Fig. 4. Assessment of BAK-mediated necrosis during MI/R.

B. Representative images and quantification of necrosis assessed by staining with Evans blue dye administered pre-mortem at the time of reperfusion by itself (top) or combined with post-mortem TTC staining (bottom) in hearts of wild type (black) and Bak−/− (red) mice subjected to sham operation (harvested 24 h later) or 45 min ischemia and 4 h or 24 h reperfusion (N= 3 sham per genotype, 6 MI/R 4 h per genotype, 5 MI/R 24 h per genotype male mice). B. Isolated adult mouse cardiomyocytes from wild type (black) and Bak−/− (red) mice treated with the indicated concentrations of H₂O₂ and cell death scored 30 min later by ethidium homodimer staining of nucleus. C. Isolated adult mouse cardiomyocytes from wild type (black) and Bak−/− (red) mice treated with the indicated concentration of ionomycin and cell death scored 30 min later with ethidium homodimer. For B. and C, N = 3 independent experiments, each in duplicate with 2–3 fields scored per replicate. D, E. Two independent calcium retention assay experiments using cardiac mitochondria isolated from wild type or Bak−/− mice (N=3 mice of each genotype in each experiment). CsA and ABT-737 denote pretreatment of mitochondria with cyclosporine A (2 μ) or ABT-737 (at the indicated concentration) for 20 min. Data – mean ± SEM. Statistic: ANOVA with Tukey’s post-test. * P < 0.05, ** P < 0.01, **** P < 0.0001.