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. 2004 Oct 21;561(Pt 3):851–859. doi: 10.1113/jphysiol.2004.069419

Acetyl group availability influences phosphocreatine degradation even during intense muscle contraction

James A Timmons 1, Dumitru Constantin-Teodosiu 2, Simon M Poucher 3, Paul L Greenhaff 2
PMCID: PMC1665386  PMID: 15498812

Abstract

We previously established that activation of the pyruvate dehydrogenase complex (PDC) using dichloroacetate (DCA) reduced the reliance on substrate-level phosphorylation (SLP) at the onset of exercise, with normal and reduced blood flow. PDC activation also reduced fatigue development during contraction with reduced blood flow. Since these observations, several studies have re-evaluated our observations. One study demonstrated a performance benefit without a reduction in SLP, raising a question mark over PDC's role in the regulation of ATP regeneration and our interpretation of fatigue mechanisms. Using a model of muscle contraction similar to the conflicting study (i.e. tetanic rather than twitch stimulation), we re-examined this question. Using canine skeletal muscle, one group was infused with saline while the other was pretreated with 300 mg (kg body mass)−1 DCA. Muscle biopsies were taken at rest, peak tension (1 min) and after 6 min of tetanic electrical stimulation (75 ms on−925 ms off per second) and blood flow was limited to 25% of normal values observed during contraction. DCA reduced phosphocreatine (PCr) degradation by 40% during the first minute of contraction, but did not prevent the almost complete depletion of PCr stores at 6 min, while muscle fatigue did not differ between the two groups. During intermittent tetanic stimulation PCr degradation was 75% greater than with our previous 3 Hz twitch contraction protocol, despite a similar rate of oxygen consumption at 6 min. Thus, in the present study enhanced acetyl group availability altered the time course of PCr utilization but did not prevent the decline towards depletion. Consistent with our earlier conclusions, DCA pretreatment reduces muscle fatigue only when SLP is attenuated. The present study and our met-analysis indicates that enhanced acetyl group availability results in a readily measurable reduction in SLP when the initial rate of PCr utilization is ∼1 mmol (kg dry mass)−1 s−1 or less (depending on intrinsic mitochondrial capacity). When measured early during an uninterrupted period of muscle contraction, acetyl group availability is likely to influence SLP under any condition where mitochondria are responsible for a significant proportion of ATP regeneration.

Daily life is typically characterized by intermittent locomotion (see Girard et al. 2001) such that fatigue mechanisms which operate under such conditions are highly relevant. Despite a century of research, understanding of the metabolic regulation during transitions from rest to work remains incomplete (Krogh & Lindhard, 1920; Margaria et al. 1963; Sahlin et al. 1988 see Tschakovsky & Hughson, 1999). There is an unquestionable physiological lag in the response to changes in work rate and this is embodied within the concept of the ‘oxygen deficit’ (OD). The OD was thought to reflect the component of muscle ATP regeneration during the rest-to-work transition period derived from PCr and glycolytic ATP regeneration (referred to as substrate-level phosphorylation or SLP; Saltin et al. 1990). In 1996, we made an interesting observation that increasing mitochondrial acetyl group availability through activation of the pyruvate dehydrogenase complex (PDC) using dichloroacetate (DCA) reduced the OD by 50% during restricted blood flow conditions (Timmons et al. 1996a). This was unexpected since reliance on SLP during contraction with impaired blood flow is substantially greater than under normal blood flow conditions (Timmons et al. 1996b) and PDC was not conceived at this time as a ‘flux generating step’. We and others have been able to reproduce our findings during submaximal exercise in human (with and without blood flow being reduced) and in various animal models of muscle fatigue (Timmons et al. 1996a, 1997, 1998a,b; Howlett et al. 1999a; Parolin et al. 2000; Roberts et al. 2002; Howlett & Hogan, 2003; Rossiter et al. 2003). Others have used intense exercise, often with an intermittent exercise protocol, and their results have challenged the universality of the acetyl group deficit (Howlett et al. 1999b; Evan et al. 2001; Bangsbo et al. 2002; Savasi et al. 2002, Watt et al. 2002).

