Amlodipine inhibits doxorubicin-induced apoptosis in neonatal rat cardiac myocytes
Satoshi Yamanaka, MD*,
Tetsuya Tatsumi, MD, PhD*,*,
Jun Shiraishi, MD*,
Akiko Mano, MD*,
Natsuya Keira, MD*,
Satoaki Matoba, MD, PhD*,
Jun Asayama, MD, PhD ,
Shinji Fushiki, MD, PhD ,
Henry Fliss, PhD and
Masao Nakagawa, MD, PhD*
* Second Department of Medicine, Kyoto, Japan
Department of Dynamic Pathology, Kyoto Prefectural University of Medicine, Kyoto, Japan
Department of Clinical Pharmacology, Kyoto Pharmaceutical University, Kyoto, Japan
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada
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Figure 1 (A) Histochemical determination of cell viability and apoptotic myocytes. Cardiac myocytes were treated with serum-free DMEM alone (a and e), 10–6 mol/l of doxorubicin (b and f), 10–6 mol/l of doxorubicin in the presence of 10–6 mol/l of amlodipine (c and g), or 10–6 mol/l of doxorubicin in the presence of 10–6 mol/l of nifedipine (d and h) for 14 h. The cells were then labeled with calcein acetoxymethyl ester and ethidium homodimer-1 (a–d) or stained with Hoechst 33258 (e–h) and visualized by fluorescence microscopy, as described in the Methods section. Photomicrographs are representative of at least three experiments for each experiment (acetoxymethyl ester and ethidium homodimer-1: magnification x 200; Hoechst 33258: magnification x 400). (B) The effect of calcium channel antagonists on myocyte apoptosis. Myocytes were treated with 10–6 mol/l of doxorubicin in the presence or absence of either amlodipine (10–9 to 10–5 mol/l) or nifedipine (10–6 mol/l) for 14 h. Fluorescent-stained nuclei of apoptotic myocytes were analyzed morphologically and expressed as the percentage of total nuclei, as described in Methods. Control myocytes were incubated in serum-deprived DMEM without any chemicals (n = 6). *p < 0.0001 vs. control. p < 0.0001 vs. doxorubicin.
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Figure 2 Detection of apoptosis by annexin V–FITC binding. Cardiac myocytes were treated with serum-free DMEM alone (a), 10–6 mol/l of doxorubicin (b), 10–6 mol/l of doxorubicin in the presence of 10–6 mol/l of amlodipine (c), or 10–6 mol/l of nifedipine (d) for 14 h. (A) The cells were then stained with annexin V–FITC and visualized by fluorescence microscopy, as described in the Methods section. Photomicrographs are representative of at least three experiments for each experiment (magnification x 400). (B and C) Quantification of apoptosis in myocytes was determined by flow cytometric analysis. Cells were harvested, stained with annexin V–FITC, and analyzed as described in Methods. The percentage of apoptotic myocytes was determined by calculating the ratio of annexin V–FITC binding cells (M1) to total cells (n = 6). *p < 0.001 vs. control. p < 0.001 vs. doxorubicin.
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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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