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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 14, 2005 as doi:10.1096/fj.04-3535fje. |
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,1
* Faculty of Kinesiology and
Faculty of Medicine, University of Calgary, Calgary, Canada; and
Department of Pediatrics, McMaster University, Hamilton, Canada
1Correspondence: Faculty of Kinesiology University of Calgary, 2500 University Dr. NW, Calgary, Alberta T2N 1N4, Canada. E-mail: hepple{at}ucalgary.ca
SPECIFIC AIMS
Long-term caloric restriction (CR) is one of only a few interventions that slow biological aging. There is growing evidence that CR is beneficial in slowing the progress of the age-associated loss of muscle known as sarcopenia. To further extend our understanding of the benefits of CR for skeletal muscle health with aging, the purpose of the current study was to determine whether CR would attenuate the nearly 50% reduction in mass-specific maximal rate of aerobic metabolism (
O2max) seen earlier in the distal hindlimb skeletal muscles of senescent Fischer 344 x Brown Norway F1-hybrid rats.
PRINCIPAL FINDINGS
1. Caloric restriction prevented the age-associated decline in skeletal muscle aerobic function
The left distal hindlimb skeletal muscles of 8- to 10-month-old (young adult) and 35-month-old (senescent) ad libitum fed (AL) and CR Fischer 344 x Brown Norway F1-hybrid rats were pump-perfused in situ to match the rate of muscle mass-specific oxygen delivery between groups. Briefly, this involved surgically isolating the left iliac artery and vein, and advancing catheters into the respective femoral artery and vein. The hindlimb was perfused with bovine erythrocytes reconstituted in a Krebs-Henseleit buffer (pH 7.4) containing 4.5% bovine serum albumin, 5 mM glucose, 100 mU·mL–1 insulin, and 0.15 mM pyruvate, to a hematocrit of 
43%.
Before entering the hindlimb, the perfusion medium was gassed with 95% oxygen and 5% carbon dioxide to yield an average oxygen content of 20.7 ± 0.2% by volume. The rate of perfusion was controlled by a peristaltic pump and selected based on the mass of the distal hindlimb muscles in the contralateral leg (determined before initiating perfusion) to allow matching of muscle blood flow between groups. To permit measurement of contractile function in the gastrocnemius-plantaris-soleus muscle group, the Achilles tendon was severed (with a piece of the calcaneus intact) and secured with noncompliant silk thread to a force transducer. Maximal tetanic contractions were elicited by electrical stimulation (200 ms trains at 100 pulses/s, each 0.05 ms in duration; 6 V) of the sciatic nerve for 1 min at each of 7.5, 15, 30, and 60 tetani per min, and for 2 min at 90 tetani per min. Blood samples were obtained anaerobically every 30 s and analyzed for PO2, PCO2, oxygen saturation, hemoglobin, and lactate. Blood flow to the distal hindlimb muscles was assessed by infusion of colored microspheres (15 µm diameter) into the hindlimb immediately after the contraction bout, and was used to calculate the rate of muscle convective oxygen delivery (convective oxygen delivery=arterial oxygen contentxblood flow) to these muscles.
There was a marked decline in maximal mass-specific force output of the gastrocnemius-plantaris-soleus muscle group in the 35-month-old (6.5±1.0 N·g–1) vs. 8- to 10-month-old (12.9±2.1 N·g–1) AL rats. In contrast, there was no decline in 8- to10-month-old (13.2±1.3 N·g–1) vs. 35-month-old (13.9±0.8 N·g–1) CR rats. Despite matching muscle oxygen delivery between groups, the mass-specific O2max was 46% lower in 35-month-old (281±54 µmol·min·100 g–1) than 8- to 10-month-old (524±13 µmol·min·100 g–1) AL rats. On the other hand, there was no decline in mass-specific O2max between 8- to 10-month-old (490±42 µmol·min·100 g–1) and 35-month-old (484±49 µmol·min·100 g–1) CR rats (Fig. 1
), showing that CR preserves skeletal muscle aerobic function with aging. Similarly, although there was a marked decline in mass-specific peak lactate efflux during contractions between 8 and 10 months (879±72 µmol·min·100 g–1) and 35 months of age (307±59 µmol·min·100 g–1) in AL rats, there was a much smaller decline between 8 to 10 months (900±69 µmol·min·100 g–1) and 35 months of age (740±58 µmol·min·100 g–1) in CR rats, suggesting that CR also better maintains glycolytic metabolic function with aging. Collectively, these results show that CR results in a remarkable preservation of skeletal muscle contractile and metabolic (ATP-generating capacity) capacity with aging.
