Chronic ethanol exposure induces cardiac fibroblast transdifferentiation via ceramide accumulation and oxidative stress.

AIMS: Excessive alcohol consumption is associated with cardiac dysfunction and the development of myocardial fibrosis. In this study, we aimed to investigate the direct impacts of ethanol on myocardial fibroblasts and elucidate the underlying mechanism responsible for chronic ethanol-induced myocardial fibrosis. METHODS: Rat primary cardiac fibroblasts exposed to ethanol for 24 h and C57BL/6J mice fed on Lieber-DeCarli diet to establish an ethanol intoxication model in vitro and in vivo, respectively. Histological analyses, molecular biology techniques, and analytical chemistry methods were then conducted. RESULTS AND CONCLUSION: In vivo and vitro experiments revealed that chronic ethanol exposure induced increased myocardial fibrosis and augmented the transdifferentiation of myocardial fibroblasts. Simultaneously, it elicited an upregulation in the production of long-chain and very-long-chain ceramides in cardiac fibroblasts. The excessive accumulation of ceramide leads to elevated levels of intracellular oxidative stress, culminating in the activation of TGF-ß-SMAD3 signaling and the development of fibrosis. Intervention of these pathways with pharmacological inhibitors in vitro or in vivo inhibited fibrosis. In conclusion, ethanol increased ceramides and reactive oxygen species (ROS) in cardiac fibroblasts, resulting in the activation of TGF-ß-SMAD3 signaling, transdifferentiation of fibroblasts, and myocardial fibrosis.

Inhibition of Ceramide Synthesis Attenuates Chronic Ethanol Induced Cardiotoxicity by Restoring Lysosomal Function and Reducing Necroptosis.

AIMS: Chronic alcohol misuse could cause alcoholic cardiomyopathy (ACM), and the specific mechanisms remained largely unknown. In this study, we aimed to explore the effects of endogenous ceramides on chronic ethanol-induced myocardial injury or cell loss (e.g. necroptosis). METHODS: We established chronic alcohol intoxication models in vivo (male C57BL/6 mice) and in vitro (H9c2 cardiomyoblasts). The ceramide profiles were analyzed in mice myocardium and cultured cardiomyocytes. Further research on the role of ceramides and underlying signaling pathways was carried out in H9c2 cells. RESULTS AND CONCLUSIONS: The ceramide profiles analysis revealed increased long and very long-chain ceramides in alcoholic myocardium and ethanol-treated cardiomyocytes. Next, we proved that endogenous ceramide inhibition could reduce necroptosis and alleviate cardiomyocytes injury as suggested by decreased levels of p-RIPK1, p-RIPK3 and p-MLKL proteins and cardiac injury factors expression. Furthermore, we found that lysosomal dysfunction also contributed to alcohol-induced cardiac damage and inhibiting ceramide biosynthesis could repaired this to some extent. Cells studies with exogenous C6 ceramide confirmed the pleotropic roles of ceramide in myocardial damage by causing both necroptosis and lysosomal dysfunction. Finally, our data suggested that lysosomal dysfunction could sensitize cardiomyocytes to induction of necroptosis due to the restriction on degradation of RIPK1/RIPK3 proteins. In conclusion, chronic ethanol treatment boosted myocardial ceramide synthesis in animal hearts and cultured cardiomyocytes. Moreover, ceramides exerted crucial roles in the intrinsic signaling pathways of alcohol-induced cardiotoxicity. Targeting ceramide biosynthesis to simultaneously attenuate necroptosis and lysosomal dysfunction might be a novel strategy for preventing alcoholic cardiotoxicity.