Figure 7: Parkin transgenic mice are resistant to cardiac aging.

(a) Representative electron micrographs of the heats of 20-month-old aged mice; original magnification × 5,000; scale bar, 2 μm. The magnified photograph shows an autophagic vacuole containing mitochondria; original magnification, × 12,000; scale bar, 500 nm. (b,c) Mitochondria incorporated into an autophagic vacuole (b) and abnormal mitochondria (c) were quantified blindly from 8–10 images from different fields (original magnification × 5,000) (n=3 per group). (d,e) The sequential state III oxygen consumption rate of heart isolated mitochondria was assessed using the Seahorse XF24 Analyser. With the initial presence of 5 μg mitochondria per well, 0.5 mM NADH and 4 μM FCCP, injections were performed as follows: port A, 50 μl of 20 μM rotenone (2 μM final); port B, 55 μl of 100 mM succinate (10 mM final); port C, 60 μl of 40 μM antimycin A (4 μM final); port D, 65 μl of 100 mM ascorbate plus 1 mM TMPD (10 μM and 100 μM final, respectively). Representative results of aged littermates are shown (d) and oxygen consumption by each complex was calculated from four independent experiments, five replicates per sample, and four samples per experiment (n=8 per group, e). (f) NADH-driven H₂O₂ production was determined using Amplex Red. Results are shown from five hearts analysed in duplicate. (g) Maximum dP/dt was examined using cardiac catheterization with graded dobutamine infusion in WT and Parkin Tg aged littermates (n=8 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. (i) Real-time PCR assessing the expression of cell cycle inhibitors and proinflammatory cytokines (n=8 per group). (j) Representative immunoblots assessing the expression of cell cycle inhibitors are shown; Data are shown as the means±s.d. *P<0.05; **P<0.01 (two-tailed unpaired Student’s t-test). (k) Proposed model for mitochondrial compromise in heart failure.