Overexpression of TRPC3 increases apoptosis in response to ischemia/reperfusion but not TNF‐α in adult mouse cardiomyocytes

Abstract

Capacitative calcium entry (CCE), attributed to transient receptor potential (TRP) proteins, has been shown to be important in regulating cardiomyocyte hypertrophy. Increased cytosolic Ca2+ also plays a critical role in mediating cell death in response to ischemia/reperfusion (I/R) injury. Therefore we tested the hypothesis that overexpression of TRPC3 in cardiomyocytes will increase sensitivity to I/R injury. Adult cardiomyocytes isolated from wild-type (WT) mice and from mice over expressing TRPC3 in the heart (TG) were subject to 90 min ischemia and 3hr reperfusion. After I/R viability was 51±1% in WT and 42±5% in TG (p<0.05). There was no significant difference in necrosis between WT and TG groups; however, apoptosis assessed by annexin-V was markedly increased in TG (32±1%) compared to WT (21±3%; p<0.05). Treatment with SKF96365 (0.5μM), an inhibitor of CCE, significantly improved cellular viability (54±4%) and decreased apoptosis (15±4%) in TG group, whereas the L-type Ca2+ channel inhibitor verapamil (10μM) had no effect. Calpain-mediated cleavage of α-fodrin was increased ~3-fold in the TG group following I/R compared to WT (p<0.05). In contrast to I/R, there was no difference between WT and TG groups in apoptosis induced by TNF-α (10ng/ml for 2 and 18 hrs). These results suggest that Ca2+ entry via TRP may play a role in cardiomyocyte apoptosis following I/R due at least in part by increased calpain activation. (Supported by NIH grants HL076165, HL079364, HL-077100).