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1 Faculty of Kinesiology
2 Faculty of Medicine, University of Calgary, Alberta, Canada.
Address correspondence to Russell T. Hepple, PhD, Faculty of Kinesiology, University of Calgary, 2500 University Dr. NW, Calgary, AB, Canada T2N 1N4. E-mail: hepple{at}ucalgary.ca
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Abstract |
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O2 and lactate production be reduced, but also that contractile economy would be altered with aging. Peak tension was lower in LMA (42%) and SEN (71%) versus YA. O2 and lactate efflux was progressively lower with increasing age. Estimated adenosine triphosphate per N of force was increased in LMA (35%) and reduced in SEN (31%) versus YA. Myosin heavy chain (MHC) analysis by sodium dodecyl sulphate–polyacrylamide gel electrophoresis showed a lower MHC type IIb and higher MHC type IIa/IIx in SEN versus YA. Therefore, whereas contractile economy is impaired in LMA, it is improved in SEN, and this latter effect may be due in part to reduced type IIb MHC.
One of the factors complicating the examination of these issues in vivo is a decline in skeletal muscle convective O2 delivery with aging, due to a reduced cardiac output (13,14) that is likely further exacerbated by impaired blood flow distribution (15,16). As such, we employed a pump-perfused rat hind-limb preparation to facilitate measurements of O2 and lactate efflux during high-intensity tetanic contractions at similar rates of skeletal muscle convective O2 delivery between age groups and thereby permit us to test the hypothesis that aging results in altered contractile economy. To help address the potential mechanism by which contractile economy may be altered with aging, we also determined MHC distribution in homogenates of the plantaris and gastrocnemius muscles.
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METHODS |
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O2 and lactate efflux) and the contractile responses were monitored throughout a 4-minute high-intensity contraction bout. The animals in this subgroup were taken from three groups of male Fischer 344 x Brown Norway F1-hybrid (F344BN) rats obtained from the United States National Institute on Aging, representing young adult (8–9-month-old), late middle-aged (28–29-month-old), and senescent (36-month-old) animals (n = 6 in each group unless specified otherwise).
Hind-Limb Perfusion Procedures
After anesthetizing the animal with pentobarbital sodium (i.p. 65–75 mg/kg), the right iliac artery and vein were ligated and the right gastrocnemius–plantaris–soleus muscle group was removed, trimmed free of fat and connective tissue, and weighed. A cross-section through the midbelly of the right plantaris (
100 mg) and gastrocnemius (300–400 mg) muscles was then frozen in liquid nitrogen and stored at –80°C for subsequent MHC analysis (below), biochemistry [see companion paper (2)], or morphology (data not shown). Following this, all of the animals were prepared for surgical perfusion of the left hind limb, as described in the companion paper (2).
Contraction Protocol
The procedures used in the contraction protocol are described in our companion paper (2). It is also relevant that a previous study in our laboratory showed that this contraction protocol yields the highest O2 and lactate efflux values for this preparation (17). In addition to drawing blood samples during the contraction bout, blood samples were obtained every 30 seconds for 4 minutes following contractions in 3 x 8–9-month-old, 4 x 28–29-month-old, and 4 x 36-month-old animals to monitor the rate at which lactate efflux declined after contractions. O2 across the hind limb was determined from the product of blood flow and the arteriovenous O2 content difference. Similarly, lactate efflux was calculated as the product of blood flow and the arteriovenous lactate content difference. Normalization of O2 and lactate efflux to the mass of the contracting muscles was performed as described in our companion paper (2). The amounts of ATP produced via aerobic metabolism and from lactate generation from 2–4 minutes during the contraction bout were estimated as done previously by Hood and colleagues (18). Briefly, it was assumed that 6 µmol of ATP was produced per µmol of O2 consumed, and that 1.5 µmol of ATP was produced per µmol of lactate evolved. From this, the estimated ATP cost of producing force was calculated as the quotient of total ATP produced (by the aerobic system and lactate production) and the force production (in N).
