Despite blunted glycolytic flux in older compared to young adults during FF, older adults increased glycolytic flux to a level similar to that of young during ISC, when oxidative phosphorylation was no longer a viable source of ATP. This metabolic adaptation suggests that glycolytic function remains intact in the ankle dorsiflexor muscles of heathly older adults. As expected, the greater fatigability of young compared to old during FF was related to inhibitory metabolite accumulation during contractions. The mechanisms of the age-related fatigue resistance during ISC are less clear. While our results provide further evidence for the role of muscular energetics in the age-related differences in muscle fatigue, they also suggest that increased metabolic economy may be a potential mechanism allowing older muscle to engage in maximal work with lower glycolytic flux compared to young muscle.
Muscle oxidative capacity
The rate constant of PCr recovery was similar in young and older subjects, as was the calculated oxidative capacity, Qmax. Although others have reported that oxidative capacity is impaired with old age in vivo (McCully et al. 1993; Taylor et al. 1997; Conley et al. 2000), the results of the present study are consistent with previous reports that oxidative capacity is preserved in the tibialis anterior (Kent-Braun & Ng, 2000; Lanza et al. 2005), forearm (Kutsuzawa et al. 2001), and plantarflexor muscles of older adults (Chilibeck et al. 1998). Studies in vitro reveal similar discrepancies with some reports of reduced oxidative enzyme activities (Coggan et al. 1992; Pastoris et al. 2000), increased mitochondrial DNA alterations (Short & Nair, 2001), and reduced mitochondrial protein synthesis (Rooyackers et al. 1996), while others found no effects of age on mitochondrial function by a variety of methods (Grimby et al. 1982; Aniansson et al. 1986; Barrientos et al. 1996). The diversity of muscle groups under investigation may be a primary explanation for these discrepant results. Indeed, numerous studies demonstrate that neural (Galea, 1996), structural (Grimby et al. 1982), mechanical (Lanza et al. 2003), and enzymatic (Houmard et al. 1998) changes with age exhibit muscle-group specificity. This consideration, as well as the impact of varying health characteristics and physical activity patterns on age-related changes in oxidative capacity, is reviewed elsewhere (Russ & Kent-Braun, 2004).
Pathways of ATP synthesis
The oxidative ATP synthesis rates were similar in young and older subjects during FF, as observed previously (Lanza et al. 2005), and were comparable to the rates reported in previous studies using similar methods (Boska, 1991; Jubrias et al. 2001; Lanza et al. 2005). The similarity in ATPOX across age groups in the present study is consistent with our observations of preserved oxidative capacity with ageing. Phosphocreatine recovery is typically attributed exclusively to oxidative ATP synthesis, based on demonstrations that PCr does not recover in the absence of oxygen (Quistorff et al. 1993; Nakagawa et al. 2005). This dogmatic view has been challenged recently by reports of a transient portion of PCr recovery that can be attributed to anaerobic glycolysis (Crowther et al. 2002b; Lanza et al. 2006). As a result, attempts have been made to adjust the calculation of oxidative flux to account for this transient glycolysis. In two studies, glycolytic PCr resynthesis was quantified during ischaemic contractions and used to derive correction factors that were then applied during equivalent free-flow contractions (Jubrias et al. 2001; Lanza et al. 2006). However, these corrections assumed equal glycolytic flux during the postcontraction interval during FF and ISC, which may be inappropriate in the present study, where glycolytic flux was higher during ISC than FF in older subjects. Therefore, we did not correct for the glycolytic portion of PCr recovery in this study, and as a result have likely overestimated ATPOX. This issue remains unresolved until more is known about the contribution of glycolysis to the postcontraction recovery of PCr.
The rates of ATP synthesis from net PCr hydrolysis in the creatine kinase reaction were similar in young and older subjects during FF and ISC. We have previously shown that ATPCK is unaffected by old age during FF (Lanza et al. 2005). Using a steady-state saturation transfer MRS method, Horska et al. (2000) also found that flux through the creatine kinase reaction does not differ by age. In contrast to these studies in vivo, experiments using biopsy tissue have revealed significant age-related reductions in creatine kinase activity in vitro (Kaczor et al. 2006; Gelfi et al. 2006). Although the effects of ageing on the temporal ATP-buffering capacity of the creatine kinase reaction have not been studied extensively, the literature to date suggests that this pathway is unaffected by the ageing process in vivo.
