Low-after-high glucose down-regulated Cx43 in H9c2 cells by autophagy activation via cross-regulation by the PI3K/Akt/mTOR and MEK/ERK1/2signal pathways.
OBJECTIVE
Hypoglycemia in diabetes is a strong predictor of cardiovascular events. High-glucose have been reported to alter connexin43 expression and to promote autophagy in cardiomyocytes. We investigated whether low-after-high glucose would influence connexin43 expression and autophagy in H9c2 cells.


METHODS
H9c2 cells were incubated in 33.3 mM glucose for 24 h followed by 2.5 mM glucose for 2, 4, 6, or 12 h with or without chloroquine (autophagy inhibitor), U0126 (MEK1/2inhibitor) or LY294002 (PI3K inhibitor). Cells incubated in 5.5, 33.3, 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 lactate dehydrogenase 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. Connexin43 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-ERK1/2. Chloroquine suppressed autophagy and reversed the downregulation of connexin43. U0126 inhibited ERK activation and decreased autophagy proteins expression but increased connexin43 expression. LY294002 suppressed p-Akt, activated autophagy, and decreased connexin43 expression. Interestingly, MEK1/2inhibition also increased p-Akt expression, but inhibition of PI3K led to p-ERK downregulation.


CONCLUSIONS
Culturing H9c2 cells under low-after-high glucose downregulated connexin43 by promoting autophagy through a mechanism involving the PI3K/Akt/mTOR and MEK/ERK1/2signaling pathways.
Department of Cardiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
Yaguang
Y
Bi
Department of Cardiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
Guangyu
G
Wang
Department of Cardiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
Xiangdong
X
Liu
Department of Cardiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.
Meng
M
Wei
Department of Cardiology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China. zhangqingyong6th@126.com.
Qingyong
Q
Zhang
D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D002460:Cell Line; D018031:Connexin 43 / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects*; D005947:Glucose / Q000008:administration & dosage*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
D018031:Connexin 43; D005947:Glucose; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases
Autophagy; Cx43; H9c2 cell; Low-after-high glucose; MEK/ERK1/2; PI3K/Akt/mTOR