BackgroundEpigallocatechin-3-gallate (EGCg) with its potent anti-oxidative capabilities is known for its beneficial effects ameliorating oxidative injury to cardiac cells. Although studies have provided convincing evidence to support the cardioprotective effects of EGCg, it remains unclear whether EGCg affect trans-membrane signalling in cardiac cells. Here, we have demonstrated the potential mechanism for cardioprotection of EGCg against H2O2-induced oxidative stress in H9c2 cardiomyoblasts.ResultsExposing H9c2 cells to H2O2 suppressed cell viability and altered the expression of adherens and gap junction proteins with increased levels of intracellular reactive oxygen species and cytosolic Ca2+. These detrimental effects were attenuated by pre-treating cells with EGCg for 30 min. EGCg also attenuated H2O2-mediated cell cycle arrest at the G1-S phase through the glycogen synthase kinase-3β (GSK-3β)/β-catenin/cyclin D1 signalling pathway. To determine how EGCg targets H9c2 cells, enhanced green fluorescence protein (EGFP) was ectopically expressed in these cells. EGFP-emission fluorescence spectroscopy revealed that EGCg induced dose-dependent fluorescence changes in EGFP expressing cells, suggesting that EGCg signalling events might trigger proximity changes of EGFP expressed in these cells.Proteomics studies showed that EGFP formed complexes with the 67 kD laminin receptor, caveolin-1 and -3, β-actin, myosin 9, vimentin in EGFP expressing cells. Using in vitro oxidative stress and in vivo myocardial ischemia models, we also demonstrated the involvement of caveolin in EGCg-mediated cardioprotection. In addition, EGCg-mediated caveolin-1 activation was found to be modulated by Akt/GSK-3β signalling in H2O2-induced H9c2 cell injury.ConclusionsOur data suggest that caveolin serves as a membrane raft that may help mediate cardioprotective EGCg transmembrane signalling.
In this study, we showed the putative mechanism for the cardio‐protection of (−)‐epigallocatechin‐3‐gallate (EGCg). H9c2 cardiac myoblasts exposed to H2O2 decreased cell viability by increasing reactive oxygen species and cytosolic Ca2+, and imposed cell cycle arrest at G1‐S phase via the signaling pathway for glycogen synthase kinase‐3β/β‐catenin/cyclin D1. All these H2O2‐induced cardiac cell injuries could be counteracted by EGCg pretreatment. To determine the binding of EGCg to H9c2 cells, EGFP (enhanced green fluorescence protein) was ectopically expressed in the cells. In overexpressed cells EGFP formed the protein complexes with 67 kD laminin receptor, caveolin‐1 and ‐3, γ‐actin, myosin IX, and vimentin. EGFP fluorescence measurements showed that EGCg bound to intact, Triton X‐100 soluble fraction, and Triton X‐100 insoluble fraction of H9c2 cells with a dissociation constant (Kd) of ~ 86, ~126, and ~42 μM, respectively. H9c2 cells pretreated with H2O2 increased the binding affinity of EGCg (Kd = 47μM). Using a rat model of myocardial infarction involving left anterior descending coronary artery ligation we showed involvement of caveolin‐3 in the cardio‐protection of green tea polyphenols against myocardial ischemia. Taken together, caveolins might serve as rafts for intracellular signaling to mediate cardiac protection of EGCg.
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