To evaluate the effects of the Prop1^-/- mutation on heart muscle mitochondrial ETC function, we compared the enzyme activities of CI-CV, as well as the coupled activities of CI-III and CII-III, for young (4-5 mo), middle-aged (10-12 mo) and aged (20-26 mo) wild type (WT) and Ames dwarf mice (Figures 1 and 2). The data show that a decrease in the enzyme activities of CI-CV occurs with age and that both WT and dwarf mice exhibit this characteristic.

Rotenone-sensitive CI activity decreased by ~15% and ~19% from young to middle-age, and by old age this activity decreased by ~29% and 33% in WT and dwarf, respectively (Figure 1A). No differences were noted between the WT and dwarf CI activities at any age, thus indicating that the significant decline in CI activity is an age-associated characteristic shared by both WT and Ames mice.

Malonate-sensitive CII activity also progressively declined in WT by ~17% in middle-age and ~28% by old age (Figure 1B). In contrast, no decline occurred in dwarf CII activity from young to middle-age, and the activity decreased by ~28% in old age (Figure 1B). Thus, the decrease in CII activity is also a characteristic of aging, although the delay in the dwarf may be a characteristic of its longevity. The CII enzyme activity was also higher in dwarf mice compared to WT only at middle age (~28%).

Antimycin A (AA)-sensitive CIII activity in WT mice showed a continuous age-associated decline, i.e., an ~19% decline by middle age and ~28% by old age, while dwarf mice declined by ~21% in middle age and remained at this level of activity in old age (Figure 1C). No differences were seen between WT and dwarf CIII activity at any age. These changes are thus a characteristic of aging that also occurs in the long-lived Ames mice.

KCN-sensitive CIV activity in the WT mice remained unchanged at middle age and then decreased by ~24% in old animals, while dwarf mice showed a continuous decline with aging - an ~29% decline in middle age and ~42% by old age (Figure 1D). This marked decrease in CIV activity in dwarf compared to WT mice suggests lower rates of oxygen consumption that may be a prolongevity characteristic.

Oligomycin-sensitive CV activity in both WT and dwarf mice decreased only in old age, by ~16% and ~21%, respectively (Figure 1E). In comparison, the dwarf CV activity was higher than in the WT at young and middle age (~12%), suggesting a higher level of ATP production in dwarf mice at these ages.

Overall, most ETC enzyme activities in WT and dwarf heart muscle showed a tendency towards an age-associated decline in activity, suggesting that this is a general physiological characteristic of aging shared by both WT and long-lived Ames mice. However, the only significant difference in activity occurred with the dwarf CIV at middle and old age, and involved the rate at which CIV activity declined, suggesting that these lower levels of enzyme function in the dwarf mice may be a characteristic of longevity. These results also raise the question of the nature of the physiological basis for decline of ETC enzyme activity in WT vs. dwarf mitochondria.

In contrast to the general trend of age-associated loss of heart muscle enzyme activity, there was an increase in CI-CIII coupled activity in both WT and dwarf mice, although the increase was significantly higher in the dwarfs (Figure 2A).Thus, the higher CI-CIII coupled activity of dwarf mice at young (~22%), middle (~17%) and old age (~30%) suggests a tighter coupling of CI-CIII associated with the longevity phenotype at all ages (Figure 2A). On the other hand, the coupled CII-III activity showed an age-associated decline in activity specific to the WT - an ~15% decline in middle age and ~25% decline by old age (Figure 2B). In contrast, no significant age-related change in coupled CII-CIII activity was seen in dwarf mice. These data suggest that the higher level of coupled CII-CIII activity, and failure of this activity to decline with age in the dwarf, may be a characteristic of the longevity phenotype.

To evaluate the physiological effects of the Prop1^(−/−) mutation on skeletal muscle mitochondrial ETC function, we chose to study the pectoralis (red), an aerobic muscle with high levels of mitochondria, and the quadriceps (white), an anaerobic muscle with lower levels of mitochondria. The pectoralis is a highly aerobic muscle consisting of slow twitch fibers (type 1), high mitochondrial content and high myoglobin levels, which improve the delivery of oxygen. We compared the enzymatic activities of the pectoralis (Figures 3, 4) and quadriceps (Figures 5, 6) ETC CI-CV as well as their CI-III and CII-III coupled activities for all three ages in both WT and dwarf mice. The data clearly show different enzyme activity profiles between WT vs. dwarf mice. All of the enzyme activities declined with age in both WT and dwarf; by old age this decline was ~19% and ~28% in CI; ~23% and ~32% in CII; ~41% and 28% in CIII; ~48% and ~46% in CIV; and ~55% and ~52% in CV, respectively (Figure 3A-E). These data are consistent with our previous studies which showed an age-associated decline in CI-CV activities in C57BL/6 male mice indicating that this is not due to genetic background [37]. There were only minor differences between the dwarf and WT in CII and CIII activities. However, in CIV, the dwarf had a lower activity at both young and middle age compared to WT (by ~13% and ~25%, respectively), though by old age their activities are similar (Figure 3D). Our data suggest that the decline in pectoralis CI-CV activities is an age-associated characteristic of both WT and dwarf pectoralis, and that the more rapid decline in activity in the dwarf may be a prolongevity characteristic (which also occurs in dwarf heart muscle).

