Abstract
PROBLEM STATEMENT: Palmitate is a known cardiac lipotoxin that blunts cardiomyocyte contractile function and induces apoptosis, likely via accumulation of the lipotoxic ceramide. Ceramide is a sphingolipid and localizes to caveolae, which are lined in the inner membrane leaflet by caveolin proteins. In this study, we investigated the effects of palmitate on caveolin proteins and on endothelial Nitric Oxide Synthase (eNOS), a signaling mediator that binds to caveolin-3, the muscle-specific caveolae scaffolding protein. APPROACH AND RESULTS: Mice fed a high palmitate diet for 12 weeks showed pathologically increased coronary flow in the ex vivo Langendorff heart especially at low extracellular calcium concentrations. In these hearts, eNOS Ser1177 phosphorylation was increased compared to standard or high fat control diet hearts. This suggested that eNOS, a potent vasodilator in the heart, is affected by palmitate. In vitro experiments showed that exposure of HL-1 cardiomyocytes to palmitate causes translocation of eNOS from the plasma membrane to a perinuclear location and causes an 80% decrease in Thr495 phosphorylation. This corresponded with a 41% decrease in NO production. To determine the mechanism of the loss of plasma membrane bound eNOS, we investigated the effect of palmitate on caveolin-3 and found decreased caveolin-3 protein levels by 70% compared to control cells. The remaining 30% of caveolin-3 was localized to a perinuclear location. In contrast to previous studies, palmitate did not cause apoptosis in cardiomyocytes. CONCLUSION: Overall, we show for the first time that a high palmitate diet leads to loss of caveolin-3 in cardiomyocytes and to coronary dysfunction of the mouse heart, via uncoupling of eNOS.