Project Summary/Abstract Heart failure is a leading cause of morbidity and mortality around the world, and the leading cause of heart failure with reduced ejection fraction (HFrEF) is coronary artery disease. The mitogen-activated protein kinases (MAPK) are an essential signal transduction cascade that play a central role in both cell growth and cell death. The MEK1-ERK1/2 branch of the MAPK pathway has been shown to promote both physiologic and pathologic growth in the heart, as well as protect against apoptotic cell death after ischemia/reperfusion (I/R) injury. We have previously demonstrated that the transcription factor ETS2, a member of E26 transformation-specific sequence (ETS)-domain family, is phosphorylated and activated by Erk1/2 upon hypertrophic stimulation. Going forward, our preliminary data reveal that cardiomyocyte- specific loss of ETS2 results in increased susceptibility to ischemic injury in both I/R and permanent ligation (myocardial infarction) models of heart failure. Connexin43 (Cx43), the predominant gap junction channel- forming protein in cardiomyocytes, has been suggested to play a role in both ischemic damage and ischemic preconditioning. Our preliminary data show that ETS2 activates Cx43 transcription and that Cx43 is downregulated in the absence of ETS2. We will test the hypothesis that the ERK1/2/ETS2 pathway protects against I/R injury in part through the upregulation of Cx43. Aim 1: To determine the role of the ERK1/2-ETS2 pathway in I/R injury. Our preliminary data suggest a model in which ETS2 protects against I/R injury. We will confirm and extend this using loss- and gain-of- function approaches in vivo and in vitro. We will track the timing of ETS2 activation by ERK and the response of each acutely and in long-term remodeling. Aim 2: To determine the impact of ETS2 on Cx43 expression and function in I/R injury. Our data suggest that Cx43 plays a protective role in ischemic injury. Our preliminary data also suggest that ETS2 is a direct transcriptional regulator of Cx43 gene expression. We will confirm and extend these findings using both loss- and gain-of-function approaches. We will also determine the role of ETS2 in Cx43-mediated cardioprotection in IPC. Aim 3: To determine the downstream targets and interactors of ETS2 under conditions of cardiac stress. We will profile genome-wide cardiac gene expression using RNAseq and ChIPseq to determine ETS2 downstream gene targets in both acute and long-term I/R injury. We will use immunoprecipitation and mass spectrometry to unveil novel protein interactions.