Role And Molecular Mechanism Of Group Ⅰ Metabotropic Glutamate Receptors In Ventricular Arrhythmia Related To Connexin43

BackgroudGroup I metabotropic glutamate receptors, mGluR1 and mGluR5, are associated with sympathetic nerve activity. Sympathetic nerve stimulation exerts a crucial effect on modulating phosphorylation status and distribution of connexin43 (Cx43) in rat heart. Hence, mGluRl and mGluR5 have an indirect effect on regulating the function of gap junction channels, which is affected by the availability of Cx43 protein. Additionally, it has been demonstrated that mGluR1/5 are present in ventricular myocardium in particular intercalated disks where Cx43 is the principal component of ventricular gap junction channels. We, therefore, hypothesized that mGluR1/5 might regulate Cx43 phosphorylation and gap junctional intercellular communication (GJIC) directly, independent of sympathetic nerve stimulation.Objective1. To explore the role of group Ⅰ metabotropic glutamate receptors, mGluR1/mGluR5, in connexin43 phosphorylation and inhibition of gap junctional intercellular communication;2. To further study the molecular mechanism of role of mGluRl/5 in Cx43 phosphorylation and GJIC inhibition.Methods1. Use the technology of Western blotting and Immunofluorescence microscopy to document the presence of mGluR1 and mGluR5 in H9c2 cardiomyoblast cells;2. addition of the selective mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine hydrate (DHPG) induced Cx43 phosphorylation and GJIC inhibition in both concentration-dependent (0.1,1,10,100,1000 μM) and time-dependent (5,10,15, 30,60,120 min) manner. Use the technology of Western blotting to ensure the optimum of time and concentration;3. Whether pre-incubation of cells with mGluRl antagonist LY367385 and mGluR5 antagonist MPEP before application of DHPG produced a marked inhibition of agonist-induced Cx43 phosphorylation and GJIC inhibition. Use the technology of Western blotting to identify the phosphorylation status of Cx43;4. PD98059, a specific inhibitor of MEK1 activating directly ERK1/2, was used to assess possible involvement of ERK1/2 in phosphorylation response of Cx43 and suppression of GJIC. Use the technology of Western blotting to identify the phosphorylation status of Cx43 and ERK1/2;5. GF109203X, a potent and selective inhibitor of PKC, was used to assess possible involvement of ERK1/2 in phosphorylation response of Cx43 and suppression of GJIC. Use the technology of Western blotting to identify the phosphorylation status of Cx43 and ERKl/2;6. Use the technology of scrape loading to determine GJIC with different intervention conditions for H9c2 cardiomyoblast cells.Results1. Presence of mGluRl/5 in rat cardiomyoblast cell line H9c2 The specific 142 and 130 kDa bands corresponding to molecular weights of mGluRl and mGluR5, respectively, were detected. The content of mG1uR1 is higher than this ofmGluR5;2. Effects of mGluRl/5 agonist DHPG on Cx43 phosphorylation pattern and total level and on GJICThe administration of mGluRl/5 agonist DHPG can induce an relative increase in Cx43-P2 band level and reduced Cx43-P0 and Cx43-Pl band levels. In addition, total amount of Cx43 protein was reduced gradually compared to that of group NC with increasing DHPG concentration. GJIC was inhibited obviously with 100μM DHPG for 15 min compared with that of negative control when Cx43 phosphorylation was most obvious;3. Effects of mGluRl/5 antagonists on DHPG-induced Cx43 phosphorylation and GJIC inhibitionMG1uR1 antagonist LY367385 and mG1uR5 antagonist MPEP were examined to determine which of these two receptors might be mainly involved in DHPG effect. Preincubation of cells with LY367385 (100 1M) for 15 min before application of DHPG (1001M,15 min) produced a marked inhibition of agonist-induced Cx43 phosphorylation activation and inhibition of GJIC, however, MPEP (101M,15 min) pretreatment failed to block them. These data indicate that Cx43 phosphorylation and GJIC inhibition is mediated mainly by mG1uR1;4. Effect of PKC inhibitor on DHPG-stimulated Cx43 and ERK1/2 phosphorylation response and GJIC inhibitionPre-incubation of the PKC inhibitor at increasing concentrations had little or no effect on DHPG-induced phosphorylation level of ERK1/2 and Cx43. Meanwhile, the PKC inhibitor even at increasing concentrations was unable to affect DHPG-induced GJIC drop;5. Effect of the MEK1 inhibitor on DHPG-stimulated Cx43 and ERK1/2 phosphorylation response and GJIC inhibitionDHPG-induced Cx43 phosphorylation was attenuated significantly by 60 min exposure of cells to increasing concentrations of PD98059, while DHPG-induced phosphorylation level of ERK1/2 also decreased by application of PD98059. PD98059 (60 min) also blocked the DHPG-induced drop of GJIC with increasing concentrations.Conclusion1. It has been proved that the presence of mGluRl and mGluR5 in H9c2 cardiomyoblast cells;2. This study indicates that group I mGluRs induce Cx43 phosphorylation, as shown in Western blot analysis, which contributes to decreased GJIC;3. The receptor that plays a major role in DHPG-induced Cx43 phosphorylation and GJIC inhibition is mG1uR1;4. These findings confirm in the rat cardiomyoblast cell line H9c2 that molecular mechanisms of DHPG-induced Cx43 phosphorylation are consistent with DHPG-induced inhibition of GJIC via ERK1/2;5. PKC pathway is not involved in the process of DHPG-induced Cx43 phosphorylation and GJIC inhibition.