"What makes some rats live so long?" The mitochondrial contribution to longevity through balance of mitochondrial dynamics and mtDNA content

Abstract

Extremely interesting for aging research are those individuals able to reach older ages still with functions similar to those of younger counterparts. We examined liver samples from ad libitum-fed old (28-month-old, AL-28) and ad libitum-fed very old (32-month-old, AL-32) rats for a number of markers, relevant for mitochondrial functionality and mitochondrial DNA (mtDNA) content. As for the mtDNA content and the protein amounts of the citrate synthase and the antioxidant peroxiredoxin III there were no significant changes in the AL-32 animals. No significant longevity-related change was found for TFAM amount, but a 50% reduction in the amount of the Lon protease, responsible for turnover of TFAM inside mitochondria, characterized the AL-32 rats. No longevity-related change was observed also for the amounts of the mtDNA repair enzymes OGG1 and APE1, whereas the intra-mitochondrial amount of the cytochrome c protein showed a 50% increase in the AL-32 rats, indicating a likely reduced initiation of the intrinsic apoptotic pathway. Totally unexpected was the doubling of two proteins, very relevant for mitochondrial dynamics, namely MFN2 and DRP1, in the AL-32 rats. This prompted us to the calculation of all individual fusion indexes that grouped together in the AL-32 rats, while in the AL-28 animals were very different. We found a strong positive correlation between the fusion indexes and the respective mtDNA contents in two AL-28 and four AL-32 rats. This supports the idea that the limited prevalence of fusion above a still active fission should have ensured a functional mitochondrial network and should have led to a quite narrow range of high mtDNA contents, likely the best-suitable for extended longevity. Our findings strongly suggest that, among the multiple causes leading to the longevity of the AL-32 rats, the maintenance of an adult-like balance of mitochondrial dynamics seems to be very relevant for the regulation of mtDNA content and functionality.

Keywords: Aged liver mitochondria; Fusion index correlation with mtDNA content; Long-living rats; Mitochondrial dynamics; mtDNA and related proteins.

Fig. 1

Effect of extended aging on mtDNA content in rat liver. Extended aging did not induce any change in the mtDNA content. The histogram shows the mean value of the ratio mtDNA/nuclear DNA, determined by qRT-PCR, in AL-32 rats compared to AL-28 rats. Bars represent the mean (±SE) obtained, respectively, from analysis in triplicate of total nucleic acids from each AL-28 and AL-32 rat. The comparison was made with respects to the value of the AL-28 rats fixed as 1. n = number of analyzed animals.

Fig. 2

Effect of extended aging on citrate synthase and peroxiredoxin III (Prx III) amounts in rat liver. Extended aging did not induce any significant change in citrate synthase and Prx III amounts. A. Representative western blot carried out in four rats from each assayed group. The bands from top to bottom show, respectively, the signals from citrate synthase, Prx III and VDAC. B. The histogram shows the relative amounts of citrate synthase and Prx III in AL-32 rats, determined by densitometry analysis of the results from triplicated western blots experiments, compared to AL-28 rats. The densitometric value of OD units of every citrate synthase and Prx III band was related to the number of OD units of the respective band of VDAC for each analyzed sample. Bars represent the mean ((±SE) of the relative (citrate synthase/VDAC, Prx III/VDAC) values obtained from each AL-28 and AL-32 rat. Comparisons were made with respects to the value of the AL-28 rats, fixed as 1. n = number of analyzed animals.

Fig. 3

Effect of extended aging on TFAM and Lon amounts in rat liver. Extended aging did not induce any significant change in TFAM amount and a 50% decrease in Lon amount. A. Representative western blot carried out in four rats from each assayed group. The bands from top to bottom show, respectively, the signals from Lon, TFAM and VDAC. B. The histogram shows the relative amounts of TFAM and Lon in AL-32 rats, determined by densitometry analysis of the results from triplicated western blots experiments, compared to AL-28 rats. The densitometric value of OD units of every TFAM and Lon band was related to the number of OD units of the respective band of VDAC for each analyzed sample. Bars represent the mean ((±SE) of the relative (TFAM/VDAC, Lon/VDAC) values obtained from each AL-28 and AL-32 rat. Comparisons were made with respects to the value of the AL-28 rats, fixed as 1. **p < 0.01 versus the value of the AL-28 rats; n = number of analyzed animals.

Fig. 4

Effect of extended aging on OGG1, APE1 and cytochrome c (Cyt c) amounts in rat liver. Extended aging did not induce any change in OGG1 and APE1 amounts and induced a 50% increase in Cyt c amount. A. Representative western blot carried out in four rats from each assayed group. The bands from top to bottom show, respectively, the signals from APE1, OGG1, Cyt c and VDAC. B. The histogram shows the relative amounts of APE1, OGG1 and Cyt c in AL-32 rats, determined by densitometry analysis of the results from triplicated western blots experiments, compared to AL-28 rats. The densitometric value of OD units of every APE1, OGG1 and Cyt c band was related to the number of OD units of the respective band of VDAC for each analyzed sample. Bars represent the mean ((±SE) of the relative (APE1/VDAC, OGG1/VDAC, Cyt c/VDAC) values obtained from each AL-28 and AL-32 rat. Comparisons were made with respects to the value of the AL-28 rats, fixed as 1. *p < 0.05 versus the value of the AL-28 rats; n = number of analyzed animals.

Fig. 5

Effect of extended aging on MFN2 and DRP1 amounts in rat liver. Extended aging induced a doubling in MFN2 and DRP1 amounts. A. Representative western blot carried out in four rats from each assayed group. The bands from top to bottom show, respectively, the signals from DRP1, MFN2 and VDAC. B. The histogram shows the relative amounts of DRP1 and MFN2 in AL-32 rats, determined by densitometry analysis of the results from triplicated western blots experiments, compared to AL-28 rats. The densitometric value of OD units of every DRP1 and MFN2 band was related to the number of OD units of the respective band of VDAC for each analyzed sample. Bars represent the mean ((±SE) of the relative (DRP1/VDAC, MFN2/VDAC) values obtained from each AL-28 and AL-32 rat. Comparisons were made with respects to the value of the AL-28 rats, fixed as 1. *p < 0.05 versus the value of the AL-28 rats; n = number of analyzed animals.

Fig. 6

Correlation between the value of fusion index and the mtDNA content. A strong positive correlation was found between the fusion index value and the mtDNA content in four out of five animals from the AL-32 group and two from the AL-28 rats (see Table 2 for individual fusion indexes). The RT-PCR quantification of the mtDNA content in AL-28 and AL-32 rats, all normalized to β-actin, was performed according to the Pfaffl mathematical model and the values resulted from the calculation of the formula 2^−ΔCT (Pfaffl 2001). Pearson’s test was performed and demonstrated a highly significant correlation (p < 0.005, correlation coefficient: 0.8931) in the AL-32 group (n = 4,) and AL-28 rats (n = 2) (filled line). The outlier from the AL-32 group and one animal from the AL-28 group shared the lowest fusion indexes together with the highest mtDNA contents, whereas the AL-28 rats with the highest fusion indexes were characterized by quite low mtDNA contents.