The Effects Of DIDS On Tunicamycin-induced Cardiomyocyte Apoptosis

Apoptosis, also known as programmed cell death (PCD), includes death receptor and mitochondrial pathways; while endoplasmic reticulum stress (ERS)-induced apoptosis has also been recognized and plays important roles in a series of cardiovascular diseases. So to inhibit ERS and attenuate apoptosis seems to be an attractive therapeutic target for cardiovascular diseases.The early stage of apoptosis requires sustained cell shrinkage, also named apoptotic volume decrease (AVD). A lot of studies showed that the efflux of Cl- and concomitant with the egress of water from the cell is an early prerequisite and ahead of activation of caspases. The inhibition of chloride channel blocker (DIDS) reduced AVD and inhibited apoptosis.In this study, we proposed to establish ERS-induced apoptotic model by Tunicamycin(Tm)in cultured neonatal rat cardiomyocytes and investigate if DIDS exerts a cardioprotective effect on ER stress-induced apoptosis. Then we primarily explored the mechanism of DIDS on ERS-induced apoptosis. Aims:1.To establish endoplasmic reticulum stress induced-apoptotic model by Tm in primary cultured neonatal rat cardiomyocytes.2.To detect the effect of chloride channel blocker DIDS on Tm-induced apoptosis of cardiomyocytes.3.The effects of chloride channel blocker DIDS on the levels of protein markers of ERS in Tm-treated cardiomyocyte apoptosis.Methods:1.Primary cultured neonatal rat cardiomyocytes were prepared and ERS- induced apoptotic model was established by Tm. Cultured caridiomyocytes were divided into four teams: the control group (Ctrl), Tm group (Tm), DIDS treatment group (Tm+DIDS) and DIDS group (DIDS).2.Cell viability was detected by the colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and apoptosis was assessed by flow cytometry analysis.3.The protein levels of GRP78 and CHOP induced by Tm were assessed by western blotting.4.The changes of cell viability, apoptosis and chaperon GRP78, signal molecular CHOP induced by Tm were detected in the absence or presence of chloride channel blocker DIDS.Results:1.Compared with the normal control, cell viability was significantly decreased in Tm group, which was time and concentration-dependent (P<0.05, n=12). The optimal concentration of tunicamycin treatment was 100ng/ml, and action time of tunicamycin was 72h. 2.The character of cell death was detected by MTT assay and flow cytometry analysis. The treatment of Tm (100ng/ml, 72h) produced 57.4±3.2 % (n=12) of cellular viability and 25.9±5.8% (n=6) of apoptotic population in cardiomyocytes. These indicate that the main death pattern-induced by Tm was apoptotic death.3 . The protein levels of GRP78 and CHOP upregulated at 6h when cardiomyocytes were exposed to Tm. After Tm treatment for 24h, the upregulation of GRP78 and CHOP reached the maximum.4.Compared with Tm group, cell viability of DIDS+Tm group was significantly improved, which were 57.4±3.2%和80.4%±4.2%( P<0.05, n=12), respectively.5 . Compared with Tm group, apoptotic rate of DIDS+Tm group was significantly declined, which were 25.9±5.8% and 14.3%±2.9%(P<0.05,n=6),respectively.6.Compared with the normal group, the protein levels of GRP78 and CHOP treated with Tm increased 3-fold and 6.5 fold, respectively. While the expressions of GRP78 and CHOP markedly downregulated when cardiomyocytes were treated with Tm and DIDS (p<0.05, n=6).Conclusion:1. Tm-induced apoptotic model in cultured neonatal rat cardiomyocytes were successfully established. The optimal concentration and time of Tm treatment was 100ng/ml, 72h, respectively.2. Chloride channel blocker DIDS significantly reduced apoptosis induced by Tm, which markedly reduced cell injury induced by endoplasmic reticulum stress.3. The inhibition of Tm-induced apoptosis by chloride channel blocker DIDS could be related with downregulated expressions of GRP78 and CHOP, which showed that ERS-induced by Tm was attenuated by DIDS.