H Yu1, Q Liu1, Y Guo1, Y Xia1, S Luo1. 1. Department of Cardiology, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
Abstract
OBJECTIVE: To investigate the effect of palmitic acid (PA) on autophagy in neonatal rat cardiomyocytes (NRCMs) and explore the underlying mechanism. METHODS: NRCMs were isolated and cultured for 24 h before exposure to 10% BSA and 0.1, 0.3, 0.5, or 0.7 mmol/L PA for 24 h. After the treatments, the expressions of Parkin, PINK1, p62, LC3Ⅱ/ LC3Ⅰ, cGAS, STING and p-IRF3/IRF3 were detected using Western blotting and the cell viability was assessed with CCK8 assay, based on which 0.7 mmol/L was selected as the optimal concentration in subsequent experiments. The effects of cGAS knockdown mediated by cGAS siRNA in the presence of PA on autophagy-related proteins in the NRCMs were determined using Western blotting, and the expressions of P62 and LC3 in the treated cells were examined using immunofluorescence assay. RESULTS: PA at different concentrations significantly lowered the expressions of Parkin, PINK1, LC3 Ⅱ/LC3 Ⅰ and LC3 Ⅱ/LC3 Ⅰ+Ⅱ (P < 0.05), increased the expression of p62 (P < 0.05), and inhibited the viability of NRCMs (P < 0.05). Knockdown of cGAS obviously blocked the autophagy-suppressing effect of PA and improved the viability of NRCMs (P < 0.05). CONCLUSION: PA inhibits autophagy by activating the cGAS-STING-IRF3 pathway to reduce the viability of NRCMs.
OBJECTIVE: To investigate the effect of palmitic acid (PA) on autophagy in neonatal rat cardiomyocytes (NRCMs) and explore the underlying mechanism. METHODS: NRCMs were isolated and cultured for 24 h before exposure to 10% BSA and 0.1, 0.3, 0.5, or 0.7 mmol/L PA for 24 h. After the treatments, the expressions of Parkin, PINK1, p62, LC3Ⅱ/ LC3Ⅰ, cGAS, STING and p-IRF3/IRF3 were detected using Western blotting and the cell viability was assessed with CCK8 assay, based on which 0.7 mmol/L was selected as the optimal concentration in subsequent experiments. The effects of cGAS knockdown mediated by cGAS siRNA in the presence of PA on autophagy-related proteins in the NRCMs were determined using Western blotting, and the expressions of P62 and LC3 in the treated cells were examined using immunofluorescence assay. RESULTS: PA at different concentrations significantly lowered the expressions of Parkin, PINK1, LC3 Ⅱ/LC3 Ⅰ and LC3 Ⅱ/LC3 Ⅰ+Ⅱ (P < 0.05), increased the expression of p62 (P < 0.05), and inhibited the viability of NRCMs (P < 0.05). Knockdown of cGAS obviously blocked the autophagy-suppressing effect of PA and improved the viability of NRCMs (P < 0.05). CONCLUSION: PA inhibits autophagy by activating the cGAS-STING-IRF3 pathway to reduce the viability of NRCMs.
Authors: Norimichi Koitabashi; Thomas Danner; Ari L Zaiman; Yigal M Pinto; Janelle Rowell; Joseph Mankowski; Dou Zhang; Taishi Nakamura; Eiki Takimoto; David A Kass Journal: J Clin Invest Date: 2011-05-02 Impact factor: 14.808