Blood Flow Distribution
Blood flow distribution in the gastrocnemius–plantaris–soleus muscle group was assessed as described in our companion paper (2).
MHC Analysis
Analysis of MHC protein expression was initially performed with plantaris muscle samples from 2 x 8–9-month-old, 2 x 28–29-month-old, and 3 x 36-month-old animals. Frozen samples taken from the midbelly of plantaris muscle (100 mg) were thawed on ice, and homogenized in 1 ml of extraction buffer #1 (250 mM sucrose, 100 mM KCl, 5 mM EDTA, 10 mM Tris-base pH 6.8) and 10 µl of protease arrest (Sigma Aldrich, St. Louis, MO) in a glass tissue grinder (Kimble/Kontes, Vineland, NJ) at 230 rpm for 3 minutes. The homogenate was then centrifuged at 3000 x g for 10 minutes and the supernatant discarded. The pellet was resuspended with 1 ml of buffer #2 (150 mM KCl, 10 mM Tris-base pH 6.8, 0.5% Triton-X) and 10 µl of protease arrest and homogenized for 3 minutes at 230 rpm. The homogenate was centrifuged again at 3000 x g for 10 minutes, and the supernatant discarded. The remaining pellet was then resuspended in 1 ml of buffer #3 (150 mM KCl, 10 mM Tris-base pH 7.0) and homogenized, centrifuged, and resuspended three more times to wash the solution. After the final spin, the pellet was resuspended with a volume of buffer #3 that covered the pellet, and 9 volumes of a glycerol buffer and 0.5 µl of ß-mercaptoethanol were added to the suspension before being vortexed. Protein sample buffer (28% glycerol, 2.8% SDS, 1.2% bromophenol blue with 50% v/v 4 x stacking buffer [6.1% Tris-base, 0.4% SDS, pH 6.8]) was mixed with the sample in a ratio of 1:4 and boiled for 4 minutes. Next, 20 µl of sample was loaded onto 3% sodium diodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) minigels containing 30% (v/v) glycerol, and electrophoresis was performed for 30 hours at a constant 100 V (19). Gels were then stained with Coomassie Blue, scanned (HP 6300C scanner; Palo Alto, CA) into JPEG files, and the staining intensity of individual bands was quantified using image analysis (Sigma Scan Pro, ver. 5.0; St. Louis, MO). Since this method did not fully separate the bands for type IIa and IIx MHC, the data for plantaris muscles are presented as IIx and IIa together (i.e., IIx/a). To overcome this limitation, an alternative method was employed to separate all MHC proteins from muscle; however, as there was insufficient tissue remaining from the plantaris muscle samples, we performed this latter analysis using gastrocnemius muscle samples from the same group of animals.
Frozen samples taken from the midbelly of gastrocnemius muscle of 4 x 8–9-month-old, 3 x 28–29-month-old, and 4 x 36-month-old animals were pulverized in liquid nitrogen and homogenized using a polytron homogenizer in 7 volumes of buffer (0.3 M NaCl, 0.1 M NaH2PO4, 50 mM Na2HPO4, 10 mM EDTA, 5 mM DTT pH 6.5) containing protease arrest. The homogenate was then left on ice for 15 minutes, with occasional vortexing to facilitate extraction. The sample was centrifuged at 10,000 x g for 20 minutes and the supernatant was mixed with an equal volume of glycerol and stored at –80°C. Protein concentration was determined (20) and aliquots mixed 1:1 with 2 x sample buffer (20% glycerol, 125 mM Tris-HCl pH 6.8, 4% SDS, 1% ß-mercaptoethanol, 0.2% bromophenol blue). Approximately 0.5 µg of protein was loaded onto 6% SDS-PAGE minigels containing 40% (v/v) glycerol, and electrophoresis was subsequently performed for 17 hours at a constant 70 V (19). Gels were then silver-stained (Bio-Rad silver stain kit; Hercules, CA), scanned into JPEG files, and the staining intensity of individual bands were analyzed by image analysis, as described above.
