Mechanical Stretch Induces Apoptosis Regulator TRB3 in Cultured Cardiomyocytes and Volume-Overloaded Heart

The expression of TRB3 (tribbles 3), an apoptosis regulated gene, increases during endoplasmic reticulum (ER) stress. How mechanical stress affects the regulation of TRB3 in cardiomyocytes during apoptosis is not fully understood. An in vivo model of aorta-caval shunt in adult rats demonstrated the increased TRB3 protein expression in the myocardium. The tumor necrosis factor-alpha (TNF-α) antagonist etanercept reversed the TRB3 protein expression and cardiomyocyte apoptosis induced by AV shunt. An in vitro model of cyclic stretch in neonatal rats was also used to investigate TRB3 expression. We hypothesized that cardiomyocyte apoptosis induced by cyclic stretch is TRB3 dependent. Neonatal rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. Cyclic stretch significantly increased TRB3 protein and mRNA expression. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125, TNF-α antibody and etanercept 30 min before stretch reversed the induction of TRB3 protein induced by stretch. Cyclic stretch induced the DNA-binding activity of growth arrest and DNA damaged inducible gene-153 (GADD153) by electrophoretic mobility shift assay. SP600125, JNK siRNA, TNF-α antibody and etanercept abolished the binding activity induced by stretch. TRB3 promoter activity was enhanced by stretch and TRB3-mut plasmid, SP600125, TNF-α antibody and etanercept attenuated TRB3 promoter activity induced by stretch. Exogenous administration of TNF-α recombinant protein to the non-stretched cardiomyocytes increased TRB3 protein expression similar to that seen after stretch. Cyclic stretch induced cardiomyocyte apoptosis is inhibited by TRB3 siRNA and etanercept. The stretch-induced TRB3 is mediated by TNF-α、JNK and GADD153 pathway. These results indicate that TRB3 plays an important role in stretch-induced cardiomyocyte apoptosis.TRIB3（tribbles 3）作为一种凋亡调控基因，其表达在内质网（ER）应激过程中显著上调。目前，机械应激在心肌细胞凋亡过程中对TRIB3表达调控的影响尚未完全阐明。本研究通过成年大鼠主动脉腔静脉分流体内模型，证实心肌组织中TRIB3蛋白表达水平升高；肿瘤坏死因子-α（TNF-α）拮抗剂依那西普可逆转主动脉腔静脉分流诱导的TRIB3蛋白表达上调及心肌细胞凋亡。本研究同时构建新生大鼠心肌细胞周期性牵张体外模型，以探究TRIB3的表达调控机制，并提出假说：周期性牵张诱导的心肌细胞凋亡依赖于TRIB3。将接种于柔性膜基底的新生大鼠心肌细胞以60次/分钟的频率进行真空牵张，使细胞伸长至最大长度的20%。结果显示，周期性牵张可显著上调TRIB3的蛋白及mRNA表达水平。在牵张前30分钟加入c-jun氨基末端激酶（JNK）抑制剂SP600125、TNF-α抗体及依那西普，可逆转牵张诱导的TRIB3蛋白表达上调。采用电泳迁移率变动分析检测发现，周期性牵张可诱导生长阻滞和DNA损伤诱导基因153（GADD153）的DNA结合活性；而SP600125、JNK小干扰RNA（siRNA）、TNF-α抗体及依那西普均可消除牵张诱导的该结合活性。周期性牵张可增强TRIB3的启动子活性；而TRIB3突变质粒、SP600125、TNF-α抗体及依那西普可削弱牵张诱导的TRIB3启动子活性。向未接受牵张的心肌细胞外源性添加重组TNF-α蛋白，可模拟牵张状态下的TRIB3蛋白表达上调效应。周期性牵张诱导的心肌细胞凋亡可被TRIB3小干扰RNA（siRNA）及依那西普抑制。综上，牵张诱导的TRIB3表达通过TNF-α、JNK及GADD153通路介导。本研究结果表明，TRIB3在周期性牵张诱导的心肌细胞凋亡中发挥关键作用。