When the ‘acetyl group deficit’ was originally discovered (Timmons et al. 1996a) it was proposed that DCA could be reducing SLP through the combined effect of enhancing flux through PDC and/or by providing a ‘stock pile’ of acetyl groups for the tricarboxylic acid cycle in the form of acetylcarnitine (where reversal of the carnitine acetyl-transferase activity rapidly feeds acetyl-coenzyme A to citrate synthase). There is experimental evidence to support all of these eventualities (Timmons et al. 1996a, 1997, 1998a,b; Roberts et al. 2002). We also demonstrated that net NADH accumulation was greater from rest to steady state following DCA pretreatment (Timmons et al. 1997). This indicates that enhanced substrate flux through the Krebs cycle or PDC occurred during the first 60 s of contraction, thus yielding a greater redox drive to the electron transport chain. In our studies, the only real assumption was that DCA resulted in additional oxygen consumption during the rest-to-exercise phase. It has now been confirmed that DCA can speed up intramuscular oxygen consumption during the rest-to-work transition period in perfused single muscle fibres (Howlett & Hogan, 2003).

To precipitate fatigue that might mimic that found in peripheral vascular disease patients, we limited blood flow to 25% of normal, during contraction (Timmons et al. 1996b). We used a 3 Hz twitch stimulation protocol (Timmons et al. 1996b) which achieves an oxygen consumption of ∼80% of maximum, if blood flow is fully intact (it yields only 25% V̇O2max of in practice). The positive characteristics of this canine model include the avoidance of protracted muscle sampling: muscle sampling occurs without the cessation of contraction and it avoids the possibility of inactive (unrecruited) muscle fibres ‘contaminating’ (Constantin-Teodosiu et al. 1996) the metabolic profile of the muscle biopsy sample. We established that reduced SLP following DCA treatment preceded the reduction in muscle fatigue changes, demonstrating that the muscle fatigue appeared to be of metabolic origin (Timmons et al. 1997).

More recently DCA has been shown to improve muscle function during tetanic muscle contraction (with intact blood flow) independent of any changes in SLP (Grassi et al. 2002). Thus an aim of the present study was to re-evaluate these findings using a tetanic stimulation protocol. The protocol was designed to precipitate PCr degradation during the rest-to-steady state period, at a rate of ∼1 mmol (kg dry mass)−1 s−1. This was because in a parallel met-analysis of the available literature, DCA did not appear to reduce SLP during exercise above this intensity. Thus, a second major aim of this study was to establish whether enhanced acetyl group delivery altered PCr degradation during this intense stimulation protocol. We specifically hypothesized that acetyl group availability may reduce the rate of PCr degradation early during contraction. To confirm this we took a muscle biopsy at the point of peak tension, prior to any fatigue, and without interrupting the stimulation protocol. This would enable us to confirm that, during an intense stimulation protocol, acetyl group availability still limited oxidative ATP regeneration.

Methods

Surgical procedures

After an overnight fast, each dog (Animal Breeding Unit, AstraZeneca Pharmaceuticals, Alderley Park, UK; body mass (mean ± s.e.m., 14.7 ± 1.5 kg, n = 10) was premedicated with morphine (10 mg i.m.) 30 min prior to the induction of anaesthesia with sodium pentobarbitone (45 ± 1 mg kg−1) followed by continuous infusion at 0.10 ± 0.01 mg kg−1 min−1, both i.v. (Sagatal, Rhône Merieux, Harlow, UK). The trachea was intubated and the dogs were artificially ventilated (24 cycles min−1, tidal volume 13–15 ml kg−1, model 16/24, Palmer Bioscience, London, UK). Muscle and rectal temperatures were maintained close to 37°C and between 36°C and 38°C, respectively. The right brachial artery was cannulated and systemic blood pressure was recorded using a pressure transducer (PDCR 75, Druck Ltd, Barendrecht, The Netherlands) and an eight-channel chart recorder (Graphtec Linearcorder, Mk 8 WR3500, Nantwich, UK). The left brachial artery and vein were cannulated for collection of arterial blood samples for monitoring blood pH, PCO2 and PCO2 (280 Blood Gas System, Ciba-Corning, Medfield, MA, USA) and venous infusion of heparin, saline or DCA. Blood glucose and lactate were analysed as previously described (Timmons et al. 1998a).

