Russell T. Hepple

Mitochondrial structure and function are disrupted by standard isolation methods.

Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca2+ handling and apoptotic signaling. Two classical methods exist to study mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intracellular components. Despite this, isolated mitochondria remain the most commonly used method to infer in vivo mitochondrial function. In this study, we directly compared measures of several key aspects of mitochondrial function in both isolated mitochondria and permeabilized myofibers of rat gastrocnemius muscle. Here we show that mitochondrial isolation i) induced fragmented organelle morphology; ii) dramatically sensitized the permeability transition pore sensitivity to a Ca2+ challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H2O2 production. These alterations are qualitatively similar to the changes in mitochondrial structure and function observed in vivo after cellular stress-induced mitochondrial fragmentation, but are generally of much greater magnitude. Furthermore, mitochondrial isolation markedly altered electron transport chain protein stoichiometry. Collectively, our results demonstrate that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. Our work and that of others underscores the importance of studying mitochondrial function in tissue preparations where mitochondrial structure is preserved and all mitochondria are represented.

Mitochondrial functional impairment with aging is exaggerated in isolated mitochondria compared to permeabilized myofibers

Mitochondria regulate cellular bioenergetics and apoptosis and have been implicated in aging. However, it remains unclear whether age‐related loss of muscle mass, known as sarcopenia, is associated with abnormal mitochondrial function. Two technically different approaches have mainly been used to measure mitochondrial function: isolated mitochondria and permeabilized myofiber bundles, but the reliability of these measures in the context of sarcopenia has not been systematically assessed before. A key difference between these approaches is that contrary to isolated mitochondria, permeabilized bundles contain the totality of fiber mitochondria where normal mitochondrial morphology and intracellular interactions are preserved. Using the gastrocnemius muscle from young adult and senescent rats, we show marked effects of aging on three primary indices of mitochondrial function (respiration, H2O2 emission, sensitivity of permeability transition pore to Ca2+) when measured in isolated mitochondria, but to a much lesser degree when measured in permeabilized bundles. Our results clearly demonstrate that mitochondrial isolation procedures typically employed to study aged muscles expose functional impairments not seen in situ. We conclude that aging is associated with more modest changes in mitochondrial function in sarcopenic muscle than suggested previously from isolated organelle studies.

Determinants of VO2 max decline with aging: an integrated perspective

Aging is associated with a progressive decline in the capacity for physical activity. Central to this decline is a reduction in the maximal rate of oxygen utilization, or VO2 max. This critical perspective examines the roles played by the factors that determine the rate of muscle oxygen delivery versus those that determine the utilization of oxygen by muscle as a means of probing the reasons for VO2 max decline with aging. Reductions in muscle oxygen delivery, principally due to reduced cardiac output and perhaps also a maldistribution of cardiac output, appear to play the dominant role up until late middle age. On the other hand, there is a decline in skeletal muscle oxidative capacity with aging, due in part to mitochondrial dysfunction, which appears to play a particularly important role in extreme old age (senescence) where skeletal muscle VO2 max is observed to decline by approximately 50% even under conditions of similar oxygen delivery as young adult muscle. It is noteworthy that at least the structural aspects of the capillary bed do not appear to be reduced in a manner that would compromise the capacity for muscle oxygen diffusion even in senescence.

Long-term caloric restriction abrogates the age-related decline in skeletal muscle aerobic function

The purpose of this study was to determine the effect of long‐term caloric restriction (CR) on the age‐associated decline of skeletal muscle aerobic function. Skeletal muscle maximal aerobic performance (VO2max) was assessed in ad libitum (AL) and CR rats aged 8–10 months and 35 months using a pump‐perfused hindlimb model to match oxygen delivery to muscle mass between groups. Whereas there was a 46% decline in muscle mass‐specific VO2max between 8–10 mo (524±13 µmol•min−1•100 g−1; mean±se) and 35 mo (281±54 µmol min−1•100 g−1) in AL rats, not only did CR rats begin at the same point in 8–10 mo old rats (490±42 µmol•min−1•100 g−1), we found no decline in 35 mo old CR animals (484±49 µmol•min−1•100 g−1). Interestingly, although most markers of oxidative capacity began at a lower point in young adult CR animals, CR rats exhibited a higher in situ activity of complex IV at VO2max. This activity allows the young adult CR animals to exhibit normal aerobic capacity despite the lower oxidative enzyme activities. In stark contrast to the 19–41% decline in activities of citrate synthase, complexes I–III, and complex IV in homogenates prepared from the plantaris muscle and mixed region of gastrocnemius muscle with aging in AL rats, no age‐related decline was found in CR animals. Thus, our results showed that CR preserves aerobic function in aged skeletal muscles by facilitating a higher in situ function of complex IV and by preventing the age‐related decline in mitochondrial oxidative capacity.

