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-specifi...