Statistics
Comparisons of body mass, muscle mass, perfusion conditions, and initial tension development between groups were done using one-way analysis of variance with a Bonferroni post hoc multiple comparison test. Two-way analysis of variance was used for comparisons of the metabolic and contractile responses during the contraction bout between groups (age x time), and for comparing myosin heavy chain composition (age x myosin isoform), with a Bonferroni post hoc multiple comparison test. To assess whether lactate release was impaired with aging, lactate efflux at 90 seconds postcontraction was compared with that observed at the end of contractions by two-way repeated measures analysis of variance (age x time).
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RESULTS |
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O2 (Figure 2) and lactate efflux (Figure 3) were progressively reduced with increasing age (8–9 months old > 28–29 months old > 36 months) during the 4-minute contraction bout. Whereas the lactate efflux had fallen significantly 90 seconds after the contraction bout in the 8–9-month-old and 28–29-month-old animals, lactate efflux 90 seconds postcontraction was not different than that observed at the end of the contraction bout in the senescent muscles (p =.32). The O2 cost of contractions and the estimated ATP cost of contractions were higher in the 28–29-month-old, but lower in the 36-month-old, versus 8–9-month-old animals (Figure 4).
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DISCUSSION |
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O2 and lactate production, but also alterations in contractile economy. Consistent with this hypothesis, we observed that O2 and lactate efflux during a 4-minute high-intensity tetanic contraction bout were progressively reduced with increasing age. Furthermore, the estimated ATP cost of contractions was increased in late middle-aged animals but reduced in senescent skeletal muscles, compared with that of young adult rats. We also observed significant shifts in MHC isoform distribution in plantaris and gastrocnemius muscles of the senescent animals. Specifically, there was a higher type IIa/x MHC and a lower type I and type IIb MHC in the plantaris muscle of senescent animals, whereas there was a higher type IIa MHC and a lower type IIb MHC in the gastrocnemius muscle of senescent versus young adult anmals. These alterations in MHC composition may be contributing in part to the lower estimated ATP cost of contractions in senescence, due the lower cost of sustaining tetanic force for slower MHC isoforms. We conclude that alterations in contractile economy help to compensate for a dramatic reduction in aerobic and glycolytic ATP-generating capacity in senescent skeletal muscles. In particular, the average tension development (N) during 2–4 minutes of the contraction bout in the senescent animals was only 40% lower than late middle-aged animals, despite an average 72% lower O2 (µmol/min) and 73% lower lactate efflux (µmol/min) in senescent muscles during this period. In view of this dramatic reduction in ATP-generating capacity and a marked reduction in muscle mass (44%) between late middle age and senescence, an improved contractile economy may be critical to preserving the capacity for locomotor activity in senescence.
Contractile Performance
A reduction in skeletal muscle contractile function with aging is well established [reviewed in (21)]. In particular, previous studies have noted a reduced maximal force-generating capacity both in human (22–24) and rodent (25,26) skeletal muscles. Denervation of skeletal muscle fibers is thought to contribute only a small part to this decline in force production with aging (27). As such, a reduced force-generating capacity is also seen in isolated fiber bundles or individual muscle fibers (or portions thereof) from aged humans (28–30) and rats (26,31,32). In contrast to the well-established impairment in maximal force-generating capacity of aged muscles, the degree of metabolic and contractile dysfunction during repeated muscle contractions that can be ascribed to alterations within the muscles has remained unclear because a reduction in skeletal muscle blood flow with aging in humans (15,33,34), and particularly the more oxidative muscles in rats (16,35), puts the aged muscles at a disadvantage in vivo. Thus, to help address this issue we used a pump-perfused rat hind-limb preparation to permit us to examine the contractile responses in aged skeletal muscles at similar rates of convective O2 delivery between animals of different ages. In view of the aforementioned issues, it is also pertinent that the blood flow distribution among the individual muscles of the gastrocnemius–plantaris–soleus muscle group was not different between age groups [see Figure 1 in companion paper (2)].
