Apelin Ameliorates High Glucose-Induced Downregulation of Connexin 43 via AMPK-Dependent Pathway in Neonatal Rat Cardiomyocytes
Li Xiaoting1, Yu Lu1, Gao Jing1, Bi Xukun1, Zhang Juhong2, Xu Shiming3, Wang Meihui4, Chen Mengmeng1, Qiu Fuyu1, Fu Guosheng1,*
1Department of Cardiology, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 2Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 3Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China 4Biomedical Research Center, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
Diabetes Mellitus is a common disorder, with increasing risk of cardiac arrhythmias. Studies have shown that altered connexin expression and gap junction remodeling under hyperglycemia contribute to the high prevalence of cardiac arrhythmias and even sudden death. Connexin 43 (Cx43), a major protein that assembles to form cardiac gap junctions, has been found to be downregulated under high glucose conditions, along with inhibition of gap junctional intercellular communication (GJIC). While, apelin, a beneficial adipokine, increases Cx43 protein expression in mouse and human embryonic stem cells during cardiac differentiation. However, it remains unknown whether apelin influences GJIC capacity in cardiomyocytes. Here, using Western blotting and dye transfer assays, we found that Cx43 protein expression was reduced and GJIC was impaired after treatment with high glucose, which, however, could be abrogated after apelin treatment for 48 h. We also found that apelin increased Cx43 expression under normal glucose. Real-time PCR showed that the Cx43 mRNA was not significantly affected under high glucose conditions in the presence of apelin or high glucose and apelin. High glucose decreased the phosphorylation of AMPKα; however, apelin activated AMPKα. Interestingly, we found that Cx43 expression was increased after treatment with AICAR, an activator of AMPK signaling. AMPKα inhibition mediated with transfection of siRNA-AMPKα1 and siRNA-AMPKα2 abolished the protective effect of apelin on Cx43 expression. Our data suggest that apelin attenuates high glucose-induced Cx43 downregulation and improves the loss of functional gap junctions partly through the AMPK pathway.
Li Xiaoting,Yu Lu,Gao Jing, et al. Apelin Ameliorates High Glucose-Induced Downregulation of Connexin 43 via AMPK-Dependent Pathway in Neonatal Rat Cardiomyocytes[J]. Aging and disease,
2018, 9(1): 66-76.
Figure 1. Effects of apelin-13 on Cx43 expression in NRCMs
Apelin-13 reversed the Cx43 downregulation triggered by high glucose in a concentration- (A) and time-dependent (B) manner. The expression of Cx43 significantly decreased under high glucose condition (30 mM, 72 h). The addition of apelin-13 (100 nM, 48 h) apparently alleviated the Cx43 reduction induced by high glucose. (C) Incubation with 100 nM apelin-13 only for 48 h increased the Cx43 protein expression. (D) The effects of high glucose and/or apelin-13 on Cx43 mRNA level were quantified by real-time PCR. CON: normal glucose (5.5 mM); HG: high glucose (30 mM); APLN: apelin-13 (100 nM). *P<0.05, **P<0.01, ***P<0.001. Data are the mean ± SD.
Figure 2. Effects of apelin-13 on high-glucose induced GJIC in NRCMs
The GJIC capacity of NRCMs was evaluated by dye transfer assay. (A) Cells were left untreated in the control group. (B) Cells were incubated with 30 mM glucose for 72 h. (C) Cells were initially treated with 30 mM glucose for 24 h and then co-treated with 100 nM apelin-13 for the following 48 h. (D) The statistical data of net LY dye transfer area indicated that high glucose significantly reduced the LY transfer area and that apelin-13 rescued the high glucose-induce transfer area to a normal level. CON: normal glucose (5.5 mM); HG: high glucose (30 mM); APLN: apelin-13 (100 nM). *P<0.05, **P<0.01, ***P<0.001. Data are the mean ± SD.
Figure 3. The activity of AMPK induced by high glucose and apelin in NRCMs
(A) Cells were separately treated with 30 mM glucose for 7.5 min, 15 min, 30 min and 60 min. The p-AMPKα/AMPKα ratio declined significantly after high glucose treatment for 7.5 min. (B) The phosphorylation of AMPKα increased after the administration of apelin-13 for 30min. (C) Cells were pre-treated with 30 mM glucose for 24h, and the level of pAMPKα/AMPKα increased at 30 min after adding apelin. CON: normal glucose (5.5 mM); HG: high glucose (30 mM); APLN: apelin-13 (100 nM). *P<0.05, **P<0.01, ***P<0.001. Data are the mean ± SD.
Figure 4. The effect of AICAR on Cx43 and pAMPK expression in NRCMs
(A) Cells incubated under normal glucose (CON), osmotic control (MNT), high glucose (HG), high glucose with apelin (HG+APLN) and AICAR were harvested for western blotting. (B) and (C) Compared with control group, the Cx43 expression and pAMPK/AMPKα ratio in osmotic control group were not significantly different. The expression and of Cx43 and pAMPK in high glucose group (30 mM, 72h) was significantly reduced compared with control group, and co-treatment with apelin (100 nM, 48h) relieved the downregulated Cx43 and pAMPK expression. Compared with control group, pAMPKα and Cx43 expression was significantly increased in AICAR group.CON: normal glucose (5.5 mM); MNT: normal glucose (5.5 mM) and mannitol (24.5 mM); HG: high glucose (30 mM); APLN: apelin-13 (100 nM). AICAR: an AMPK activator (0.5 mM). *P<0.05, **P<0.01, ***P<0.001. Data are the mean ± SD.
Figure 5. AMPKα suppression inhibited Cx43 expression up-regulated by apelin-13
(A) Western blotting was examined to reveal the expression of p-AMPKα, AMPKα and Cx43 in cells transfected with siRNA-NC and siRNA-AMPKα1/α2. During the transfection of siRNA, cells were cultured under normal glucose. After 48 h, cells were cultured in high glucose conditions for 72 h with/without apelin (100 nM, 48 h). (B) siRNA-AMPKα1/α2 significantly suppressed the expression of p-AMPKα and AMPKα compared with negative control siRNA. (C) Compared with the high glucose group, apelin-13 addition could increase the p-AMPKα/AMPKα ratio in cells transfected with siRNA-NC, while siRNA-AMPKα1/α2 reduced the p-AMPKα/AMPKα ratio. (D) In cells transfected with negative control siRNA, Cx43 expression was reduced under high glucose and retained with apelin-13 co-treatment. The effect of apelin-13 on Cx43 was abolished by siRNA-AMPKα1/α2. SiRNA-NC: negative control siRNA; *P<0.05, **P<0.01, ***P<0.001. Data are the mean ± SD.
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