Palmitate impairs the autophagic flux to induce p62-dependent apoptosis through the upregulation of CYLD in NRCMs.

Abstract The most abundant saturated free fatty acid such as palmitate (PA), can accumulate in cardiomyocytes and induce lipotoxicity. CYLD is a known regulator in the development of cardiovascular disease and an important mediator of apoptosis. The role of CYLD in PA-induced cardiomyocyte apoptosis is not completely known. Here, we showed that PA treatment resulted in a concentration- and time-dependent effect on neonatal rat cardiomyocytes (NRCMs) apoptosis. PA impaired autophagy by significantly increasing the expression levels of LC3-II, Beclin 1, and also p62 in NRCMs. The autophagy flux was measured by detecting the fluorescence in the cells with Ad-mCherry-GFP-LC3B, a decrease in red puncta and a significant increase in yellow puncta in response to PA stimulation indicated that PA impairs the autophagic flux at the late stage of autophagosome-lysosome fusion. We further found knocked down of p62 by siRNA significantly decreased the expression level of cleaved caspase-3, decreased the apoptosis rate, also alleviated the loss of mitochondrial membrane potential, and decreased AIF and Cyt C releasing from mitochondria into the cytoplasm in the PA-treated NRCMs. From this, we considered that p62 accumulation was responsible for mitochondria-mediated apoptosis in PA-treated NRCMs. In addition, PA-induced a strong elevation of CYLD, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced apoptosis and restored the autophagic flux in NRCMs. Knockdown of CYLD activation of the Wnt/β-catenin pathway to restore the autophagic flux and reduce the accumulation of p62 in PA- stimulated NRCMs, while an inhibitor of the Wnt/β-catenin pathway reversed this effect. Thus, our findings provide new insight into the molecular mechanism of PA toxicity in myocardial cells and suggest that CYLD may be a new therapeutic target for lipotoxic cardiomyopathy.