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Aerobic and Lactate Production Responses
Our companion paper (2) and previous work from our laboratory (1) demonstrates that skeletal muscles from late middle-aged animals exhibit a reduced muscle mass-specific O2max during high-intensity tetanic contractions even when supplied with similar rates of muscle mass-specific convective O2 delivery as young adult animals. The current results extend these observations in late middle-aged animals to show that aerobic metabolism is lower throughout a 4-minute high-intensity contraction bout, and demonstrate an even more profound decrease in aerobic function in senescent muscles. Similarly, we also observed a marked reduction in lactate efflux during contractions in both the late middle-aged and senescent animals. The persistence of age-related differences after normalization of absolute values of O2 and lactate efflux to the mass of the contracting muscles (i.e., muscle mass-specific O2 and lactate efflux) demonstrates that these lower values cannot be explained by atrophy of the skeletal muscles with aging alone, although reduced quantity of muscle clearly plays a very significant role in senescence.
In a prior investigation (1), lactate efflux was only monitored for 3 minutes during contractions, and it is possible that the lower lactate efflux in the late middle-aged animals may have reflected an impaired lactate transport capacity with aging, rather than reduced lactate production. To help address this, in the current study we followed lactate efflux throughout the contraction bout and for 4 minutes into recovery. Lactate efflux was maintained at near-peak levels for 90 seconds into recovery in the senescent animals, whereas lactate efflux in the other two groups had declined significantly by this point, strongly suggesting an impaired lactate transport in senescent muscles relative to the other groups. Consistent with this, a reduced lactate transport capacity has been seen previously in sarcolemmal "giant vesicles" obtained from muscles of older rats (37). Thus, the lactate efflux observed during contractions in the senescent group is most likely underestimating the rate of anaerobic glycolysis. However, the integrated lactate efflux over the 4 minutes of contractions and the 4 minutes of recovery is still markedly lower in the senescent animals (2.46 ± 0.41 mM/100 g) versus the young adult animals (5.06 ± 0.42 mM/100 g; p <.05), suggesting that lactate trapping alone cannot account for the lower lactate efflux during contractions in the senescent muscles, and that lactate production is in fact reduced in the senescent muscles. The extent to which these reduced aerobic metabolic and lactate production responses are an effect versus a cause of the reduced contractile function with aging is difficult to determine. However, given the significant role of mitochondrial dysfunction in the impaired aerobic responses of these aged muscles (2), it is likely that the impairment of aerobic function is playing a crucial role in limiting the contractile responses of the aged muscles.
Contractile Economy
The ATP cost of maintaining isometric force is a function of several factors, including the rate of cross-bridge turnover. In this regard, work by Crow and Kushmerick (38) using isolated muscles showed that slow-twitch muscles had a significantly lower ATP cost of maintaining isometric force than fast-twitch muscles. More recent work in single muscle fibers shows that the ATP cost of generating force differs as a function of MHC isoform expression [type I < type IIa 
type IIx < type IIb (10–12)]. In this regard, alterations in fiber type and MHC composition with aging are well known, although highly variable from muscle to muscle [e.g., (8)]. On this basis, we hypothesized that aging would be associated with alterations in skeletal muscle contractile economy. To test this hypothesis, the ATP generated during contractions was estimated from the aerobic (O2) and lactate efflux responses, as done previously (18). Whereas it is acknowledged that these calculations do not take into account energy contributions from phosphocreatine hydrolysis or breakdown of stored ATP, we performed this analysis from 2 to 4 minutes during the contraction bout because it represents a time when the vast majority of ATP required would be provided by the aerobic and glycolytic pathways. Thus, these exclusions do not compromise the major conclusions.
