Differential vulnerability to oxidative stress in rat cardiac myocytes versus fibroblasts
Xiaomin Zhang, MD, PhDa,
Gohar Azhar, MDa,
Koichiro Nagano, MD, PhDa and
Jeanne Y. Wei, MD, PhD*,a
a Gerontology Division, Department of Medicine, Beth Israel Deaconess Medical Center and the Division on Aging, Harvard Medical School, Boston, Massachusetts, USA
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Figure 1 (A) Cell survival of rat cardiac myocytes versus cardiac fibroblasts after hydrogen peroxide (H2O2) (300 to 800 µM). Neonatal rat cardiac myocytes and fibroblasts were cultured for 5 days before exposure to H2O2 for 4 h. n = 6 experiments at each time point (two-way analysis of variance [ANOVA], p < 0.01). Results are expressed as mean ± SEM. t test was used for individual comparisons. **p < 0.01. (B) Graph showing time course of the effect of H2O2 (100 µM) on survival of neonatal cardiac myocytes and fibroblasts. Cells were exposed to H2O2 (100 µM) for a period of up to 72 h before cell survival analysis was performed. n = 4 experiments for both cardiac myocytes and fibroblasts at each time point (two-way ANOVA, p < 0.01). Results are expressed as mean ± SEM. t test was used for individual comparisons. *p < 0.05, **p < 0.01. (C) Deoxyribonucleic acid (DNA) ladder electrophoresis of neonatal cardiac myocytes treated with H2O2 (in concentrations from 25 to 800 µM) for 60 min. The DNA fragmentation ladder is most intense at 100 and 300 µM H2O2, and becomes much less visible at lower concentrations (25 and 50 µM) as well as higher (500 and 800 µM) concentrations.
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Figure 2 (A) Representative Western blot showing effect of (H2O2) (300 to 800 µM) on the activation and phosphorylation of the mitogen-activated protein kinases (MAPK) in neonatal rat cardiac myocytes and fibroblasts. Cells were exposed to different concentrations of H2O2 for a period of 30 min before protein extraction and analysis. (B–E) Densitometric evaluation of phosphoextracellular signal-regulated kinases (Erk) 1 (B), phospho Erk 2 (C), phospho p38 MAPK (D) and phospho JNK (E) activation after H2O2. n = 3 at each dose (two-way analysis of variance (ANOVA), p < 0.01). Results are expressed as mean ± SD. t test was used for individual comparisons. *p < 0.05, myocyte versus fibroblast. (F) Ratios of phosphorylated p38 to phosphorylated ERK 2 and phosphorylated c-jun NH2 terminal kinase (JNK) to phosphorylated ERK 2 in cardiac myocytes and cardiac fibroblasts after treatment with H2O2 (300, 500, 800 µM) for 30 min. Ratios are based on means of n = 3, at each dose and after standardization of the controls to a value of 1 (two-way ANOVA, p < 0.02). M = myocyte, F = fibroblast. t test was used for individual comparisons. *p < 0.05, myocyte versus fibroblast.
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Figure 3 Representative Western blot showing the effect of menadione (vitamin K) on the activation and phosphorylation of the mitogen-activated protein kinases (MAPK) in neonatal rat cardiac myocytes and fibroblasts. Cells were exposed to different concentrations of menadione (50, 100, 200 µM) for a period of 30 min before protein extraction and analysis. Erk = extracellular signal-regulated kinases; JNK = c-jun NH2 terminal kinase.
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Figure 4 (A) Representative Western blot of bcl2 and bax protein expression in the cardiac myocyte and cardiac fibroblast after exposure to different dosages of hydrogen peroxide (H2O2) (300, 500, 800 µM) for 30 min. The experiment was repeated at least three times, with n = 3, for both cardiac myocytes and fibroblasts with similar results. C = control cells, not exposed to oxidative stress and assayed after 30 min. Coomassie blue staining was used to confirm equal loading. (B) Densitometric analysis of bcl2 and bax protein in the neonatal cardiomyocytes and fibroblasts (n = 3 each dose; two-way analysis of variance, p < 0.01). Results are expressed as mean ± SD. t test was used for individual comparisons. *p < 0.05, myocyte versus fibroblast for differences in bax protein expression (bax-M vs. bax-F). M = myocyte, F = fibroblast.
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