The Effects Of Low-after-high Glucose Culture On Autophagy And Expression Of Connexin43 In H9c2 Cardiomyocytes

Objective:Hypoglycemia is a very common complication in diabetic patients and is a strong predictor of cardiovascular events.High-glucose has been reported to alter connexin43?Cx43?expression and to promote autophagy in cardiomyocytes.We investigated whether low-after-high glucose would influence Cx43 expression and autophagy level in H9c2 cells.Methods:H9c2 cells were incubated in 33.3 mM glucose for 24 h followed by 2.5mM glucose for 2,4,6,or 12 h with or without chloroquine?autophagy inhibitor?,U0126(MEK_1/2 inhibitor)or LY294002?PI3K inhibitor?.Cells incubated in 5.5 mM,33.3 mM or 2.5 mM glucose with or without inhibitors and in the presence of mannitol were used as controls.Protein expression was assayed by western blot,apoptosis was assayed by flow cytometry,cell proliferation was determined by MTT assays,and cytotoxicity was assayed by LDH measurement.Results:Cytotoxicity and early apoptosis were increased and cell proliferation was decreased after exposure to low-after-high glucose,and these results were reversed by chloroquine and U0126 but were aggravated by LY294002.Cx43 expression was downregulated in a time-dependent manner and was accompanied by upregulated expression of LC3-II,Beclin-1,p62,p-Akt,p-mTOR and p-ERK_1/2.Chloroquine suppressed autophagy and reversed the downregulation of Cx43.U0126 inhibited ERK activation and decreased autophagy proteins expression but increased Cx43expression.LY294002 suppressed p-Akt,activated autophagy,and decreased Cx43expression.Interestingly,MEK_1/2 inhibition also increased p-Akt expression,but inhibition of PI3K led to p-ERK downregulation.Conclusion:Culturing H9c2 cells under low-after-high glucose downregulated Cx43expression by promoting autophagy through a mechanism involving the cross regulation of PI3K/Akt/mTOR and MEK/ERK_1/2 signaling pathways.