Abstract Body: Introduction: To maintain homeostasis, cardiomyocytes (CMs) primarily rely on fatty acid β-oxidation for ATP production facilitated by substrate uptake via fuel transporters. The glucose transporter, GLUT4, and fatty acid translocase, CD36, are directed to the cardiomyocyte sarcolemma in response to insulin signaling and AMP-activated protein kinase (AMPK) signaling. In adverse conditions like acute hypoxia and ischemia, these cells undergo a metabolic shift to favor glycolysis. Previous reports showed that under these conditions, CD36 moved away from the sarcolemma as GLUT4 was delivered to the cell membrane, contributing to a shift from fatty acid oxidation to glycolysis. Investigating CD36 and GLUT4 membrane delivery in cardiomyocytes is key to better understanding cardiac energy metabolism under normal and disease conditions. Prior work in adipocytes, and our own studies in skeletal muscle showed that the exocyst trafficking complex is critical for membrane delivery of GLUT4 and CD36 in response to insulin and AMPK. Our objective was to determine if in cardiomyocytes GLUT4 and CD36 trafficking, and subsequent fuel uptake is dependent on the exocyst.
Methods and Results: We demonstrate that the exocyst is recruited to GLUT4 and CD36-vesicles in cultured H9C2 cells in response to stimuli with proximity ligation assays. Using on-cell Western analysis and fuel uptake assays we also show that the exocyst is essential for induced GLUT4 and CD36 cell surface delivery and subsequent energy substrate uptake, as inhibition of the exocyst attenuates stimulated surface translocation of both fuel transporters and subsequent fluorescent fuel analog uptake.
Our earlier work revealed that protein levels of exocyst subunits are increased in murine heart tissue following myocardial infarction. To examine the role of the exocyst in fuel uptake under ischemic conditions, we used an in vitro model of simulated ischemia, and found similar alterations in exocyst subunit expression in this model at both mRNA and protein levels. This suggests an increased demand for exocyst activity.
Conclusion: Further research will explore how ischemia impacts exocyst-mediated trafficking and if the exocyst contributes to the glycolytic shift under ischemia in cardiomyocytes.