Interestingly, the CI-CIII coupled activity showed no age-associated changes (Figure 4A), thus being unique in its resistance to aging. In previous studies we found similar resistance in the pectoralis of aged C57BL/6 mice [37]. On the other hand, there were ~33% declines in CII-CIII coupled activity in both WT and dwarf mice (Figure 4B), suggesting that this is a general characteristic of aging, not affected by longevity. Interestingly, we demonstrated in our previous studies that the CII-CIII activity of C57BL/6 mice is not affected by aging, suggesting differences that may be due to genetic background [37].

We chose to study the mitochondrial ETC activities of the quadriceps because it consists of fast-twitch type I fibers whose physiological characteristics include fewer mitochondria. The high levels of glycolytic enzymes enable these fibers to respire anaerobically. The data in Figure 5 show that there was a significant decrease in WT CI and no effect on CII, CIII and CV activity; there was a noticeable but not quite statistically significant drop in CIV activity by old age. In the dwarf quadriceps, there was a significant decrease in CI activity and a dramatic decline in CIV activity associated with aging, i.e., ~14% decline in middle age and ~32% decline by old age (Figure 5D). Thus, as with the pectoralis the CIV activity of the dwarf quadriceps exhibited a specific sharp decrease in activity, suggesting that this is a characteristic of longevity.

With respect to the CI-CIII and CII - CIII coupled activities the data showed essentially no change in the WT and only a minor decrease in CI - CIII in the dwarf (Fig. 6A, 6B).

Overall, the two physiologically and functionally different skeletal muscles showed certain unique profiles that emphasize the differences in ETC enzyme activities with aging in both WT and dwarf mice. The difference in CIV activity of the dwarf vs. the WT was the only major difference associated with longevity.

We chose to study the mitochondrial ETC activities of the kidney because its high urea levels cause high levels of endogenous oxidative stress [38]. The kidney is also prone to relatively high levels of hydroxynonenol (HNE) adduct formation in mitochondrial proteins [39]. In our previous studies we showed a direct relationship between increased protein modification and decreased enzyme function for CI, CII and CV, suggesting increased endogenous oxidative stress with aging due to mitochondrial dysfunction [40]. In the current studies we evaluated the physiological effects of the Prop1^−/− mutation on kidney mitochondrial ETC function. We measured the enzymatic activities of CI-CV as well as the coupled activity of CI-III and CII-III for all three ages in WT and dwarf mouse kidneys (Figures 7 and 8). In contrast to the heart and skeletal muscles, we found major differences in CI, CII, CIII and CV enzyme activities between WT and dwarf mouse kidneys. In WT kidney, no changes were seen in CI and CIII activities (Figure 7A and 7C); and there was an age-related decline in function in WT CII (~23% in middle age and ~28% by old age, Figure 7B); in CIV (~14% by old age, Figure 7D); and CV (~29% in middle age and no further decline in old age, Figure 7E).

On the other hand, the dwarf kidney showed no age-related changes in CI and CIV activities (Figure 7A and 7D, although these activities were significantly higher than in WT), whereas the other enzyme functions (CII, CIII, CV) declined with age. In particular, CII and CIII function decreased at old age by ~17% and ~32%, respectively (Figure 7B and 7C). CV activity also showed a continuous decline with aging, such that the activity decreased by ~28% in middle age and ~35% at old age (Figure 7E).

Our data show that the overall enzyme activities of the kidney ETC complexes were significantly higher at all ages in dwarf than in WT mice. In fact, the CIII and CV activities were more than two-fold higher in dwarf at young age compared to the WT. Thus, although the WT and dwarf activities decreased progressively with aging, the significant differences between them suggest that the higher levels of activity in the dwarf may be an organ-specific characteristic of longevity.

The data in Figure 8A show an age-associated increase in WT CI-CIII coupled activity (~26%). On the other hand, the CI-CIII coupled activity in the young dwarf kidney is significantly higher than in the WT (by ~2.5 fold) and progressively declines with age; the dwarf activity remains higher at all ages. Similarly, CII-CIII coupled activity in the WT kidney, which is significantly lower than in the dwarf, declines by ~40% in middle age, but no further in old age (Figure 8B). As with the CI-CIII coupling, the CII-CIII coupled activities in dwarf kidneys declined with age; CI-CIII activity decreased by ~10% in middle and ~30% in old age (Figure 8A), and CII-CIII activity decreased by ~17% and ~35% in middle and old age, respectively (Figure 8B).

