The role and mechanism of endoplasmic reticulum stress key factor PREK in myoblast apoptosis under stress loading

Abstract

PURPOSE: This study was aimed to figure out the way that cyclic-stretch influenced the apoptosis of myoblasts and evaluate the importance of PERK and its possible mechanism involved. METHODS: L6 rat myoblasts were cultured in vitro and mechanical stimulation model was constructed successfully. The myoblasts were imposed tension for 0, 2, 6, 12 and 24 hours respectively by multi-channel cell stress loading system. The force value was 15% cell deformation and the frequency was 10 cycles/min. Each cycle was consisted of stretch for 3 seconds and relaxation for 3 seconds, and the group without tension was used as the control group. The apoptotic myoblasts were dyed by DAPI and observed through fluorescence microscopy to detect the apoptosis rate; the mRNA levels of PERK and CHOP in different groups were detected by real-time PCR and protein levels of PERK and p-PERK in different groups were detected by Western blot. PERK inhibitor was used to clear the role of PERK in apoptosis induced by cyclic-stretch and clarify the relationship between the endoplasmic reticulum stress and apoptosis induced by cyclic-stretch. SPSS 17.0 software package was used to analyze the data statistically. RESULTS: DAPI nuclear stain showed that cyclical tensile stress can induce apoptosis in vitro cultured myoblast. Apoptosis rate showed a trend of rising gradually over time, peaked at 24 h. After dealt with the inhibitor of PERK, the apoptosis rate of the 24 h group under the cyclic stretch showed no difference compared with the control. The results of real- time PCR showed that the mRNA of CHOP was increased with the extension loading time, while the mRNA of PERK showed no difference compared with the control. Western blot results showed that the protein level of p-PERK was increased with the extension of loading time, while the expression of PERK showed no difference compared with the control group. When PERK inhibitor added, the mRNA level of CHOP along with the protein expression level of p-PERK showed no significant difference compared to the control. CONCLUSIONS: PERK signaling pathway is involved in the apoptosis of myoblasts induced by cyclic stretch, and the possible mechanism may be closely related to the phosphorylation of PERK.