All experiments were carried out in full accordance with the United Kingdom Home Office Animals (Scientific Procedures) Act of 1986, and approved by the AstraZeneca local ethics committee.

Both gracilis muscles were vascularly isolated, leaving only the main arterial and venous blood flows intact. The distal tendon of each muscle was attached to an isometric force transducer (Grass FTC 10, Quincy, Medfield, MA, USA). The popliteal artery was catheterized for recording gracilis muscle perfusion pressure. Heparin was infused (Multiparin, 1 U (kg body weight)−1 min−1) for the duration of the experiment. Each animal was infused with either 45 ml saline (n = 5) or 300 mg kg−1 DCA (n = 5, in 45 ml saline) over a period of 45 min. The femoral artery supplying each gracilis muscle was cannulated proximally and distally and attached sequentially to a perfusion pump (Minipuls 3, Gilson, Villiers Le Bel, France). The resting muscle blood flow was fixed by setting the perfusion pump at ∼6.5 ml min−1. We have previously established this as being equivalent to resting flow rate in the gracilis muscle (Timmons et al. 1996a). This flow rate was maintained for the duration of the experiment and equates to ∼20% of the normal flow observed when this muscle was stimulated to contract with the blood supply intact (Timmons et al. 1996a). The dose of DCA used in the present study completely transforms PDC to its active form, PDCa, in vivo (Timmons et al. 1997). At the end of the protocol, the animals were humanely killed using a lethal dose of anaesthetic, followed by saturated potassium chloride.

Muscle stimulation parameters

The resting length of the muscle was altered to obtain a standard resting tension. Sixty minutes after the onset of DCA or saline infusion, muscle contraction was induced via electrical stimulation of the obturator nerve (Grass S88 stimulator, Quincy, Medfield, MA, USA). Muscle stimulation was carried out using a tetanic stimulation protocol (75 ms on, once every second, at 10 V (pulse width of 0.2 ms, 30 Hz). This protocol, which results in complete muscle fibre recruitment, was applied for 6 min in total. The stimulation procedure was then repeated with the contralateral muscle.

Muscle sampling and analysis

Each group consisted of five animals and hence ten separate stimulation protocols. A resting muscle biopsy was taken, by superficial excision of the gracilis, using a scalpel, 1 h after the onset of infusion. After 1 min and 6 min of stimulation a thin piece of muscle was excised, during contraction, and rapidly frozen. The number of biopsies obtained at each time point was between 5 and 8 since for each animal, only one resting biopsy was taken for resting metabolite analysis (n = 5) and on a couple of occasions a 6 min biopsy was not successful.

All biopsy samples were then divided into two portions and stored under liquid nitrogen. Subsequently, one portion was freeze dried, dissected free from visible connective tissue and blood, powdered and extracted in 0.5 m perchloric acid containing 1 mm EDTA. Following centrifugation, the supernatant was neutralized with 2.2 m KHCO3 and used for spectrophotometric determination of ATP, PCr, creatine and lactate (Harris et al. 1974). The extract was also used for the determination of free carnitine and acetyl-carnitine by enzymatic assays using radioisotopic substrates, as previously described (Cederblad et al. 1990). Freeze-dried muscle powder was used for the determination of muscle glycogen (Harris et al. 1974). Muscle citrate, malate and fumarate were analysed enzymatically (Bergmeyer, 1974) using a fluorometer (Hitachi F-2000).

Statistics and calculations

All data are reported as mean ± s.e.m. Comparisons between treatments for both absolute concentrations and changes from rest were carried out using analysis of variance (ANOVA). When a significant F value was found a Tukey post hoc test was used to locate the differences. Significance was accepted at the 5% level.

