Experimental Study Of Protective Effects Of Salubrinal On Cardiocyte Apoptosis Induced By Tunicamycin

Ischemia can trigger the apoptosis of cardiomyocyte, if the condition persists, progressive loss of cardiomyocyte were ensued, and even develop into heart failure. Recent studies have shown that in addition to caspase dependent apoptotic pathway, there also exists the endoplasmic reticulum stress-induced apoptotic pathway. Under cellular stress conditions, such as hypoxia,glucose deprivation and drugs, the accumulation of unfolded/misfolded proteins, disrupts the proper function of ER and initiates the unfolded protein response (UPR) to cope with this adverse situation. Activated PERK phosphorylates eukaryotic translation initiation factor 2 subunit a (eIF2a), which inhibited global protein synthesis and leads to protection of cells,However, prolonged ER stress with the failure of cellular protective mechanisms by UPR will eventually activate the expression of CHOP/GADD153 and caspase-12, two molecular chaperones in ERS apoptosis pathway, and finally results in cell apoptosis.Tunicamycin (tunicamycin, TM), an inhibitor of glycosylation of proteins, can induce cellular apoptosis. Salubrinal was identified as a selective inhibitor of phosphatases, through inhibiting the dephosphorylation of eIF2a, which result in a halt of protein synthesis and permit cells to recover from endoplasmic reticulum (ER) stress. But in terms of cardiomyocyte apoptosis induced by TM and protective effect by salubrinal has not been reported. Here, we examined whether TM initiates the apoptosis of cardiomyocyte through activating the death pathway of ER stress, and investigated the role and signaling pathways,which salubrinal act on the apoptosis of myocardiomyocyte induced by tunicamycin.1. To investigate tunicamycin in the induction of cardiomyocyte apoptosis and the possible pathway of apoptosis.To investigate the relationship between tunicamycin and cardiomyocyte apoptosis, neonatal rat cardiomyocyte treated by tunicamycin was detected by MTT assay,DNA Ladder and flow cytommetry. Real time PCR and western blot methods were used to observe the expression of GRP78, eIF2a, phosphorylation of eukaryotic initiation factor 2-a(P-eIF2a) and caspase-12. The results showed that:①Tunicamycin can induce cardiomyocyte apoptosis in a concentration-dependent and time-dependent manner;②Unfolded protein response (UPR),a cytoprotective response, was detected by the induction of GRP78, and P-eIF2a in the primary stage, laterlly the ER stress response switched from UPR to a pro-apoptotic response as demonstrated by the upregulation of CHOP/GADD153 and processing of caspase-12.2. To investigate the protective effects and related mechanisms of salubrinal on neonatal rat cardiomyocyte apoptosis induced by tunicamycinMTT, flow cytometry, TUNEL were used to detect the protective effects of salubrinal on cardiomyocyte apoptosis induced by tunicamycin,and the expressions of GRP78, eIF2a, P-eIF2a, CHOP/GADD153 and cleaved caspase-12 during the apoptosis were detected by high content screening, Hoechst staining and Western blot. We found that①Within the range of 100μM, salubrinal was non-toxic to cardiomyocyte;②The expression of P-eIF2a protein in cardiomyocyte treated by salubrinal was increased in a concentration-dependent manner.③Salubrinal does not cause endoplasmic reticulum stress by itself, and plays protective roles in the cardiomyocyte apoptosis induced by tunicamycin in a concentration-dependent manner;④The protective effect of salubrinal on tunicamycin-induced cardiomyocyte apoptosis, may through inhibiting the dephosphorylation of eIF2a and donwn-regulating the expressions of CHOP/GADD153 and processing of caspase-12.