An increase in cytosolic Ca2+ via a capacitative calcium entry (CCE)-mediated pathway, attributed to members of the transient receptor potential (TRP) superfamily, TRPC1 and TRPC3, has been reported to play an important role in regulating cardiomyocyte hypertrophy. Increased cytosolic Ca2+ also plays a critical role in mediating cell death in response to ischemia-reperfusion (I/R). 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 overexpressing TRPC3 in the heart were subjected to 90 min of ischemia and 3 h of reperfusion. After I/R, viability was 51 ± 1% in WT mice and 42 ± 5% in transgenic mice (P < 0.05). Apoptosis assessed by annexin V was significantly increased in the TRPC3 group compared with WT (32 ± 1% vs. 21 ± 3%; P < 0.05); however, there was no significant difference in necrosis between groups. Treatment of TRPC3 cells with the CCE inhibitor SKF-96365 (0.5 μM) significantly improved cellular viability (54 ± 4%) and decreased apoptosis (15 ± 4%); in contrast, the L-type Ca 2+ channel inhibitor verapamil (10 μM) had no effect. Calpain-mediated cleavage of α-fodrin was increased approximately threefold in the transgenic group following I/R compared with WT (P < 0.05); this was significantly attenuated by SKF-96365. The calpain inhibitor PD-150606 (25 μM) attenuated the increase in both α-fodrin cleavage and apoptosis in the TPRC3 group. Increased TRPC3 expression also increased sensitivity to Ca2+ overload stress, but it did not affect the response to TNF-α-induced apoptosis. These results suggest that CCE mediated via TRPC may play a role in cardiomyocyte apoptosis following I/R due, at least in part, to increased calpain activation. Copyright © 2008 the American Physiological Society.