Figure 4

DOX-induced degeneration, apoptosis and cell death in the heart. (a) Electron micrographs of cardiomyocytes showing marked DOX-induced degenerative changes (left panel) that were significantly attenuated by G-CSF (right panel). Bars, 1 μm. (b) TUNEL assay. Photomicrographs showing TUNEL-positive cells in the myocardium: left panel, a TUNEL-positive cardiomyocyte; right panel, a TUNEL-positive nonmyocyte. Arrows point to TUNEL-positive cells. Bars, 20 μm. Graphs show the TUNEL assay-based apoptotic index among cardiomyocytes (left) and nonmyocytes (right) at 14 days after the treatments. (c) The graph shows the apoptotic index of cardiomyocytes at 1, 3 and 7 days after DOX treatment. (d) Western analysis of caspase-3 expression. The active (cleaved) form of caspase-3 was not detectable in hearts from any of the groups. (e) Evans blue staining of myocardium from the heart treated with DOX. Fluorescent microphotograph shows Evans blue was exclusively confined in vessel lumens, but never in myocardial parenchyma, in hearts from any group. Bar, 20 μm. (f) Survival rate of cultured cardiomyocytes treated with saline (S) or DOX (D) and monitored with Trypan blue dye exclusion for the indicated intervals.