Figure 6: p53-mediated mitophagy deficiency and biological cardiac aging.

We used 20-month-old heterozygous littermates surviving grossly tumor free. (a,b) The sequential state III oxygen consumption rate of heart isolated mitochondria (300 μg) was monitored with a fibre-optic system. Substrates and inhibitors were added sequentially at the final concentrations indicated as follow. FCCP, 1 μM carbonylcyanide-p-trifluoromethoxyphenylhydrazone; M/P, 5 mM malate+5 mM pyruvate; Rot, 100 nM rotenone; Suc, 5 mM succinate; AA, 50 nM antimycin A; TMPD/Asc, 0.4 mM N,N,N′,N′-tetramethyl-p-phenylenediamine +1 mM ascorbate; KCN, 5 mM potassium cyanide. Representative results are shown (a) and oxygen consumption by each complex was calculated (n=5–8 per group, b). (c,d) Mitochondrial particle size (c) and membrane potential (d) were determined using flow cytometry for TMRE in cardiac mitochondria preparations. Results are shown from 4–8 hearts analysed in duplicate. (e) H₂O₂ production with 0.5 mM NADH, complex I-linked substrate, was determined in the absence or presence of 2 μM complex I inhibitor rotenone using Amplex Red (n=5–8 per group). (f) Myocardial high-energy phosphate metabolism under 8γ dobutamine loading was measured biochemically with HPLC (n=5 per group). (g) Maximum dP/dt was examined using cardiac catheterization with graded a dobutamine infusion (n=5 per group). (h) Photographs showing heart sections after SA-β-gal staining; original magnification, × 200; scale bar, 40 μm. SA-β-gal-positive cells were quantified (n=4 per group). Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section. HT indicates heterozygous; DHT double heterozygous. Data are shown as the means±s.d. *P<0.05; **P<0.01; ***P<0.001 (two-tailed unpaired Student’s t-test).