Upregulation of connexin 43 and apoptosis‑associated protein expression by high glucose in H9c2 cells was improved by resveratrol via the autophagy signaling pathway

Wang,Guang‑Yu; Bi,Ya‑Guang; Liu,Xiang‑Dong; Han,Jun‑Feng; Wei,Meng; Zhang,Qing‑Yong

doi:10.3892/mmr.2017.6953

Upregulation of connexin 43 and apoptosis‑associated protein expression by high glucose in H9c2 cells was improved by resveratrol via the autophagy signaling pathway

Authors:
- Guang‑Yu Wang
- Ya‑Guang Bi
- Xiang‑Dong Liu
- Jun‑Feng Han
- Meng Wei
- Qing‑Yong Zhang
View Affiliations

Affiliations: Department of Cardiology, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai 200233, P.R. China, Department of Endocrinology and Metabolism, Shanghai Sixth People's Hospital, Shanghai Jiao Tong University, Shanghai 200233, P.R. China
Published online on: July 12, 2017 https://doi.org/10.3892/mmr.2017.6953
Pages: 3262-3268
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The expression of connexin43 (Cx43) protein and the apoptotic rate of cardiomyocytes may be regulated by autophagy and associated with diabetic cardiomyopathy. It is possible that the beneficial effect of resveratrol on diabetic cardiomyocytes occurs via the autophagy pathway. However, it remains to be elucidated whether resveratrol treatment may attenuate the hyperglycemia‑induced remodeling of Cx43 and apoptosis through the regulation of autophagy. H9c2 cardiac cells were incubated with 5.5 and 25 mM glucose, 25 mM glucose with chloroquine (50 µM), and 25 mM glucose with or without resveratrol (10, 25 µM) for 24 h. H9c2 cells were also incubated with 25 µM resveratrol in the presence of chloroquine (50 µM). Cell viability was determined using an MTT cell survival assay. Cytotoxicity was determined by quantification of the release of lactate dehydrogenase. The expression of Cx43, autophagic maker proteins [Beclin‑1, p62 and microtubule‑associated protein 1 light chain 3 (LC3)], apoptosis maker proteins (B‑cell lymphoma‑2 and Bcl‑2 associated X protein), AMP‑activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were determined using western blotting. Resveratrol treatment led to reduced Cx43 expression levels compared with the 25 mM glucose treatment and significantly reduced the expression of apoptosis‑associated proteins in H9c2 cells under hyperglycemic conditions. Autophagy was increased as indicated by the upregulation of Beclin‑1 and p62 expression and the reduced LC3‑II/LC3‑I ratio. AMPK expression was increased, whereas mTOR expression was reduced in the resveratrol treatment groups. Treatment with chloroquine reversed effect of resveratrol. In conclusion, administration resveratrol may protect H9c2 cells against hyperglycemia‑induced Cx43 upregulation and apoptosis, which may be mediated through the induction of the autophagy signaling pathway.

