Abstract 464: Dynamic Regulation of the Epidermal Growth Factor Receptor Protein Interactome via β1-adrenergic Receptor-mediated Transactivation in Cardiomyocytes

Abstract

Heart Failure (HF) is a chronic, progressive condition wherein the heart cannot perfuse adequate amounts of blood and oxygen to meet the demands of metabolizing tissues. Gold standard HF therapeutics currently consist of a small repertoire of neurohormone modulators that still result in ~50% mortality within 5 years. Our ongoing goal is to develop new signaling modalities as refined HF therapeutics. We have shown that β1-adrenergic receptor (β1AR) stimulation rapidly induces epidermal growth factor receptor (EGFR)-dependent protein signaling responses in various regions of cardiomyocytes (CM). We hypothesize that differences in the subcellular localization and protein interactomes of EGFR following differential ligand stimulation modulate CM functions in distinct manners. To test this, we infected neonatal rat ventricular myocytes (NRVM) with adenoviruses encoding HA-β1AR and Flag-EGFR, stimulated them with vehicle, isoproterenol or EGF for 5 min and performed mass spectroscopy analysis of the Flag-EGFR immunoprecipitates. While hundreds of protein interactions were identified, one of the highly enriched subcellular groups belonged to mitochondrial proteins, for which there is no information regarding the role of EGFR in their regulation in CM. Via fractionation and digestion analyses, we have confirmed EGFR localization to the outer mitochondrial membrane in CM and have begun to assess the impact of EGFR on mitochondrial function. Transmission electron microcopy analysis of mitochondrial morphology show that CM-specific EGFR knockdown (CM-EGFR-KD) increases mitochondrial circularity and mitochondria/area without changes in other parameters including perimeter or cristae density, suggesting altered bioenergetic potential. Indeed, extracellular flux (Seahorse) analysis of CM-EGFR-KD versus control CM revealed that CM-EGFR-KD increased maximal and reserve capacities and a trend toward increased ATP production. Elucidating the mechanism(s) of EGFR localization to and association with proteins at CM mitochondria, as well as its impact on mitochondrial function normally or in response to catecholamine stimulation will provide insight into the potential utility of targeting mitochondrial EGFR to improve HF outcomes.