FIG. 6.

p53 mediates MAO-A-dependent cardiomyocyte necrosis. (A–D) Neonatal cardiomyocytes were untransduced (multiplicity of infection [MOI] 0) or transduced with AdeMAO-A at MOI 5. (A) Quantification of MAO-A activity by luminescence (n=3). (B) Lactate dehydrogenase (LDH) release after treatment with 50 μM NE for 24 h (n=8–9). (C) LDH release in response to 500 μM tyramine for 24 h in the presence of 10 μM Clorg (Tyr+clorg), 5 mM NAC (Tyr+NAC), or 1 mM Trolox (Tyr+Trolox), when indicated (n=4–5). (D) Reactive oxygen species generation with DCFDA probe in response to 500 μM tyramine for 2 h in the presence of NAC (5 mM), Clorg (10 μM), or 1 mM Trolox (Tyr+Trolox), when indicated (n=9) in untransduced (MOI 0) or AdeMAO-A-transduced (MOI 5) cells. (E,F) Analysis of total or phosphor(ser15)-p53 levels by immunoblot in AdeMAO-A-transduced neonatal cardiomyocytes (MOI 5) stimulated with 500 μM tyramine for the indicated time in the presence of NAC or Trolox, when indicated (n=3–5). GAPDH was used as a loading control. (G) LDH release in AdeMAO-A-transduced cardiomyocytes (MOI 5) transfected with Scr or p53 siRNA for 48 h, and stimulated with 500 μM tyramine for 24 h (n=7). Immunoblot illustrates silencing of p53 protein at 48 and 72 h after siRNA transfection. (H) LDH release in AdeMAO-A-transduced cardiomyocytes (MOI 5) stimulated with 500 μM tyramine for 24 h in the presence of 20 μM pifithrin, when indicated (n=6–8) *p<0.05, **p<0.01, ***p<0.001, ^§p<0.05, ^§§p<0.01 versus indicated values or Ct.