A, Merged images of sectioned fish ventricles co-stained with TUNEL (green) and Mef2 (red) at 4 weeks after 20 ug/gbm DOX injection with or without rapamycin (0.2 μmol/L) treatment. Insets are images of higher magnification. Arrows: Mef2+/TUNEL+ cells. Scale bar=20 μm. B, Merged images of primary cultured CMs co-stained with TUNEL (green), Mef2 (red) and DAPI (blue) after 2 h of DOX (5 μmol/L) treatment with or without rapamycin (0.2 μmol/L) co-treatment. Arrows: TUNEL+/Mef2+ cells; arrowheads: TUNEL+/Mef2-cells. Scale bar=20 μm. C, Quantification of apoptotic cells in sectioned ventricles to show dramatically increased apoptosis at 4 weeks, but not 3 days after 20 μg/gbm DOX injection, and rapamycin attenuates 20 μg/gbm DOX-induced apoptosis significantly. D, Quantification of apoptotic cells in sectioned ventricles to show significantly reduced apoptosis was observed in ztor+/− fish compared to that in wild type fish at 4 weeks, but not 3 days after 20 μg/gbm DOX injection. E, Quantification of the TUNEL index from (B) showed that 5 μmol/L DOX induced significant apoptosis in primary cultured CMs and that rapamycin was able to inhibit DOX-induced apoptosis in vitro. *P<0.05. ns, not significant.