果通过分析 CM 特异性 Ptpmt1，发现 Ptpmt1 cKO 小鼠表型的 CM 中 ATF4 完全丧失敲除/Atf4 杂合 (hcKO) 敲除小鼠和 CM 特异性 Ptpmt1/Atf4 双敲除 (dcKO) 小鼠。