View Figures

View References

1	He C, Zhu H, Li H, Zou MH and Xie Z: Dissociation of Bcl-2-Beclin1 complex by activated AMPK enhances cardiac autophagy and protects against cardiomyocyte apoptosis in diabetes. Diabetes. 62:1270–1281. 2013. View Article : Google Scholar : PubMed/NCBI
2	Xie Z, Lau K, Eby B, Lozano P, He C, Pennington B, Li H, Rathi S, Dong Y, Tian R, et al: Improvement of cardiac functions by chronic metformin treatment is associated with enhanced cardiac autophagy in diabetic OVE26 mice. Diabetes. 60:1770–1778. 2011. View Article : Google Scholar : PubMed/NCBI
3	Martins-Marques T, Catarino S, Marques C, Pereira P and Girão H: To beat or not to beat: Degradation of Cx43 imposes the heart rhythm. Biochem Soc Trans. 43:476–481. 2015. View Article : Google Scholar : PubMed/NCBI
4	Rohr S: Role of gap junctions in the propagation of the cardiac action potential. Cardiovasc Res. 62:309–322. 2004. View Article : Google Scholar : PubMed/NCBI
5	Vozzi C, Dupont E, Coppen SR, Yeh HI and Severs NJ: Chamber-related differences in connexin expression in the human heart. J Mol Cell Cardiol. 31:991–1003. 1999. View Article : Google Scholar : PubMed/NCBI
6	Zhou P, Zhang SM, Wang QL, Wu Q, Chen M and Pei JM: Anti-arrhythmic effect of verapamil is accompanied by preservation of cx43 protein in rat heart. PLoS One. 8:e715672013. View Article : Google Scholar : PubMed/NCBI
7	Rutledge CA, Ng FS, Sulkin MS, Greener ID, Sergeyenko AM, Liu H, Gemel J, Beyer EC, Sovari AA, Efimov IR and Dudley SC: c-Src kinase inhibition reduces arrhythmia inducibility and connexin43 dysregulation after myocardial infarction. J Am Coll Cardiol. 63:928–934. 2014. View Article : Google Scholar : PubMed/NCBI
8	Hesketh GG, Shah MH, Halperin VL, Cooke CA, Akar FG, Yen TE, Kass DA, Machamer CE, Van Eyk JE and Tomaselli GF: Ultrastructure and regulation of lateralized connexin43 in the failing heart. Circ Res. 106:1153–1163. 2010. View Article : Google Scholar : PubMed/NCBI
9	Zhang W, Zhao G, Hu X, Wang M, Li H, Ye Y, Du Q, Yao J, Bao Z, Hong W, et al: Aliskiren-attenuated myocardium apoptosis via regulation of autophagy and connexin-43 in aged spontaneously hypertensive rats. J Cell Mol Med. 18:1247–1256. 2014. View Article : Google Scholar : PubMed/NCBI
10	Lin H, Ogawa K, Imanaga I and Tribulova N: Remodeling of connexin 43 in the diabetic rat heart. Mol Cell Biochem. 290:69–78. 2006. View Article : Google Scholar : PubMed/NCBI
11	Howarth FC, Nowotny N, Zilahi E, El Haj MA and Lei M: Altered expression of gap junction connexin proteins may partly underlie heart rhythm disturbances in the streptozotocin-induced diabetic rat heart. Mol Cell Biochem. 305:145–151. 2007. View Article : Google Scholar : PubMed/NCBI
12	Howarth FC, Chandler NJ, Kharche S, Tellez JO, Greener ID, Yamanushi TT, Billeter R, Boyett MR, Zhang H and Dobrzynski H: Effects of streptozotocin-induced diabetes on connexin43 mRNA and protein expression in ventricular muscle. Mol Cell Biochem. 319:105–114. 2008. View Article : Google Scholar : PubMed/NCBI
13	Fong JT, Kells RM, Gumpert AM, Marzillier JY, Davidson MW and Falk MM: Internalized gap junctions are degraded by autophagy. Autophagy. 8:794–811. 2012. View Article : Google Scholar : PubMed/NCBI
14	Martins-Marques T, Catarino S, Zuzarte M, Marques C, Matafome P, Pereira P and Girão H: Ischaemia-induced autophagy leads to degradation of gap junction protein connexin43 in cardiomyocytes. Biochem J. 467:231–245. 2015. View Article : Google Scholar : PubMed/NCBI
15	Wei H, Liu L and Chen Q: Selective removal of mitochondria via mitophagy: Distinct pathways for different mitochondrial stresses. Biochim Biophys Acta. 1853:2784–2790. 2015. View Article : Google Scholar : PubMed/NCBI
16	Matsui Y, Takagi H, Qu X, Abdellatif M, Sakoda H, Asano T, Levine B and Sadoshima J: Distinct roles of autophagy in the heart during ischemia and reperfusion: Roles of AMP-activated protein kinase and Beclin 1 in mediating autophagy. Circ Res. 100:914–922. 2007. View Article : Google Scholar : PubMed/NCBI
17	Lichtenstein A, Minogue PJ, Beyer EC and Berthoud VM: Autophagy: A pathway that contributes to connexin degradation. J Cell Sci. 124:910–920. 2011. View Article : Google Scholar : PubMed/NCBI
18	Kanamori H, Takemura G, Goto K, Tsujimoto A, Mikami A, Ogino A, Watanabe T, Morishita K, Okada H, Kawasaki M, et al: Autophagic adaptations in diabetic cardiomyopathy differ between type 1 and type 2 diabetes. Autophagy. 11:1146–1160. 2015. View Article : Google Scholar : PubMed/NCBI
19	Liu Y, Palanivel R, Rai E, Park M, Gabor TV, Scheid MP, Xu A and Sweeney G: Adiponectin stimulates autophagy and reduces oxidative stress to enhance insulin sensitivity during high-fat diet feeding in mice. Diabetes. 64:36–48. 2015. View Article : Google Scholar : PubMed/NCBI