Consistent with the hypothesized alterations in contractile economy with aging, we observed a significant increase in the estimated ATP cost of contractions in late middle-aged animals, but a reduced estimated ATP cost of contractions in senescent animals, compared with young adult animals. Note that whereas our previous study found no difference in contractile economy between young adult and late middle-aged animals (1), this analysis was previously performed only at O2max (reducing the resolution of the measurement). Secondly, the current experiments utilized a different muscle clamp from that used in the majority of the previous set of experiments. This new muscle clamp substantially reduced the amount of force lost to the compliance of the clamp at high muscle forces, which resulted in greater forces being measured at the force transducer (particularly in the stronger young adult animals) in the current set of experiments. Lastly, our current approach utilized the total O2 during contractions less that estimated to be contributed by the noncontracting muscles, whereas our previous approach (1) utilized the total O2 during contractions less the total O2 measured at rest (not just that contributed by the noncontracting tissues). Collectively, these methodological and experimental differences account for the differences in estimates of contractile economy between the current and previous (1) study.
Note that the estimated ATP provision from lactate production in the senescent animals is likely underestimated because of the aforementioned impairment of lactate transport out of these muscles during contractions. However, even if we assume that the senescent muscles possess an equal mass-specific capacity to generate lactate as do the late middle-aged animals' muscles, this does not alter the conclusion that the estimated ATP cost of contractions in senescent muscles is lower than in young adult muscles or late middle-aged muscles. To our knowledge, the only other investigation to consider alterations in contractile economy with aging was a study by De Haan and colleagues (36), and in that study they observed no differences between 5-month-old and 22-month-old rats (i.e., rats that were considerably younger than studied here).
The higher estimated ATP cost of contractions in the late middle-aged animals is at odds with the changes in MHC isoform composition, since an increased type IIx/a MHC was the only significant change noted in homogenates prepared from the plantaris muscle of the late middle-aged animals versus young adult (no changes observed in gastrocnemius muscles at this age). On the basis that mitochondrial leakiness tends to increase with aging (39,40), the apparent increase in the ATP cost of contractions (estimated based upon an assumed constant ATP yield per µmol of O2 between age groups) may actually reflect a lower ATP yield per µmol of O2 consumed in late middle age, as reflected in the higher O2 cost of contractions observed in these animals. Based upon Rolfe and colleagues' previous observations that 34% of the total O2 measured across vigorously contracting pump-perfused rat hind-limb muscles can be ascribed to proton leak (41), we estimate that proton leak would need to increase to 58% of the total measured O2 across the contracting muscles in the late middle-aged animals to yield the observed differences in estimated O2 cost of contractions between young adult and late middle-aged animals. Why mitochondrial proton leak would be reduced between late middle age and senescence (as is suggested by the lower O2 cost of contractions in the senescent animals), however, is not clear and warrants further study. Regardless, it is clear that the degree of contractile impairment in the late middle-aged animals far exceeds the reduction in O2 and lactate efflux versus the young adult animals and, therefore, that inspection of the metabolic responses alone would lead one to underestimate the extent of contractile dysfunction in late middle age. Interestingly, a study of "normally active" 62–73-year-old men (i.e., similar relative age to the late middle-aged animals) also observed a higher O2 at a given power output during cycle exercise (42), whereas it was reduced in 55–68-year-old endurance-trained men (34) compared with activity-matched young adults. In the study with "normally active" subjects (42), it was not possible to evaluate where the "extra" O2 was being used; however, our results suggest that an increased ATP cost of maintaining force in the locomotor muscles may contribute to this phenomenon.
