Palmitate diet-induced loss of cardiac caveolin-3: a novel mechanism for lipid-induced contractile dysfunction.

Obesity is associated with an increased risk of cardiomyopathy, and mechanisms linking the underlying risk and dietary factors are not well understood. We tested the hypothesis that dietary intake of saturated fat increases the levels of sphingolipids, namely ceramide and sphingomyelin in cardiac cell membranes that disrupt caveolae, specialized membrane micro-domains and important for cellular signaling. C57BL/6 mice were fed two high-fat diets: palmitate diet (21% total fat, 47% is palmitate), and MCT diet (21% medium-chain triglycerides, no palmitate). We established that high-palmitate feeding for 12 weeks leads to 40% and 50% increases in ceramide and sphingomyelin, respectively, in cellular membranes. Concomitant with sphingolipid accumulation, we observed a 40% reduction in systolic contractile performance. To explore the relationship of increased sphingolipids with caveolins, we analyzed caveolin protein levels and intracellular localization in isolated cardiomyocytes. In normal cardiomyocytes, caveolin-1 and caveolin-3 co-localize at the plasma membrane and the T-tubule system. However, mice maintained on palmitate lost 80% of caveolin-3, mainly from the T-tubule system. Mice maintained on MCT diet had a 90% reduction in caveolin-1. These data show that caveolin isoforms are sensitive to the lipid environment. These data are further supported by similar findings in human cardiac tissue samples from non-obese, obese, non-obese cardiomyopathic, and obese cardiomyopathic patients. To further elucidate the contractile dysfunction associated with the loss of caveolin-3, we determined the localization of the ryanodine receptor and found lower expression and loss of the striated appearance of this protein. We suggest that palmitate-induced loss of caveolin-3 results in cardiac contractile dysfunction via a defect in calcium-induced calcium release.

Palmitate-Induced Translocation of Caveolin-3 and Endothelial Nitric Oxide Synthase in Cardiomyocytes.

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.