Figure 1.

Monoamine oxidase‐A (MAO‐A) and oxidative stress are increased in senescent mouse cardiomyocytes, and stimulation of MAO‐A triggers reactive oxygen species (ROS)‐dependent DNA damage response (DDR) in H9C2 cells. (a) Immunoblots of MAO‐A, MAO‐B and 4‐HNE protein adducts in adult ventricular myocytes from young (3 months) and old (20 months) mice. Quantifications of the ratios to GAPDH are shown in the lower panel (N = 4). (b) MAO‐A activity in isolated cardiomyocytes from young (3 months) and old (20 months) mice (N = 4). (c) MAO‐A‐mediated DCFDA oxidation in response to 500 μM Tyr for 2 hr in adult ventricular myocytes from young (3 months) and old (20 months) mice (N = 4). (d) Immunoblots of p53, p21 and p15/p16 proteins in adult ventricular myocytes from young (3 months) and old mice (20 months) mice. Quantifications of the ratios to GAPDH are shown in the lower panel (N = 4). (e) Time‐course of DCFDA oxidation in response to 500 μM Tyr in H9C2 cells (N = 5). (f) DCFDA oxidation in response to 500 μM Tyr for 15 min alone or in the presence of clorg (10 μM), siMAO‐A siRNA or Trolox (500 μM) in H9C2 (N = 4). (g) Comet assay representing DNA damage (DNA strand breaks) in H9C2 cells treated with Tyr (500 μM) for the indicated time. Scale Bar = 10 μm. Images are representative of N = 3 experiments. (h) DNA damage foci (reflected by γH2A.X foci) in H9C2 treated with 500 μM Tyr for the indicated times. DAPI‐labelled nuclei are in blue and γH2A.X is stained in green. Scale Bar = 10 μm. Images are representative of N = 3 experiments. (i) Immunoblots of total and phosphorylated levels of H2A.X and ATM in H9C2 cells stimulated with Tyr (500 μM) for the indicated time. Actin was used as a loading control. Quantifications of the ratios of γH2A.X to total H2A.X and phospho‐ATM to total ATM are shown in the lower panels (N = 4). Data are expressed as the mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 vs. young mice or control; §§p < 0.01, §§§p < 0.001 vs. Tyr)