On the other hand, the lower estimated ATP cost of contractions and lower O2 cost of contractions seen in the senescent muscles could reflect the influence of a switch toward slower and more economical (i.e., lower ATP requirement for sustaining tetanic force) MHC isoforms. Consistent with this notion, we observed a significant increase in type IIa/x or type IIa at the expense of type IIb MHC isoforms in homogenates prepared from both the plantaris and gastrocnemius muscles of the senescent animals. Note that the fiber type distributions in these two muscles are representative (43) of the whole of the contracting muscles in our preparation [which includes the gastrocnemius–plantaris–soleus muscle group, tibialis anterior muscle, and deep tibial muscles (44)]. Although previous studies indicate a nearly 50% lower ATP cost of sustaining isometric tension in IIa versus type IIb fibers in rat locomotor skeletal muscles (10), which would go a long way towards explaining the observed differences in estimated ATP cost of contraction, a cautionary note is that the fall in force by 2 minutes, regardless of age, was between 46% and 70%. Based upon this high degree of fatigue, it is unclear what proportion of the type IIb fibers would still be contributing to the contractile and metabolic responses during the 2–4-minute period when contractile economy was estimated. If type IIb fibers make only a modest contribution to these responses during this period, the role that the alterations in MHC isoform distribution play in the reduction in the ATP cost of contractions in the senescent muscles could be relatively minor. Further work is required to clarify these issues.
Notwithstanding the aforementioned uncertainties, the implications of the changes in estimated ATP cost of contractions between late middle age and senescence for locomotor activity are striking. In particular, the decline in estimated ATP provision by the aerobic and glycolytic metabolic pathways (absolute values: 70%) in senescent versus late middle-aged muscles is far in excess of the difference in force production between these groups (40%). Thus, in the absence of a reduction in ATP cost of contractions between late middle age and senescence, the senescent muscles would only be able to generate enough ATP to sustain a force output of 2.1 N (versus the 4.5 N that was observed). In the context of a 500 g rat (0.5 N), this difference in force output would have a significant impact on the locomotor ability of the animal. The extent to which these findings in a rat model of aging apply to the aging human warrants consideration. In this regard, we have previously noted (2) that the estimated decline in skeletal muscle mass in senescent F344BN rats is very similar to that seen in senescent humans (i.e., 
80 y). Thus, if our observations in senescent F344BN rats represent a compensatory mechanism to the severe muscle atrophy and metabolic impairment evident at this age, a similar process may occur in the frail elderly. Future studies should consider this possibility.
Summary
We observed marked reductions in the contractile and metabolic (O2 and lactate efflux) responses to a 4-minute high-intensity tetanic contraction bout with increasing age in skeletal muscles of F344BN rats pump-perfused at similar rates of convective O2 delivery. The reductions in these major ATP-generating pathways were evident even after normalizing for the atrophy of the skeletal muscles with aging, showing that this alteration cannot be explained by muscle atrophy alone. Interestingly, estimation of the ATP cost of generating tetanic force from 2 to 4 minutes during the contraction bout suggested that contractile economy was lower in late middle-aged animals, but higher in senescent animals, compared with young adult animals. In conjunction with these changes, we also observed a significant reduction in type IIb MHC and significant increases in type IIa/x (plantaris muscle) or type IIa (gastrocnemius muscle) MHC in senescence, which may help explain the lower estimated ATP cost of contractions in this group. In conclusion, an improved contractile economy in senescent skeletal muscles helps to compensate for a dramatic reduction in the capacity to generate ATP via aerobic metabolism and lactate production.
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Acknowledgments |
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Footnotes |
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Received March 31, 2004
Accepted July 25, 2004
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References |
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O2max. J Gerontol Biol Sci. 2004;59A:1099-1110.
O2 max is unaffected by altering the temporal pattern of stimulation frequency in rat hindlimb in situ. J Appl Physiol. 2003;95:705-711.This article has been cited by other articles:
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  A. C. Betik, D. J. Baker, D. J. Krause, M. J. McConkey, and R. T. Hepple Exercise training in late middle-aged male Fischer 344 x Brown Norway F1-hybrid rats improves skeletal muscle aerobic function Exp Physiol, July 1, 2008; 93(7): 863 - 871. [Abstract] [Full Text] [PDF]
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 I. R. Lanza, R. G. Larsen, and J. A. Kent-Braun Effects of old age on human skeletal muscle energetics during fatiguing contractions with and without blood flow J. Physiol., September 15, 2007; 583(3): 1093 - 1105. [Abstract] [Full Text] [PDF]
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p <.05 versus 8–9-month-old group