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The mitochondrial theory of ageing proposes that the accumulation of oxidative damage to mitochondria leads to mitochondrial dysfunction and tissue degeneration with age. However, no consensus has emerged regarding the effects of ageing on mitochondrial function, particularly for mitochondrial coupling (P/O). One of the main barriers to a better understanding of the effects of ageing on coupling has been the lack of in vivo approaches to measure P/O. We use optical and magnetic resonance spectroscopy to independently quantify mitochondrial ATP synthesis and O2 uptake to determine in vivo P/O. Resting ATP demand (equal to ATP synthesis) was lower in the skeletal muscle of 30-month-old C57Bl/6 mice compared to 7-month-old controls (21.9 ± 1.5 versus 13.6 ± 1.7 nmol ATP (g tissue)−1 s−1, P= 0.01). In contrast, there was no difference in the resting rates of O2 uptake between the groups (5.4 ± 0.6 versus 8.4 ± 1.6 nmol O2 (g tissue)−1 s−1). These results indicate a nearly 50% reduction in the mitochondrial P/O in the aged animals (2.05 ± 0.07 versus 1.05 ± 0.36, P= 0.02). The higher resting ADP (30.8 ± 6.8 versus 58.0 ± 9.5 μmol g−1, P= 0.05) and decreased energy charge (ATP/ADP) (274 ± 70 versus 84 ± 16, P= 0.03) in the aged mice is consistent with an impairment of oxidative ATP synthesis. Despite the reduced P/O, uncoupling protein 3 protein levels were not different in the muscles of the two groups. These results demonstrate reduced mitochondrial coupling in aged skeletal muscle that alters cellular metabolism and energetics.
The mitochondrial theory of ageing proposes that oxidative damage to mitochondria leads to mitochondrial dysfunction and tissue degeneration with age (Miquel et al. 1980). Research into the effects of ageing on mitochondria has primarily focused on identifying oxidative damage and biochemical defects in the electron transport chain (ETC) in isolated mitochondria and cells. There is a striking lack of data on the effects of ageing in vivo, particularly data addressing the coupling of ATP synthesis to O2 uptake (P/O). This is in spite of the fact that mitochondrial coupling is now recognized to be important in the integration of cell energetics (Nicholls, 2004), production of reactive oxygen species (ROS) (Brand, 2000; Nicholls, 2004), and cell death (Stoetzer et al. 2002; Heerdt et al. 2003) – all of which have been demonstrated to be important factors in the ageing process. The absence of appropriate in vivo approaches is one of the primary reasons for this gap in our knowledge. This study uses a newly developed method to directly measure reduced mitochondrial coupling in vivo in aged mice.
Modulation of the P/O is primarily through changes in the proton leak through the inner mitochondrial membrane. An increase in proton leak reduces the ATP produced per O2 consumed, thereby impairing the ability of the cell to meet the energetic demand of the tissue. The increased proton leak will also lead to reduced resting proton-motive force (Δp) (Brand, 2000), which has been demonstrated to sensitize the cell to the induction of apoptosis (Stoetzer et al. 2002; Heerdt et al. 2003). Evidence that oxidative damage to mitochondrial lipids increases proton leak (Brookes et al. 1998), and therefore more uncoupled respiration, suggests that increased mitochondrial uncoupling may provide a link between the effects of age on oxidative damage, cellular energetics and cell death. This provides an additional mechanism, distinct from electron transport chain dysfunction, for the role of mitochondria in loss of function and degeneration with age.
Due to a lack of appropriate in vivo approaches, the relatively few groups (in relation to those measuring mtDNA damage and ETC dysfunction) that have addressed mitochondrial proton leak in ageing tissues have done so in isolated mitochondria and cells. Functional assays performed in isolated organelles and cells may not reflect in vivo function because mitochondrial coupling and proton leak are modulated by many cellular factors (e.g. flux rates, oxygenation, metabolite levels) (Gnaiger et al. 2000) and damage to mitochondria can occur during the isolation process (Anson et al. 2000). We suggest that a direct in vivo measurement of mitochondrial P/O is the necessary next step to understand the role of proton leak and reduced coupling in the degenerative pathologies of ageing.
This work implements a strategy that combines the physiological relevance of in vivo analysis with the ability to measure mitochondrial coupling. We use a combination of optical and magnetic resonance techniques developed in our laboratories to independently measure O2 and ATP fluxes in intact skeletal muscle (Marcinek et al. 2004). This approach allows us not only to determine the effects of age on mitochondrial coupling, but also to determine how reduced coupling affects in vivo energetics and resting metabolism. We find significantly lower P/O in aged mouse skeletal muscle associated with a decrease in resting ATP demand and a lower ATP/ADP. These results indicate that reduced mitochondrial efficiency significantly alters the bioenergetics and metabolism in ageing skeletal muscle.