ABSTRACT
AIM: To detect the effects of resveratrol (RSV) on the expression of microRNA-21 (miR-21) in primarily cultured neonatal rat atrial myocytes with electric remodeling induced by rapid electrical stimulation (RES).Furthermore, to find out the possible mechanism of miR-21 regulating electrical remodeling.METHODS: The neonatal rat atrial myocytes were isolated by double-enzyme (trypsin and collagenase I) digestion and differential adhesion method.The atrial fibrillation (AF) model was induced by RES.Atrial myocytes were randomly divided into 4 groups: control group, RSV group, RES group, and RSV+RES group.To further detect whether RSV regulated electric remodeling by miR-21, except the 4 groups, we add miR-21 over-expression group and miR-21 inhibitor group: RES+negative control (NC) group, RES+miR-21 mimics group, RES+miR-21 mimics+RSV group, RES+miR-21 inhibitor group, and RES+miR-21 inhibitor+RSV group.The optimal concentration and pretreatment time of resveratrol were determined by CCK-8 assay.The expression of miR-21 and the mRNA expression of L-type calcium channels CACNA1C and CACNB2 in atrial myocytes were detected by qPCR.The protein expression of L-type calcium channels Cav1.2 and Cavβ2 in the atrial myocytes was analyzed by Western blot.RESULTS: The expression of miR-21 in RES group was significantly increased compared with control group, while preconditioning with RSV decreased the expression of miR-21.Compared with RES+miR-21 mimics group, the expression of miR-21 in RES+miR-21 mimics+RSV group was significantly decreased.Meanwhile, the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ2 were increased (P<0.05).Compared with RES group, the expression of miR-21 in RES+miR-21 inhibitor group and RES+miR-21 inhibitor+RSV group was decreased, while the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ2 were increased.However, no difference of the expression of miR-21, the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ2 among RSV+RES, RES+miR-21 inhibitor and RES+miR-21 inhibitor+RSV groups was observed (P<0.05).CONCLUSION: In AF model induced by RES, RSV may reduce electric remodeling by inhibiting the expression of miR-21 and regulating the downstream target genes.
ABSTRACT
Objective: To explore the protective mechanism of resvertrol (RSV) suppressing the rapid electrical stimulation incurred oxidative stress injury in neonatal rats cardiomyocytes. Methods: The neonatal rats cardiac ifbroblasts and myocytes were isolated by double enzyme digestion and differential adhesion method, and the cardiomyocytes were randomly divided into 5 groups:①Control (CTR) group,②Rapid electrical stimulation (RES) group,③RES+APO group, cells were pretreated with NADPH oxidase inhibitor APO,④RES+RSV group,⑤RES+AIP group, cells were pretreated with CaMKII inhibitor AIP. In order to conifrm whether RSV protection was via MsrA-OX-CaMKⅡpathway, the cells were further divided into another 3 groups:①DMSO control group,②RSV+DMSO group,③RES+ RSV+DMSO group. The best dose of RSV was measured with Kit-8 by cardiomyocytes surviving condition, the optimal electrical stimulation time was detected with ELISA by Ang II level in conditioned medium. The level of reactive oxygen species (ROS) in cardiomyocytes was detected by flow cytometry, the protein expressions of MsrA, Nox4, Nox2, P22phox, OX-CaMK II and apoptosis related cleaved caspase-3 were observed by Western blot analysis.Results:①Compared with CTR group, RES group showed increased AngII secretion with increased protein expressions of Nox4, Nox2, P22phox, OX-CaMK II and cleaved caspase-3.②Compared with RES group, the RES+APO, RES+RSV, RES+AIP groups had decreased ROS level, the ROS was even lower in RES+RSV group.③Compared with RES group, the RES+APO, RES+RSV groups presented decreased protein expressions of Nox4, Nox2, P22phox, OX-CaMK II and cleaved caspase-3, while RES+AIP group only had decreased Nox2, OX-CaMK II and cleaved caspase-3.④Compared with DMSO control group, RES+ RSV+DMSO group had the lower level of cleaved caspase-3 expression. Conclusion: RSV has protective effect on rapid electrical stimulation incurred oxidative stress injury in neonatal rats cardiomyocytes, which might be via NADPH oxidase with the increased MsrA expression .