(A) Typical image of the effects of pH gradient stress on permeabilized rat cardiomyocyte mt-cpYFP fluorescence. Upper panel, young, middle panel, old, lower panel, old+SS-31 (10 μM, 3 days) visualized after exposure of the cells to pH 7.5 and, later, to pH 5.3. The excitation is 488 nm and collection is at 505–730 nm. (B) Saponin (50 μg/ml) permeabilized cardiomyocytes expressing mt-cpYFP were exposed to progressively lower external pH. Proton permeability of old mitochondria was greater than that of young mitochondria, but preincubation of old cells with 10 μM SS-31 for 3 days enhanced the mitochondrial inner membrane resistance to the pH stress. The traces were averaged from 4 to 19 experiments. The arrows indicate the changes of pH. (C) Quantitation of the SS-31 treatment effect on the mitochondrial matrix cpYFP ratio at pH 5.3. The data are from 7 to 8 min after the pH was adjusted to 5.3. The mean value of the normalized 488/405 ratio in old group is 0.59 with a SD ±0.10 and the range is from 0.46 to 0.77. N = 5–19 rats in each group, analyzed by one-way ANOVA with Dunnett’s test. (D) SS-31 decreased the rate of cpYFP 488/405 ratio drop at pH 6.9. N = 4–10 rats in each group. The rate is calculated as indicated in Figure 2—figure supplement 3. Student’s t-test was applied to determine the statistical significance. The time dependence (N = 3–4 rats in each group) (E) and dose dependence (N = 3–14 rats in each group) (F) of SS-31 protection of mitochondrial resistance to pH gradient stress are shown and analyzed by one-way ANOVA with Fisher’s LSD test. After cardiomyocyte permeabilization, 10 μM SS-31 was added for the times shown in (E) or at the doses shown in (F) for 30 min, followed by pH stress. *p<0.05, **p<0.01 vs Young; #p<0.05, ##p<0.01 vs Old.