Glycolytic flux was lower in older compared to young subjects during FF, as reported recently (Lanza et al. 2005) and inferred from blunted intracellular acidosis during muscle contractions (Coggan et al. 1993; Taylor et al. 1997; Chilibeck et al. 1998; Kent-Braun et al. 2002). Contrary to our hypothesis, ATPGLY increased in older subjects when oxidative ATP synthesis was eliminated during ischaemia, suggesting that the functionality of glycolytic ATP synthesis in vivo remains robust with old age. Therefore, the blunted glycolytic flux observed during FF is likely to reflect the ability of older muscle to adequately meet energetic needs without increasing glycolytic flux to the same extent as young. It is important to note that we did not measure the capacity for anaerobic glycolysis, but rather the functionality of this pathway for ATP production under different conditions in vivo. It is certainly possible that the glycolytic flux we measured during maximal isometric contractions, even under ischaemic conditions, is below the upper limit that is possible in vivo. For example, dynamic contractions, which are more metabolically demanding than isometric contractions (Newham et al. 1995), may result in higher glycolytic flux than observed in the present study.
Studies of age-related alterations in glycolytic function in humans have been limited primarily to enzymatic analyses, which showed similar (Grimby et al. 1982; Coggan et al. 1992) or lower (Larsson et al. 1978; Pastoris et al. 2000) activity of glycolytic enzymes in old compared to young muscle. The ability to generate ATP glycolytically could decline with age as a result of reduced type II fibre area. By multiplying the proportional area of type I (Y, 64.5%; O, 81.1%) and type II fibres (Y, 35.5%; O, 18.9%; Jakobsson et al. 1990) by their respective PFK activities (type I, 25.8, type II, 49.4 mmol min−1 (kg dry weight)−1; Essen et al. 1975), we estimate that the reduction in overall muscle PFK activity due to reduced type II fibre proportions is not likely to exceed ∼11% in old muscle. This prediction is higher than our earlier estimations based on α-glycerol phosphate dehydrogenase (GPDH) activity; however, PFK is likely to be a better proxy for the potential for glycolytic flux than GPDH (Lanza et al. 2005). Regardless, both estimates suggest that the age-related fibre type shift cannot fully explain the observation that glycolytic flux is blunted in older compared to young under FF conditions.
Our results in vivo are in contrast to stimulated rat hind-limb experiments, which show reduced lactate production in older compared to young rats (Campbell et al. 1991; Hepple et al. 2004). However, this result was exclusive to white gastrocnemius muscle, whereas soleus, plantaris, and red gastrocnemius demonstrated no age-related impairment in glycolytic function. Thus, it seems that, at least in rat skeletal muscle, glycolytic function may be impaired with age in fast glycolytic fibres but preserved in more oxidative fibres. Nevertheless, despite the mixed composition of human tibialis anterior muscle (Jakobsson et al. 1990) we observed that glycolytic function is preserved with age in this muscle group. Further studies are warranted to determine if this result is consistent across other muscle groups with more glycolytic fibre composition.
Although our results oppose the notion of impaired glycolytic capacity as a mechanism to explain blunted glycolytic flux in old muscle, there remain numerous potential mechanisms that warrant discussion. We have previously proposed that glycolytic flux may be higher in younger subjects due to constricted blood flow during muscle contractions (Kent-Braun et al. 2002; Lanza et al. 2005). The larger, stronger muscles of younger individuals would be expected to generate greater intramuscular pressure and thus occlude blood flow to the working muscle to a greater extent than the smaller, weaker muscles of older subjects. However, this mechanism is unlikely to explain the greater glycolytic flux in young subjects in the present study since all subjects, regardless of age, generated > 60% MVC during the free-flow protocol, which is a level force beyond which skeletal muscle perfusion is fully occluded during voluntary dorsiflexion (Wigmore et al. 2004).
Activation of glycolysis during muscle contraction is believed to be regulated by a ‘dual control’ model whereby intracellular calcium (Ca2+) and metabolic by-products of muscle contraction (Pi, AMP, ADP) synergistically regulate glycolytic flux (Quistorff et al. 1993; Connett & Sahlin, 1996; Crowther et al. 2002a). Although [AMP] and [ADP] were similar in young and old during both protocols here, [Pi] was higher in the young during FF, but similar across age groups during ISC. During FF, [Pi] was at (older) or above (young) the reported Michaelis constant (Km) of glycogen phosphorylase for Pi of ∼27 mm (Chasiotis et al. 1982), consistent with the notion of Pi as a regulator of age-related differences in glycolytic flux in FF. During ISC, [Pi] increased to a similar, high (∼37 mm) level in young and old, and ATPGLY was similar in both groups.
