PROJECT SUMMARY Activating Transcription Factor 4 (ATF4), also known as cAMP-Response Element Binding Protein 2 (CREB2), belongs to the ATF/cAMP responsive element-binding (CREB) protein family of transcription factors. As a well- characterized stress-response transcription factor, ATF4 is constitutively and ubiquitously expressed at low levels but can be rapidly induced under a variety of cell-stress conditions. Previous studies have shown that ATF4 functions in diverse cell types and tissues under various physiological and pathological conditions, including cardiac diseases. However, little is known as to the specific role of ATF4 and its target genes in mammalian cardiac development. To address this gap in knowledge and to determine the role of ATF4 in cardiomyocytes (CMs), we have generated novel Atf4 CM-specific constitutive knockout (cKO) and tamoxifen- inducible CM-specific knockout (icKO) mouse lines. Our preliminary studies revealed that Atf4 cKO mice exhibited perinatal death and cardiac morphological defects, associated with reduced CM proliferation. RNA- seq and ChIP-seq analyses of embryonic ventricular tissues revealed upregulation of a series of cell cycle arrest-associated genes known to be downstream of p53, and downregulation of a series of p53-independent cardiac development/function and/or cell cycle progression associated genes, most of which harbored ATF4 binding regions, in Atf4 cKO mice. Loss of ATF4 in developing CMs also resulted in increased p53 protein levels but not Trp53 mRNA levels. Moreover, p53 ablation in Atf4 cKO mice partially restored ventricular wall thickness and ameliorated upregulation of p53 target cell cycle arrest genes at E17.5, but failed to rescue lethality beyond postnatal day 1. Conversely, inducible ablation of Atf4 in adult CMs had no effect on cardiac function or left ventricular dimension, suggesting distinct roles for ATF4 at specific stages of CM development. Taken together, the foregoing evidence leads us to the hypothesis that ATF4 plays an essential role in CM proliferation and function via p53-dependent and -independent mechanisms at specific stages of cardiac development. Accordingly, our Specific Aims are: 1. To determine the role of ATF4 in cardiomyocyte proliferation and cardiac development by analyzing Atf4 cardiomyocyte-specific knockout (Atf4 cKO) mice, and to elucidate mechanisms by which ATF4 regulates target gene pathways in a p53-dependent and/or -independent manner; and 2. To determine times at which ATF4 is required for embryonic and neonatal cardiomyocyte proliferation and function (from E7.5 to P30) by analysis of Atf4 inducible cardiomyocyte- specific knockout (icKO) mice. Our proposed studies will help us to understand the specific roles of ATF4 and p53, as well as other novel ATF4 targets in CMs at critical developing stages in vivo, as well as to determine a safe therapeutic window for the potential application of ATF4 and/or p53 inhibitors in cardiac diseases.