20	Wang B, Yang Q, Sun YY, Xing YF, Wang YB, Lu XT, Bai WW, Liu XQ and Zhao YX: Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice. J Cell Mol Med. 18:1599–1611. 2014. View Article : Google Scholar : PubMed/NCBI
21	Gurusamy N, Lekli I, Mukherjee S, Ray D, Ahsan MK, Gherghiceanu M, Popescu LM and Das DK: Cardioprotection by resveratrol: A novel mechanism via autophagy involving the mTORC2 pathway. Cardiovasc Res. 86:103–112. 2010. View Article : Google Scholar : PubMed/NCBI
22	Kanamori H, Takemura G, Goto K, Tsujimoto A, Ogino A, Takeyama T, Kawaguchi T, Watanabe T, Morishita K, Kawasaki M, et al: Resveratrol reverses remodeling in hearts with large, old myocardial infarctions through enhanced autophagy-activating AMP kinase pathway. Am J Pathol. 182:701–713. 2013. View Article : Google Scholar : PubMed/NCBI
23	Guo R and Ren J: Deficiency in AMPK attenuates ethanol-induced cardiac contractile dysfunction through inhibition of autophagosome formation. Cardiovasc Res. 94:480–491. 2012. View Article : Google Scholar : PubMed/NCBI
24	Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, Øvervatn A, Bjørkøy G and Johansen T: p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem. 282:24131–24145. 2007. View Article : Google Scholar : PubMed/NCBI
25	Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y and Yoshimori T: LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19:5720–5728. 2000. View Article : Google Scholar : PubMed/NCBI
26	Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Arozena A Acevedo, Adachi H, Adams CM, Adams PD, Adeli K, et al: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 12:1–222. 2016. View Article : Google Scholar : PubMed/NCBI
27	Wang R, Liu YY, Liu XY, Jia SW, Zhao J, Cui D and Wang L: Resveratrol protects neurons and the myocardium by reducing oxidative stress and ameliorating mitochondria damage in a cerebral ischemia rat model. Cell Physiol Biochem. 34:854–864. 2014. View Article : Google Scholar : PubMed/NCBI
28	Vilar S, Quezada E, Santana L, Uriarte E, Yánez M, Fraiz N, Alcaide C, Cano E and Orallo F: Design, synthesis, and vasorelaxant and platelet antiaggregatory activities of coumarin-resveratrol hybrids. Bioorg Med Chem Lett. 16:257–261. 2006. View Article : Google Scholar : PubMed/NCBI
29	Liu PL, Chong IW, Lee YC, Tsai JR, Wang HM, Hsieh CC, Kuo HF, Liu WL, Chen YH and Chen HL: Anti-inflammatory effects of resveratrol on Hypoxia/Reoxygenation-induced alveolar epithelial cell dysfunction. J Agric Food Chem. 63:9480–9487. 2015. View Article : Google Scholar : PubMed/NCBI
30	Ghaly HA, Boyle PM, Vigmond EJ, Shimoni Y and Nygren A: Simulations of reduced conduction reserve in the diabetic rat heart: Response to uncoupling and reduced excitability. Ann Biomed Eng. 38:1415–1425. 2010. View Article : Google Scholar : PubMed/NCBI
31	Okruhlicova L, Tribulova N, Misejkova M, Kucka M, Stetka R, Slezak J and Manoach M: Gap junction remodelling is involved in the susceptibility of diabetic rats to hypokalemia-induced ventricular fibrillation. Acta Histochem. 104:387–391. 2002. View Article : Google Scholar : PubMed/NCBI
32	Santos-Almeida FM, Girão H, da Silva CA, Salgado HC and Fazan R Jr: Cholinergic stimulation with pyridostigmine protects myocardial infarcted rats against ischemic-induced arrhythmias and preserves connexin43 protein. Am J Physiol Heart Circ Physiol. 308:H101–H107. 2015. View Article : Google Scholar : PubMed/NCBI
33	Greener ID, Sasano T, Wan X, Igarashi T, Strom M, Rosenbaum DS and Donahue JK: Connexin43 gene transfer reduces ventricular tachycardia susceptibility after myocardial infarction. J Am Coll Cardiol. 60:1103–1110. 2012. View Article : Google Scholar : PubMed/NCBI
34	Fernandes R, Girão H and Pereira P: High glucose down-regulates intercellular communication in retinal endothelial cells by enhancing degradation of connexin 43 by a proteasome-dependent mechanism. J Biol Chem. 279:27219–27224. 2004. View Article : Google Scholar : PubMed/NCBI
35	Yu L, Zhao Y, Fan Y, Wang M, Xu S and Fu G: Epigallocatechin-3 gallate, a green tea catechin, attenuated the downregulation of the cardiac gap junction induced by high glucose in neonatal rat cardiomyocytes. Cell physiol Biochem. 26:403–412. 2010. View Article : Google Scholar : PubMed/NCBI
36	Kanno S, Kovacs A, Yamada KA and Saffitz JE: Connexin43 as a determinant of myocardial infarct size following coronary occlusion in mice. J Am Coll Cardiol. 41:681–686. 2003. View Article : Google Scholar : PubMed/NCBI
37	Colosetti P, Puissant A, Robert G, Luciano F, Jacquel A, Gounon P, Cassuto JP and Auberger P: Autophagy is an important event for megakaryocytic differentiation of the chronic myelogenous leukemia K562 cell line. Autophagy. 5:1092–1098. 2009. View Article : Google Scholar : PubMed/NCBI
38	Zheng Q, Su H, Ranek MJ and Wang X: Autophagy and p62 in cardiac proteinopathy. Circ Res. 109:296–308. 2011. View Article : Google Scholar : PubMed/NCBI