Activation of an apoptotic signal transduction pathway involved in the upregulation of calpain and apoptosis-inducing factor in aldosterone-induced primary cultured cardiomyocytes.

In this study, aldosterone (ALD)-induced apoptosis of cardiomyocyte was evaluated based on the previous studies, and the roles of calpain signaling were clarified. Primary cultured rat cardiomyocytes were injured by ALD (0.01-10 μM) for varying time periods. Then, the effects of ethylene glycol tetraacetic acid (EGTA) (0.5 mM), calpeptin (2.5 μM), and spironoclactone (10 μM) were evaluated on cardiomyocytes activated by ALD. Cardiomyocytes that were injured by ALD were assayed by the MTT and LDH leakage ratio. Apoptosis was evaluated by a TUNEL assay, annexin V/PI staining, and caspase-3 activity. The expression of cleavage of Bid (tBid), calpain and apoptosis-inducing factor (AIF) was evaluated by western blot analysis. ALD increased calpain expression and caspase-3 activity and promoted Bid cleavage. It also induced the release of AIF from mitochondria into the cytosol. The upregulation of calpain, tBid and caspase-3 activity were further inhibited by treatment with EGTA in the presence of ALD. Additionally, AIF levels in the cytosol decreased due to EGTA but not due to calpeptin. This was also accompanied by a significant decrease in apoptosis. Furthermore, treatment with spironoclactone not only attenuated the pro-apoptotic effect of ALD but reversed the ALD-induced increase of calpain and AIF levels.