Dexmedetomidine Preconditioning Attenuates Oxygen Glucose Deprivation/Reperfusion Injury By Suppression Of RyR2 Phosphorylation In H9C2 Cells

Objective Myocardial ischemia/reperfusion injury(MIRI)is a common and complex physiological and pathological process,and Ca2+ regulatory disorder is one of the important causes of myocardial dysfunction and cell injury after ischemia-reperfusion.As a clinically-used sedative analgesic,the organ protection of dexmedetomidine(Dex)has been widely reported in recent years.Dex has been shown to protect myocardium from ischemia-reperfusion(I/R)injury by inhibiting ROS and activating anti-inflammatory pathways.However,it is still unclear whether it can reduce myocardial I/R injury by regulating calcium homeostasis.The purpose of this study was to investigate the protective effect of Dex preconditioning against oxygen glucose deprivation/re-oxygenation(OGD/R)injury in H9C2 cells and its regulation on intracellular calcium.Methods1.Rat cardiomyocyte cell line H9C2 cells were treated with OGD for 24 h and then re-oxygenated for 3 h to establish the OGD/R model;Dex groups: cells were exposed to different concentrations of Dex(0.1 ?mol,1 ?mol/L and 10 ?mol/L)for 1 h before OGD/R.Cells in the control group were cultured as usual.2.Cell viability and the lactate dehydrogenase(LDH)release were assayed.The calcium imaging technology was used to detect intracellular calcium.Observe the morphology of the cells under an inverted microscope.3.The expression of FKBP12.6 m RNA was detected by real-time polymerase chain reaction(RT-PCR).Western Blot was used to detect the protein expression of FKBP12.6,Caspase-3,cleaved Caspase-3,Ry R2 and the phosphorylation of Ry R2.4.Knock down FKBP12.6 to check if it can reverse the protective effect of Dex.Results1.OGD/R treatment can significantly reduce cell viability,increase LDH release and intracellular calcium concentration,and aggravate cell injury and apoptosis.2.OGD/R decreased the expression of FKBP12.6 at m RNA and protein levels,up-regulated the protein level of cleaved Caspase-3.The expression of Ry R2 protein was not statistically different,but the phosphorylation of Ry R2 Ser2814 was significantly increased in OGD/R group.3.Dex can significantly improve OGD/R cell viability,decrease LDH release and intracellular calcium.The protective effect is consistent with the concentration of Dex,and it can exert optimal protective effect when the final concentration is 1 ?mol/L.Additionally,Dex markedly increased the m RNA and the protein level of FKBP12.6 compared to group OGD/R.There was no statistical difference in Ry R2 protein expression but Dex depressed the phosphorylation of Ry R2 at site Ser2814.And the expression of cleaved Caspase-3 was decreased in the Dex group.4.Knockdown of FKBP12.6 reversed the phosphorylation of Ry R2 at S2814 and the expression of cleaved Caspase-3,which significantly abolished the DEX-mediated protective effects.Conclusion Dexmedetomidine preconditioning protects H9C2 cells against OGD/R injury,which may be associated with an inhibition of the intracellular calcium overload by up-regulation of FKBP12.6 and depression of the Ry R2 phosphorylation at site Ser2814.