Palmitate impairs the autophagic flux to induce p62-dependent apoptosis through the upregulation of CYLD in NRCMs
TOXICOLOGY(2022)
摘要
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/beta-catenin pathway to restore the autophagic flux and reduce the accumulation of p62 in PA- stimulated NRCMs, while an inhibitor of the Wnt/beta-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.
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关键词
Palmitate, Apoptosis, Autophagy, CYLD, NRCMs, Wnt/beta-catenin pathway
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