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2. Caloric restriction prevented the age-associated decline in skeletal muscle mitochondrial oxidative capacity
The plantaris muscle and the mixed region of gastrocnemius muscle of the right leg were frozen in liquid nitrogen for in vitro assays of mitochondrial oxidative capacity. Muscles were first powdered under liquid nitrogen and subsequently homogenized in a phosphate buffer. The homogenates were then vortexed and freeze-thawed three times. After the final freeze-thaw cycle, each homogenate was spun at 900 g for 3 min and the supernatant was divided into 100 µL aliquots, frozen in liquid nitrogen, and stored at –70°C until assayed. Using spectrophotometric methods, we determined the rotenone-sensitive activity of electron transport chain complexes I–III, the activity of complex IV, and citrate synthase activity. In the plantaris muscle there was a 38–41% reduction in citrate synthase, complexes I–III, and complex IV activities between 8–10 months and 35 months of age in AL rats. Although the reduction in citrate synthase activity (19%) and complexes I–III activity (29%) in the mixed region of gastrocnemius muscle was not statistically significant in 35-month-old AL rats, complex IV activity was reduced to a similar extent as in plantaris muscle (39%). In contrast to these findings in AL rats, there was no age-related decline in mitochondrial oxidative capacity in CR rats, regardless of the muscle or enzyme pathway examined. In addition, for citrate synthase activity and complex IV activity, the 8- to 10-month-old animals started at a lower point in the CR rats than the AL rats, but CR maintained this level with age.
3. The oxygen flux through complex IV in situ was higher in muscles from caloric restricted rats than ad libitum-fed rats
In vitro biochemical assays, where all enzyme substrates are provided in excess, may not reveal subtle changes in enzyme function (e.g., due to alterations in the affinity of the enzyme for its substrates). Thus, we calculated the mass-adjusted activity of complex IV in the plantaris and mixed region of gastrocnemius muscle (which together comprise 50% of the contracting muscles by mass) as a whole, then took the quotient of O2max and this aggregate complex IV activity to estimate the rate of oxygen flux through complex IV in situ. This calculation revealed a higher rate of oxygen flux through complex IV at O2max in CR than AL rats, despite matching the rate of muscle oxygen delivery between groups, and suggests a higher affinity of complex IV for oxygen in the mitochondria of CR vs. AL animals (Fig. 2
). These results are similar to previous findings showing that complex IV of mitochondria from skeletal muscles of CR mice has a higher proportion of high affinity binding sites, such that it exhibits a higher rate of activity for a given concentration of cytochrome c. Therefore, these results show that CR promotes a higher function of complex IV in situ, and that this in part contributes to the maintenance of skeletal muscle aerobic function with aging by CR.
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CONCLUSIONS AND SIGNIFICANCE
The current results extend our understanding of the beneficial effects of CR on skeletal muscle health with aging. Our results show that CR completely prevents the nearly 50% decline in skeletal muscle mass-specific maximal aerobic function seen between young adulthood and senescence in AL rats. This effect is due in part to maintenance of mitochondrial oxidative capacity with aging by CR and is facilitated by superior mitochondrial function in CR animals (Fig. 3
). As such, the current results show that, in addition to the previously established slowing of muscle atrophy with aging, CR has an even more profound effect in maintaining skeletal muscle contractile and aerobic metabolic function.
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The mechanism(s) by which CR promotes its beneficial effects remains an area of intensive study. In the context of maintained mitochondrial function with aging, a number of possibilities exist. One we are pursuing is that, by promoting protein renewal, CR may prevent the age-associated accumulation of damaged mitochondrial proteins, thereby better preserving their function.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3535fje;
This article has been cited by other articles:
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D. J. Baker, A. C. Betik, D. J. Krause, and R. T. Hepple No decline in skeletal muscle oxidative capacity with aging in long-term calorically restricted rats: effects are independent of mitochondrial DNA integrity. J. Gerontol. A Biol. Sci. Med. Sci., July 1, 2006; 61(7): 675 - 684. [Abstract] [Full Text] [PDF]
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