Overall, the most significant differences in enzyme activities between the dwarf and WT occurred in the kidney. These data suggest that ETC activities of tissues of aging complex organisms may exhibit tissue-specific physiological characteristics of longevity.

The Ames colony is maintained at the University of Texas Medical Branch at Galveston. Ames mice were generated by mating Prop1^+/- heterozygous males and females; progeny were weaned at 1 month of age and tail DNA collected to genotype Prop1^+/+ (WT), Prop1^+/- (heterozygous), and Prop1^-/- (dwarf) mice. RT-PCR was performed in a Bio-Rad iCycler using the manufacturer's protocols (Bio-Rad, Hercules, and CA) to clone a small region of the Prop1 gene; iCycler software was utilized to analyze the results. Mice were housed separately according to their genotype, and WT and dwarf mice were aged for further use. Young (4-5 months), middle-aged (10-12 months) and old (20-26 months) male WT and dwarf mice were maintained in our animal care facility with a 12h light/dark cycle and fed ad libitum on a standard chow diet before sacrifice, following all regulations of the UTMB Institutional Animal Care and Use Committee.

Micewere sacrificed by decapitation and their tissues harvested immediately, rinsed in ice-cold PBS, and prepared for mitochondrial isolation. Mitochondria were prepared from the pooled tissues of 8 young, 8 middle-aged or 8 old WT, and 12 young, 13 middle-aged, or 8 old Ames dwarf male mice. Mitochondrial isolation was performed at 4°C as described [50] with minor modifications [40]. Briefly, tissues were blended in a Brinkman Polytron PT 3000 (large blade) for 10-15 seconds in isolation buffer (250 mM sucrose, 0.5 mM EGTA, 2 mM EDTA, 10 mM HEPES-KOH, pH 7.4) containing Antipain, Chymostatin, Leupeptin and Pepstatin A (final concentration 1 mg each/mL). The blended tissues were homogenized 20X with a Teflon pestle homogenizer, and then centrifuged at 800 X g for 20 minutes. The supernatants were collected in separate tubes and the pellets re-suspended in half a volume of isolation buffer each, and homogenized and centrifuged as before. The supernatants were combined with those from the previous step and centrifuged twice more at 800 X g for 20 minutes. Each time the supernatants were transferred to new tubes and the pellets discarded. The final supernatant was centrifuged at 8000 X g for 20 minutes to pellet mitochondria. The mitochondrial pellets were washed 2X each with half volumes and centrifuged at 8000 X g. The final mitochondrial pellets were re-suspended in minimal volumes of isolation buffer, aliquoted and stored at − 80°C. Fresh aliquots were used for each analysis; sonicated mitochondria were generated with a Branson Model 250 Digital Sonifier (2 cycles, 1.8 seconds each, 6 second total time for each cycle; one minute between each cycle, amplitude 30% pulse time on for 0.3 seconds, pulse time off for 0.7 seconds).

Enzyme activities were assayed at room temperature using a Beckman Coulter DU 530 Spectrophotometer (Beckman Coulter, Brea, CA) as described [37, 43]. Citrate synthase activity was measured at 412 nm (e = 13.6 mM^-1 cm^-1) as described [51]. Rotenone-sensitive CI, malonate-sensitive CII, antimycin A-sensitive (AA) CIII, KCN-sensitive CIV, and oligomycin-sensitive CV activities, and CI-III and CII-III coupled activities were assayed as described [40, 52, 53].

All activity results are averages of 4 assays from the pooled samples from WT and dwarf mice at each age group. Citrate synthase assay results from young WT were used to calculate ratios of young to middle-aged and young to old, as well as WT to dwarf mitochondrial protein levels and these ratios were multiplied to normalize the enzyme activities for each age group in WT and dwarf. Statistical significance was calculated using the Student's t-test, with p<0.05 and p<0.001 considered significant and highly significant, respectively.

Blue-native PAGE (BN-PAGE) and SDS-PAGE were carried out by established methods [54] with minor modifications [40, 43, 54]. Briefly, a 5 to 12% acrylamide gradient was used for the first dimension BN-PAGE; imidazole was used as the buffer instead of Bis-Tris, and Criterion 10-20% 2D-well gels (Bio-Rad) were used for the second dimension (SDS-PAGE).

Immunoblot analyses were performed as described [40, 43, 55]. Intact mitochondrial ETC complex bands were visualized using antibodies against CI (NDUFA9 subunit), CII (SDHA subunit), CIII (UQCRFS1 subunit), CIV (COX1), and CV (ATP5A1 subunit; all from Molecular Probes, Eugene, OR). Antibody to the mitochondrially encoded COX1 subunit was used as a CIV-specific antibody. All other complex-specific antibodies are against nuclear encoded subunits.