Abstract 232: Role of Transient Receptor Potential Melastatin 2 in Cardiomyocyte Apoptosis Induced by TNF-α

Abstract

Mechanical trauma, such as that induced by motor vehicle crashes, represents a major medical and economic problem in the world. Identifying the mechanisms responsible for post-traumatic secondary myocardial injury is critical in order to reduce overall mortality and improve quality of life after trauma. We have previously demonstrated that mechanical trauma-induced overproduction of TNF-α plays a causative role in cardiomyocyte apoptosis via oxidative/nitrative stress. Transient receptor potential melastatin 2 (TRPM2) is a Ca 2+ permeable non-selective cation channel activated by oxidative stress, expressed in the cardiomyocytes. The present study attempted to identify whether TRPM2 is involved in TNF-α-induced cardiomyocyte apoptosis. Cardiomyocytes were isolated from adult male Sprague Dawley rats and cultured with TNF-α (10 ng/ml) for 12h. RT-PCR and semi-quantitative immunohistochemistry were used to quantify TRPM2 mRNA and protein levels respectively. Significant increases in TRPM2 mRNA and protein expression were observed in TNF-α-treated cardiomyocytes, suggesting that TRPM2 may contribute to TNF-α-induced cardiomyocyte apoptosis. To identify the effect of TRPM2 on TNF-α-induced cardiomyocyte apoptosis, cardiomyocytes were cultured with TNF-α or TNF-α + TRPM2 inhibitor (flufenamic acid (FFA) 100uM or clotrimazole 30uM), respectively. Exposure of cardiomyocytes to TNF-α for 12h induced significant apoptosis as determined by caspase-3 activation (1.7-fold increase vs. control, P < 0.01). In contrast, TNF-α-induced caspase-3 activity increases were significantly depressed by FFA and clotrimazole, respectively (P < 0.05). To further confirm the effect of TRPM2 on TNF-α-induced cardiomyocyte apoptosis, we tested the effects of TRPM2-specific small interfering RNA (siRNA). As a result, impressively, TNF-α-induced increases of caspase-3 activity and lysate nucleosomes were significantly reduced in TRPM2-specific siRNA-treated cardiomyocytes (P < 0.01). These results indicate that TRPM2 plays an important role in TNF-α-induced cardiomyocyte apoptosis. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating mechanical trauma-induced secondary myocardial injury.