Effects And Mechanism Of EGCG On Gap Junctions In Rat Cardiomyocytes Under Hyperglycemia

Diabetes mellitus is a disease that affects more than220million people in the world. The major risk for cardiovascular diseases has been widely recognized, such as coronary heart disease and diabetic cardiomyopathy. As the diabetic patients have the high incidence for cardiac arrhythmias including atrial or ventricular fibrillation and sudden death, more recent studies focus its mechanisms. In the heart, cardiac gap junctions contribute to electrical coupling between cardiomyocytes and orderly electrical conduction. Several studies suggested that altered gap junction expression or function could lead to the conduction disturbance and the high incidence of arrhythmias. Therefore, the change of gap junctions between cardiomyocytes in diabetes might be related with arrhythmia complications. It has been demonstrated that Cx43is the major connexin in ventricular cardiomyocytes, whereas Cx40and Cx45is relatively co-expressed in atrial myocytes and the conduction system. The quantity alteration or redistribution of Cx43could provide with an arrhythmogenic substrate for various arrhythmias.In diabetic patients, hyperglycemia is regarded as a major initiator for cardiac complications. However, with the development of diabetes, other miscellaneous factors like advanced glycation end products (AGEs) and hypertriglyceridemia will show their effect, which might be different from hyperglycemia alone. According to some recent studies, the amount of cardiac Cx43was either reduced in a3-week STZ-induced diabetic rat model or increased in a12-week diabetic rat model. We could not exclude the possibility that the discordant results might be due to other factors other than high glucose alone. Therefore, we aimed to explore the effect of simple factor-hyperglycemia on the cardiac gap junctions, which still lack direct evidence till now.Epigallocatechin-3gallate (EGCG), a major component of green tea extracts, has recently been found to have protective role in cardiovascular disease. Several groups showed the beneficial effects of EGCG on cardiac myocytes against ischemia/reperfusion-induced apoptosis and oxidative stress from cardiac hypertrophy. It was reported that EGCG could protect pancreatic beta cells in diabetic rats. Previous studies have also shown the pretreatment with EGCG ameliorated the Cx43-inhibitory effect of dimethylnitrosamine in Mardin-Darby canine kidney (MDCK) cells. To date, however, little is known about the effect of EGCG on the protective role of cardiac gap junctions and the underlying mechanisms.Therefore, in the present study, we aimed to investigate the expression and the function of gap junction in cultured neonatal rat cardiomyocytes under high glucose incubation, and to study the effects and the possible signal transduction pathway of EGCG co-treatment on the cardiac gap junction. Finally, we observed the effect of EGCG on Cx43in a3-week STZ-induced diabetic rat model, in which high blood glucose act as the major pathogenic factor.Part1The effect and mechanisms of high glucose on cardiac gap junctions in rat cardiomyocytesObjective:To investigate the effect and mechanisms of high glucose on cardiac gap junctions in rat cardiomyocytes. Methods:Cardiomyocytes were obtained from2day old Sprague-Dawley rats. After5days, cells were treated with high glucose (30mM). The expression of Cx43protein was detected by Western blot and immunofluorescence. The mRNA level of Cx43was measured by realtime PCR. Scrape loading dye transfer assay was used to observe the function of cardiac gap junctions. The PKC and Mitogen-activated protein kinases (MAPKs) were quantified by Western blot.Result: The Cx43protein expression was down-regulated by high glucose and had most inhibitory effect after72h. The function of gap junctions was reduced, too. The mRNA level of Cx43, however, remained unchanged. The pretreatment of leupeptin (a lysosomal inhibitor) could reverse the Cx43inhibitory effect of high glucose. High glucose activated the time-dependent phosphorylated PKC and Erk, but not P38or JNK. The PKC inhibitor (GF109203X), however, abrogated the Cx43inhibitory effect of high glucose.Conclusion:The Cx43protein expression and gap junction function could be reduced by high glucose. But the Cx43mRNA level remained unchanged, which might be due to the post-transcriptional modification. PKC pathway might be involved in the effect of high glucose on Cx43. Part2The effect and mechanisms of EGCG on reduced Cx43expression by high glucose in rat cardiomyocytesObjective: To investigate the effect and mechanisms of EGCG on reduced Cx43expression by high glucose in rat cardiomyocytes.Methods:Cardiomyocytes were obtained from2day old Sprague-Dawley rats and incubated with high glucose (30mM) for72h。 EGCG was then added into high glucose treated cardiomyocytes. Cx43expression was measured by Western blot, realtime PCR and immunofluorescence. Scrape loading dye transfer assay was used to observe the function of cardiac gap junctions. The Mitogen-activated protein kinases (MAPKs) were quantified by Western blot.Result:Addition of EGCG to high glucose treated cardiomyocytes attenuated the Cx43reduction in a dose-and time-dependent manner and also recovered the reduced function of cells coupling. The mRNA or protein level of Cx40and Cx45showed no significant change by high glucose or EGCG. Nor did the Cx43mRNA level. EGCG activated the time-dependent phosphorylated Erk, JNK and p38MAPK, but not PKC. The p38MAPK inhibitor (SB203580), however, inhibited the protective effect of EGCG.Conclusion:This study showed that EGCG could improve the gap junction function and Cx43expression inhibited by high glucose. P38MAPK pathway might partly participate in the Cx43protective effect of EGCG. Part3The effect of EGCG on cardiac gap junctions in early diabetic ratsObjective:To investigate the effect of EGCG on cardiac gap junctions in early diabetic rats.Methods:Diabetes was induced in male Sprague-Dawley rats by a single intraperitoneal injection of streptozotocin (60mg/kg). The rats were divided into3groups as normal rats, diabetic rats and diabetic rats with EGCG (50mg/kg/d) treatment. After21days, the distribution and expression of Cx43was observed by immunohistochemical staining. Result: The cardiac Cx43expression was reduced in early diabetic rats with disordered and disconnected redistribution. The Cx43remodeling could be improved by EGCG treatment.Conclusion:This study showed that EGCG could improve the downregulation and redistribution of cardiac Cx43in early diabetic rats...