Caloric restriction optimizes the proteasome pathway with aging in rat plantaris muscle: implications for sarcopenia

To gain insight into the significance of alterations in the proteasome pathway for sarcopenia and its attenuation by calorie restriction, we examined protein oxidation and components of the proteasome pathway in plantaris muscle in 8-, 30-, and 35-mo-old ad libitum-fed (AL) rats; and in 8-, 35-, and 40-mo-old calorie-restricted (CR) rats. We hypothesized that CR rats would exhibit a lesser accumulation of protein carbonyls with aging and that this would be associated with a better maintenance of skeletal muscle proteasome activity and function with aging. Consistent with this view, whereas AL rats had a significant increase in protein carbonylation with aging, there was no such increase in CR rats. Protein levels of the ubiquitin ligases MuRF1 and MAFbx increased similarly with aging in both AL and CR rats. On the other hand, chymotrypsin-like activity of the proteasome increased with aging more gradually in CR rats, and this increase was paralleled by increases in the expression of the C2 subunit in both groups, suggesting that differences in activity were not related to differences in proteasome function with aging. Interestingly, the plot of muscle mass vs. proteasome activity showed that the oldest animals in both diets had a lower muscle mass than would be predicted by their proteasome activity, suggesting that other factors explain the acceleration of sarcopenia at advanced age. Since calorie restriction better protects skeletal muscle function than muscle mass with aging (Hepple RT, Baker DJ, Kaczor JJ, Krause DJ, FASEB J 19: 1320-1322, 2005), and our current results show that this protection of function is associated with a prevention of oxidative protein damage accumulation, we suggest that calorie restriction optimizes the proteasome pathway to preserve skeletal muscle function at the expense of modest muscle atrophy.

Lower Oxidative DNA Damage Despite Greater ROS Production in Muscles from Rats Selectively Bred for High Running Capacity

Artificial selection in rat has yielded high-capacity runners (HCR) and low-capacity runners (LCR) that differ in intrinsic (untrained) aerobic exercise ability and metabolic disease risk. To gain insight into how oxygen metabolism may have been affected by selection, we compared mitochondrial function, oxidative DNA damage (8-dihydroxy-guanosine; 8dOHG), and antioxidant enzyme activities in soleus muscle (Sol) and gastrocnemius muscle (Gas) of adult and aged LCR vs. HCR rats. In Sol of adult HCR rats, maximal ADP-stimulated respiration was 37% greater, whereas in Gas of adult HCR rats, there was a 23% greater complex IV-driven respiratory capacity and 54% greater leak as a fraction of electron transport capacity (suggesting looser mitochondrial coupling) vs. LCR rats. H(2)O(2) emission per gram of muscle was 24-26% greater for both muscles in adult HCR rats vs. LCR, although H(2)O(2) emission in Gas was 17% lower in HCR, after normalizing for citrate synthase activity (marker of mitochondrial content). Despite greater H(2)O(2) emission, 8dOHG levels were 62-78% lower in HCR rats due to 62-96% higher superoxide dismutase activity in both muscles and 47% higher catalase activity in Sol muscle in adult HCR rats, with no evidence for higher 8 oxoguanine glycosylase (OGG1; DNA repair enzyme) protein expression. We conclude that genetic segregation for high running capacity has generated a molecular network of cellular adaptations, facilitating a superior response to oxidative stress.

Elevated caspase and AIF gene expression correlate with progression of sarcopenia during aging in male F344BN rats.

To establish the relationship between the progression of sarcopenia and apoptosis we examined apoptotic gene expression in plantaris muscles (Pl) from 8 mo old (n=8), 30 mo old (n=8) and 35 mo old (n=6) male rats by real-time PCR. Pl mass declined from 368 +/- 7 mg at 8 mo to 333 +/- 7 mg at 30 mo (P<0.05) and 210 +/- 15 mg at 35 mo of age (P<0.05). BAX, Bcl-2, and Apaf-1 expression decreased by 62-74% at 30 mo and by 90-96% at 35 mo of age (all P<0.05 vs 8 mo old). In contrast, the expression of Caspases 3, 8, and 9 and AIF increased 3- to 5-fold at 30 mo (NS) and 7- to 50-fold at 35 mo of age (P<0.05). There were significant (P<0.05) correlations between Pl mass and Caspase 3 (r(2)=-0.60), Caspase 9 (r(2)=-0.58), Caspase 8 (r(2)=-0.50), and AIF (r(2)=-0.48). Thus, our results show that the expression of some genes involved in apoptosis increase with aging in Pl and correlate with progression of sarcopenia (Caspase 3, Caspase 9, Caspase 8, and AIF), whereas others decline with aging (BAX, Bcl-2, and Apaf-1).

