ABSTRACT The complexity of cardiomyocyte signaling requires scaffolding proteins to coordinate the cellular processes driven by receptors and transporters. β-Arrestins are prototypical intracellular scaffold proteins that negatively regulate cardiac β-adrenergic receptor function via desensitization. Recently, a larger and more ancient family of structurally related arrestins, termed α-arrestins, has been identified, which shares functions in regulating transporter trafficking. Txnip, the best-studied member of α-arrestins, serves as an adaptor protein to facilitate endocytosis of glucose transporters (GLUTs) and suppresses glucose influx through its arrestin domains. We previously found that targeted deletion of Txnip leads to a substantial metabolic switch, directing cardiomyocytes toward enhanced glycolytic metabolism under severe ischemia. Despite the potential to identify new molecular mechanisms, the functions of other α-arrestins, Arrestin domain-containing protein (Arrdc) 1-5, remain largely undefined in the heart. Here we present preliminary data demonstrating that two α- arrestins Arrdc4 and Txnip are related to their conserved arrestin domains and share the function to inhibit GLUT1. Interestingly, this metabolic inhibition was more potent in Arrdc4 than in Txnip. Using our recently- generated Arrdc4 knockout mouse model, the data reveal exciting findings that inhibition of Arrdc4 enhances myocardial glucose uptake during hypoxia and improves outcomes after myocardial infarction. These results define the outlines of an Arrdc4-GLUT1 pathway that may provide a link between cardiac glucose metabolism and cardiomyocyte survival. This project aims to delineate the molecular nature of this pathway and tests its role in the pathogenesis of ischemic heart disease. Aim 1 tests three non-exclusive hypotheses by which Arrdc4 regulates GLUT1 function in cardiomyocytes: (a) specific arrestin domains of Arrdc4 mediate clathrin- dependent endocytosis of GLUT1; (b) Arrdc4 promotes GLUT1 ubiquitination through an E3 ligase-mediated pathway; (c) Arrdc4 and Txnip are complementary in the regulation of cardiomyocyte glucose metabolism. Aim 2 employs the Arrdc4 knockout mouse model to test the overall significance of cardioprotection against myocardial ischemia through a GLUT1-mediated mechanism in vivo. By genetically “reconstituting” the hearts of Arrdc4 knockout mice with the informative Arrdc4 mutant, this aim also tests the roles of the specific molecular mechanisms linking Arrdc4 and GLUT1 in ischemic heart disease. Furthermore, using a combination of virtual screening and cell-based assays, Aim 3 will search for the possible Arrdc4-GLUT1 interaction inhibitors that may improve energy homeostasis and enhance cardiomyocyte survival under hypoxia. These studies are highly innovative as we propose a pathway that has never been entertained as a cardiac metabolic regulator. The resulting knowledge will provide a novel mechanistic basis for understanding t...