Figure 2.
Monoamine oxidase‐A (MAO‐A) activation induces expression of cell cycle inhibitors and senescence markers in H9C2 and primary cardiomyocytes. (a) Analysis of mRNA levels of CDKi p21cip, p16ink4a and p15ink4b normalized to GAPDH by real‐time RT‐PCR in H9C2 cells stimulated with 500 μM Tyr for 72 hr (N = 4). (b) Immunoblots of phospho‐Ser15‐p53, total p53, p21 and phospho‐Rb in H9C2 stimulated with 500 μM Tyr for the indicated times. Quantifications of the ratios to actin are shown on the histograms (right panels) (N = 3). (c) Immunoblots of p21 and pRb in cells stimulated with 500 μM Tyr for 72 hr in control conditions or in the presence of clorg (10 μM) or Trolox (500 μM), when indicated. Quantifications of the ratios to actin are shown on the histogram below (N = 3). (d) Representative images and quantitative analysis of the percent of SA‐β‐gal + cells (blue staining) and cell area (vinculin staining in green) after stimulation with 500 μM Tyr for 1 week in the presence of clorg (10 μM) or Trolox (500 μM), when indicated. The number of blue cells positive for SA‐β‐gal was expressed as % of total cell number (100 cells counted for each condition, N = 4 independent experiments, Scale Bar = 100 μm). For cell area, DAPI (blue) was used to label nuclei and vinculin was used for cell size measurement (100 cells were counted for each condition, N = 4 independent experiments, Scale Bar = 10 μm). (e) Neonatal rat ventricular myocytes (NRVMs) transduced with MAO‐A adenovirus were stimulated with increasing concentrations of Tyr for 6 hr to measure DCFDA oxidation (N = 4). (f) Neonatal rat ventricular myocytes (NRVMs) transduced with MAO‐A adenovirus were stimulated with increasing concentrations of Tyr for 72 hr to measure cytotoxicity by LDH release (N = 4). (g) Immunoblots of γH2A.X and total H2A.X in neonatal rat ventricular myocytes (NRVMs) transduced with MAO‐A adenovirus and stimulated with 100 μM of Tyr for 72 hr. Quantifications of the ratios to GAPDH are shown on the histogram below (N = 6). (h) Immunoblots of p‐p53, p53, p21 and pRb in neonatal rat ventricular myocytes (NRVMs) transduced with MAO‐A adenovirus and stimulated with 100 μM of Tyr for 72 hr. Quantifications of the ratios to GAPDH are shown in the right histograms (N = 6). Data are expressed as the mean ± SEM (*p < 0.05, **p < 0.01, *** p < 0.001 vs. control; §p < 0.05, §§p < 0.01, §§§p < 0.001 vs. Tyr)