Oxidative capacity interacts with oxygen delivery to determine maximal O2 uptake in rat skeletal muscles in situ

Based on proportional changes in V̇O2,max with alterations in O2 delivery, it is widely held that O2 availability limits V̇O2,max. In contrast, reductions in V̇O2,max are also seen when mitochondrial oxidative capacity is reduced. Taken collectively, these prior results are consistent with the notion that there is not a single‐step limitation to V̇O2,max. We used a pump‐perfused rat hindlimb model to test the hypothesis that combining moderate reductions in O2 delivery and mitochondrial oxidative capacity would yield a greater reduction in V̇O2,max than seen when performing each intervention independently, demonstrating an interaction between O2 supply and mitochondrial oxidative capacity in determining V̇O2,max. Four groups of animals were studied: two in high O2 delivery conditions (hindlimb O2 delivery: 88 ± 1 μmol O2 min−1; mean ±s.e.m.) and two in moderately reduced O2 delivery conditions (66 ± 2 μmol O2 min−1). One group at each level of O2 delivery was treated with 0.1 μM myxothiazol to reduce mitochondrial oxidative capacity via competitive inhibition of NADH cytochrome c reductase. V̇O2,max in control animals (no myxothiazol) was 29 % lower in the moderately reduced O2 delivery group (592 ± 24 mmol O2 min−1 (100 g)−1); P < 0.05) than in the high O2 delivery group (833 ± 63 μmol O2 min−1 (100 g)−1). Similarly, V̇O2,max was reduced by 29 % (594 ± 22 μmol O2 min−1 (100 g)−1); P < 0.05) in myxothiazol‐treated animals in high O2 delivery conditions compared to control animals in high O2 delivery conditions. When myxothiazol treatment was combined with moderately reduced O2 delivery, V̇O2,max was reduced by an additional 18 % (484 ± 21 μmol O2 min−1 (100 g)−1); P < 0.05) compared to either intervention performed independently. These results show that O2 supply and mitochondrial oxidative capacity interact to determine V̇O2,max.

Slow twitch soleus muscle is not protected from sarcopenia in senescent rats

Although most literature suggests a relative protection of slow twitch muscle with aging, there is limited data in senescence when muscle atrophy and functional decline markedly accelerate. To address this issue we examined age-related changes in muscle mass, contractile function, mitochondrial enzyme activities, and myosin heavy chain (MHC) expression in the slow twitch soleus (Sol) and fast twitch gastrocnemius (Gas) muscle of young adult (YA) and senescent (SEN) rats. Muscle mass declined between YA and SEN in the Sol by 35% compared to 55% in the Gas muscle. After normalizing for muscle mass, tetanic force per g of muscle declined by 58% in Sol and by 36% in Gas muscle. Time-to-peak tension was increased only in the Gas (30%), whereas time-to-half relaxation was increased by 70% in Sol and 51% in Gas. Citrate synthase and complex IV activity declined in homogenates of Sol (30-36%) and red oxidative region of Gas (46-51%), but not white glycolytic region of Gas. Strikingly, the shift away from the dominant adult MHC isoform with aging was much greater in Sol (fibers positive for MHC fast: 11+/-2% in YA versus 36+/-3% in SEN) than in Gas (fibers positive for MHC slow: 12+/-1% in YA versus 26+/-3% in SEN) muscle. Collectively, these results show that the slow twitch Sol muscle undergoes large phenotypic alterations in very old age and for several measures (tetanic tension per g, time-to-half relaxation and shift in adult MHC expression) that is of greater magnitude than fast twitch muscle, underscoring the importance of including age-related changes in slow twitch muscle in seeking potential treatments for sarcopenia.

Exercise training in late middle‐aged male Fischer 344 × Brown Norway F1‐hybrid rats improves skeletal muscle aerobic function

The Fischer 344 × Brown Norway F1‐hybrid (F344BN) rat has become an increasingly popular and useful strain for studying age‐related declines in skeletal muscle function because this strain lives long enough to experience significant declines in muscle mass. Since exercise is often considered a mechanism to combat age‐related declines in muscle function, determining the utility of this strain of rat for studying the effects of exercise on the ageing process is necessary. The purpose of this study was to evaluate the plasticity of skeletal muscle aerobic function in late middle‐aged male rats following 7 weeks of treadmill exercise training. Training consisted of 60 min per day, 5 days per week with velocity gradually increasing over the training period according to the capabilities of individual rats. The final 3 weeks involved 2 min high‐intensity intervals to increase the training stimulus. We used in situ skeletal muscle aerobic metabolic responses and in vitro assessment of muscle mitochondrial oxidative capacity to describe the adaptations of aerobic function from the training. Training increased running endurance from 11.3 ± 0.6 to 15.5 ± 0.8 min, an improvement of ∼60%. Similarly, distal hindlimb muscles from trained rats exhibited a higher maximal oxygen consumption in situ (23.2 ± 1.3 versus 19.7 ± 0.8 μmol min−1 for trained versus sedentary rats, respectively) and greater citrate synthase and complex IV enzyme activities in gastrocnemius (29 and 19%, respectively) and plantaris muscles (24 and 28%, respectively) compared with age‐matched sedentary control animals. Our results demonstrate that skeletal muscles from late middle‐aged rats adapt to treadmill exercise by improving skeletal muscle aerobic function and mitochondrial enzyme activities. This rat strain seems suitable for further investigations using exercise as an intervention to combat ageing‐related declines of skeletal muscle aerobic function.