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Abstract

Introduction: Autophagy is a highly conserved process involved in lysosomal degradation of cellular contents and plays an important role in various pathophysiologic conditions. Formation of LC3B-II is a downstream product and a marker of autophagy. Tumor necrosis factor-α (TNFα) is a cytokine, which is overexpressed during the systemic inflammatory response of sepsis, trauma and ischemia and reperfusion injury and contributes to myocardial depression. AMP-activated protein kinase (AMPK) is an important homeostasis regulator, which activates autophagy and mitochondrial recovery. Hypothesis: TNFα induces autophagy through activation of AMPK in rat cardiac myocytes. Methods: H9C2 cardiac myocytes were stimulated with TNFα (100 U/ml) or medium alone for 24 hrs. Western blot analysis was used to evaluate expression of LC3B-II in the cytosol, expression of AMPK and its phosphorylated active form pAMPK in the cytosol, and PPAR gamma coactivator-1? (PGC-1?) in the nucleus. Data was analyzed by t-test. Results: Treatment with TNFα increased expression of LC3B-II in the cytosol (3.32 ± 0.15 fold increase) when compared with basal levels of cells treated with medium alone (p<0.05), thus suggesting formation of autophagosome. To determine the mode of regulation of autophagy, we evaluated the cytosol expression of the upstream kinase AMPK. TNFα also increased both total AMPK and active pAMPK (1.75 ± 0.19 and 2.61 ± 0.30 fold increase, respectively) when compared with basal levels (p<0.05). Since AMPK influences metabolic homeostasis also through regulation of the transcriptional coactivator PGC-1?, we determined the nuclear expression of PGC-1?. Treatment with TNFα increased PGC-1? expression (1.31 ± 0.18 fold increase) when compared with basal levels of cells treated with medium alone (p<0.05). Conclusions: These data indicate that TNFα induces autophagy in cardiac myocytes through activation of AMPK. This event might represent a metabolic adaptation since TNFα-induced autophagy is associated with increased expression of the energy regulator PGC-1?. Supported by NIH grants (R01 GM067202 and R01 AG027990).