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Figure 5 Loss of mitochondrial transmembrane potential and release of cytochrome c. A, Myocytes were treated with 10–6 mol/l of doxorubicin in the presence or absence of 10–6 mol/l of amlodipine for 14 h. The cells were then stained with JC-1, as described in the Methods section, and fluorescence was monitored at 527 and 590 nm (magnification x 400). Control myocytes were incubated in serum-deprived DMEM without any chemicals. (a) Control cardiac myocytes; (b) myocytes exposed to 10–6 mol/l of doxorubicin for 14 h; (c) myocytes exposed to 10–6 mol/l of doxorubicin in the presence of 10–6 mol/l of amlodipine for 14 h. B, Myocytes treated with 10–6 mol/l of doxorubicin in the presence or absence of 10–6 mol/l of amlodipine were harvested for detection of cytochrome c. Mitochondrial and cytosolic fractions were prepared, and aliquots containing 20 µg protein were subjected to Western blot analysis and probed with antibody for cytochrome c, as described in Methods. Photomicrographs are representative of at least three experiments for each experiment. C, Densitometric analysis of cytochrome c release. Levels of cytochrome c are shown as the percent change in the average from three independent experiments, as compared with control. *p < 0.01 vs. control.  p < 0.01 vs. doxorubicin.
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