Abstract 1794: Role of TNF-α in Cardiomyocyte Apoptosis after Mechanical Trauma
We have previously demonstrated that mechanical traumatic injury without circulatory shock causes cardiomyocyte apoptosis and cardiac dysfunction. The present study attempted to identify the mechanisms responsible for cardiomyocyte apoptosis induced by trauma. Cardiomyocytes were isolated from normal mice (NC) or nonfatal traumatic mice (TC) (immediately after a Noble-Collip drum trauma) and cultured with 10% plasma from normal (NP) or traumatic mice (TP, 1.5 hours after trauma). Exposure of TC to NP for 12 hours failed to induce significant apoptosis as determined by caspase-3 activation. In contrast, exposure of NC to TP resulted in an 1.9-fold increase in caspase-3 activity (P < 0.01). These results indicate that certain molecules released to plasma by trauma, not the cardiomyocyte itself, that play a causative role in post-traumatic cardiomyocyte apoptosis. To further identify the pro-apoptotic molecules present in TP, NC were cultured with a cytomix (10 ng/ml TNFα, 1 ng/ml IL-1β, and 1000 U/ml IFN-γ) or individual cytokine for 12 hours. Addition of cytomix resulted in significant cardiomyocyte apoptosis as evidenced by a 1.7-fold increase in caspase-3 activity (p < 0.01). Treatment with IL-1β or IFN-γ alone had no effect on caspase-3 activity. However, treatment with TNFα alone significantly increased caspase-3 activity (1.50±0.19-fold, p<0.05). Moreover, TP-induced caspase-3 activation was markedly inhibited by addition of an anti-TNFα antibody (p<0.05). These results demonstrated that TNFα is the primary pro-apoptotic cytokine in the TP. To further identify the pro-apoptotic signaling pathway within the cardiomyocytes that is activated by TP, cardiomyocyte nitrotyrosine (a footprint for peroxynitrite) was determined. Impressively, a greater than 10-fold increase in nitrotyrosine content was observed in TP-treated cardiomyocytes, suggesting that production of ONOO−, a highly cytotoxic and pro-apoptotic molecule, was increased in TP-treated cardiomyocytes. In conclusion, our study demonstrated for the first time that cardiomyocyte apoptosis caused by mechanic trauma is initiated by TNFα produced by the injured peripheral tissues and mediated by overproduction of cytotoxic reactive nitrogen species within the heart.