When examining the change in [Pi] in both groups from FF to ISC (Table 2), it is somewhat surprising to note the lack of increase in ATPGLY in the young, given their ∼5 mm increase in [Pi] from FF to ISC. It is possible that [Pi] in young was sufficiently above the km during FF, such that a further increase in [Pi] during ISC had little effect on ATPGLY. In contrast, the change in [Pi] from FF to ISC in the old (∼8 mm) apparently occurred at a steeper portion of the saturation curve between Pi and ATPGLY, and thus was able to further activate glycolysis in the old. Additionally, the inhibitory effects of intracellular acidosis on glycolytic flux (Hill, 1955; Chase & Kushmerick, 1988) may have been different in young and old across conditions. That is, the young may have reached an intracellular pH during both FF and ISC at which glycolysis became inhibited, while the older group may have attained this level of acidosis only during ISC.
While the differences in Pi accumulation and intracellular pH provide an attractive mechanism to explain the current results, the importance of intracellular Ca2+ in regulation of ATPGLY should not be overlooked. It is possible that the age-related decline in motor unit discharge rates (MUDR) (Kamen et al. 1995; Connelly et al. 1999) and the leftward shift in the force–frequency relationship with old age (Ng & Kent-Braun, 1999; Allman & Rice, 2004) may combine to generate high force with relatively fewer activation pulses in the old. This effect would translate to less Ca2+ release from the sarcoplasmic reticulum, lower intracellular Ca2+, and lower glycolytic flux during high-intensity contractions in older compared to young muscle. This possibility needs to be explored.
Total ATP flux
Because there was no change in [ATP] during these protocols, ATPTOT can serve as a proxy for ATP demand. We observed that ATPTOT was lower in old compared to young during FF. Although one might expect differences in absolute force production to contribute to the observed differences in the overall ATP demand, the similar strength of the two age groups, and the use of volumetric units (mm s−1), render our flux measures independent of muscle size. Thus, our data suggest that the ATP requirements of force production are lower in older muscle, possibly as a result of more economical force production due to the combination of contractile slowing and lower MUDR mentioned above.
Previous reports of age-related changes in metabolic economy are scarce and thus far limited to animal studies, which have shown increased contractile economy with age (de Haan et al. 1993; Hepple et al. 2004). The age-related shift of muscle toward a slower, more oxidative and economical fibre-type composition (Jakobsson et al. 1990; Lexell, 1995; Hepple et al. 2004) is a potential source of increased metabolic economy with age. We are unaware of any studies that have investigated age-related changes in metabolic economy in humans to date.
Force and voluntary activation
As expected, young subjects fatigued more than older subjects during FF, in agreement with some previous studies (Bemben et al. 1996; Ditor & Hicks, 2000), but in contrast to others (Davies & White, 1983; Lennmarken et al. 1985; Cupido et al. 1992). In the present study, the strong association between fatigue and [H2PO4−] during FF supports the contention of a metabolic basis for the observed fatigue resistance with old age. We hypothesized that older subjects would fatigue more than young during ISC because of an inability to increase glycolytic flux in compensation for suppressed oxidative ATP synthesis. Although all subjects fatigued more during ISC compared to FF, the fatigue resistance of older subjects persisted despite occlusion of blood flow. As discussed above, older adults were capable of increasing their reliance on substrate-level phosphorylation during ISC as necessary to meet the energetic demands of the contraction protocol. Similar to FF, we again observed a strong association between fatigue and [H2PO4−] during ISC, suggesting that metabolite accumulation may still be a potent mechanism to explain greater fatigue in young than older muscle, even in the absence of blood flow. However, there was a trend for the age-related difference in H2PO4− accumulation to be less pronounced during ISC than FF, which suggests that something other than the direct effects of accumulating metabolites on muscle force development may have been contributing to the fatigue resistance of the older subjects. Recent observations from our laboratory (Chung et al. unpublished) reveal that greater central and peripheral activation failure in the young may explain a portion of the age-related fatigue resistance during ischaemic MVCs. However, we did not measure activation during fatigue in the present study, and thus cannot ascertain the neural contribution to fatigue.
In summary, we have shown that, during maximal voluntary ankle dorsiflexion, glycolytic flux was lower in older compared to young subjects during FF, but similar across age groups during ISC. These data suggest that glycolytic function remains intact with old age in this muscle group, and that the ankle dorsiflexors of older individuals retain the ability to meet the energetic demands of contractile activity under a variety of conditions. In addition, the results point to an age-related increase in metabolic economy as a potential mechanism that may allow older muscle to engage in maximal work with less fatigue compared to young. Further study is needed to determine whether the present findings are consistent across other morphologically and functionally distinct muscle groups.