Growth-arrest-specific Protein 6 Protection From Cardiac Myocytes Apoptosis Induced By Anoxia-reoxygenation Via PI3K/Akt Survival Pathway

OBJECTIVE: To investigate the protection effect of growth-arrest-specific protein 6 on the anoxia/reoxygenation(A/R) induced primary neonatal cardiac myocytes(PCM) and H9c2 cell lines injury, and its possible relationship with PI3K/Akt pathway.METHODS: In the part One,cultured primary neonatal cardiac myocytes and H9c2 cells in vitro were disposed with anoxia/reoxygenation A/R (3 h/3 h,2 h/3 h) respectively. Cultured cardiomyocytes of neonatal Wistar rats and H9c2 cells were both randomly divided into three groups: normal control group (Control group), anoxia/reoxygenation group (A/R group) and anoxia/reoxygenation+rhGas6 group (A/R+rhGas6 group). The cardiocyte morphology and beating rate were observed under inverted microscope. The myocardial survival rate, LDH concentration and the levels of Caspase-3 activity were detected by Automatic Biochemistry Analytic Instrument. Cell apoptotic rate was evaluated by flow cytometry. In the part Two, the cultured H9c2 cells were randomly divided into four groups: normal control group (Control group), anoxia/reoxygenation group (A/R), anoxia/reoxygenation+Gas6 group (A/R+Gas6) and anoxia/ reoxygenation + Gas6 + LY294002 group (A/R+Gas6+LY294002). Cell apoptotic rate was evaluated by flow cytometry and the levels of pAkt protein were determined by Western blotting.RESULTS: Compared with that of normal group, the beating rate of primary neonatal cardiac myocytes (35±7 vs 94±3) and survival rate (48.5±4.0 vs 94.2±3.3)% were significantly decreased in A/R group (P<0.05), and L-lactate dehydrogenase(LDH) concentration (206.67±16.31 vs 81.50±5.94)IU/L, Caspase-3 activity (10.19±0.78 vs 3.23±0.41) and Cell apoptotic rate (14.51±0.82 vs 2.83±0.42)% were increased (P<0.05). In the part two, with Gas6 intervention, the beating rate (78±6 vs 35±7) and survival rate (83.5±3.6 vs 48.5±4.0)% were increased significantly (P<0.05), meanwhile L-lactate dehydrogenase(LDH) concentration (107.22±8.77 vs 206.67±16.31) IU/L,Caspase-3 activity (5.17±0.64 vs 10.19±0.78) and Cell apoptotic rate (5.17±0.64 vs 14.51±0.82) % were decreased as compared with A/R group (P<0.05). While to H9c2 cells, the survival rate (82.1±4.3 vs 43.2±3.5)% were increased significantly in rhGas6 group as compared with A/R group (P<0.05), and L-lactate dehydrogenase(LDH) concentration (110.67±11.72 vs 193.83±17.25)IU/L, Caspase-3 activity (4.63±0.50 vs 8.86±0.70) and Cell apoptotic rate (5.58±0.53 vs 12.16±0.68)%were decreased (P<0.05). In the part Two, with the intervention of rhGas6 in H9c2 cells, the Cell apoptotic rate was decreased (5.87±0.27 vs 12.14±0.56)% and the levels of pAkt protein (1.6269±0.065 vs 1.3217±0.041) were increased as compared with A/R group (P<0.05); However, the effect of Gas6 was inhibited by LY294002, the Cell apoptotic rate was increased markedly (9.92±0.41 vs 5.87±0.27)%, and the levels of pAkt protein (0.2978±0.011 vs 1.6269±0.065) were decreased in the A/R+Gas6+LY294002 group (P<0.05).CONCLUSIONS: Gas6 may protect anoxia/reoxygenation (A/R) induced primary neonatal cardiac myocytes and H9c2 cells injury, and H9c2 cells can replace of the primary cardiac myocytes in the research of the protection of Gas6 in myocardial cells as an important kind of cell lines. The protection of Gas6 from H9c2 cells apoptosis induced by anoxia/reoxygenation is possibly mediated by the activation of PI3K/Akt survival pathway via increasing the expression levels of pAkt protein.