Results

Contractile function, haemodynamics and plasma metabolites

Resting tension did not vary between the groups (range 0.6–0.7 kg (100 g muscle tissue)−1). Peak tension was 10.9 ± 0.4 kg (100 g)−1 in the control group and 10.2 ± 0.5 kg (100 g)−1 in the group pretreated with DCA. There was no difference in the extent of the fatigue demonstrated between treatments (control, 26.5 ± 1.5%, versus DCA, 22.7 ± 3.2%). Muscle force production appeared stabilized by 6 min of contraction consistent with earlier studies (Timmons et al. 1996a, 1997).

Blood flow did not differ between control and DCA treatments. As can be observed from the data in Table 1 DCA had no effect on muscle oxygen consumption, muscle lactate efflux or glucose uptake during the period in which arterio-venous samples were collected (minutes 4–5), although resting muscle glucose uptake tended to be higher following DCA. Arterial plasma glucose concentrations did not differ (control, 5.3 ± 0.2 mm; DCA, 5.8 ± 0.2 mm). Arterial free fatty acid concentrations did not differ (control, 0.17 ± 0.01 mm; DCA, 0.13 ± 0.02 mm). Arterial lactate concentration was reduced by DCA (control, 1.7 ± 0.2 mm; DCA, 0.47 ± 0.05 mm; P < 0.05). Arterial haemoglobin concentration was 14.0 ± 0.2 g dl−1 (control) and 15.5 ± 0.2 g dl−1 (DCA). Oxygen delivery did not significantly differ between the groups at any time point. Likewise, oxygen consumption did not differ between the groups. During contraction venous PCO2 measured during the 1st and 2nd minute (25 ± 2 mmHg) of contraction did not differ from the measurement made between minute 4 and the end of the contraction period (25.5 ± 2 mmHg). There were no differences in venous PCO2 between groups during contraction (DCA group, 26.2 ± 2.5 and 27.1 ± 2 mmHg, respectively).

Table 1.

Blood and plasma parameters

Control DCA
Rest Contraction Rest Contraction
Blood flow (ml min (100 g)−1) 36.1 ± 1.2 36.1 ± 1.2 34.6 ± 1.8 34.6 ± 1.8
Perfusion pressure (mmHg) 133.2 ± 4.6 59.8 ± 3.7 122.2 ± 8.0 64.6 ± 4.6
V̇CO2 (ml min−1(100 g)−1) 1.1 ± 0.2 4.1 ± 0.4 1.2 ± 0.2 4.1 ± 0.4
Glucose uptake (μmol min−1 (100 g)−1) 1.8 ± 1.6 1.2 ± 1.8 4.98 ± 2.27 1.03 ± 2.1
Lactate efflux (μmol min−1 (100 g)−1) −2.92 ± 1.6 16.4 ± 3.5 0.49 ± 0.7 14.1 ± 2.6
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Blood and plasma parameters in canine gracilis muscle at rest and during contraction (blood sample taken during minutes 4–6 of contraction period) with and without prior activation of pyruvate dehydrogenase complex (using dichloroacetate (DCA)) with a tetanic nerve stimulation protocol of 75 ms on and 925 ms off (15 V, 0.2 ms). V̇CO2, oxygen consumption.

Muscle metabolites

As presented previously, DCA pretreatment significantly increases the concentration of acetylcarnitine prior to contraction. There was a clear demarcation in the effects observed with DCA following 1 min of contraction compared with the metabolic profile observed at the end of the stimulation protocol. PCr degradation and lactate accumulation were reduced by ∼60% and ∼30%, respectively, after 1 min of the contraction following DCA pretreatment (Table 2). This was under conditions of identical force production and hence presumably ATP turnover. However, at the end of the stimulation period the concentration of PCr and lactate in the DCA group did not differ from the control group. By the end of the stimulation period the acetylcarnitine concentration did not differ between groups. The sum of the measured tricarboxylic acid (TCA) intermediates (citrate + malate + fumarate) did not differ between groups although numerically the concentration tended to be lower at all time points following DCA pretreatment.

Table 2.

Muscle metabolite concentrations

Control DCA
Rest (n = 5) 1 min (n = 8) 6 min (n = 8) Rest (n = 5) 1 min (n = 8) 6 min (n = 8)
ATP 25.7 ± 0.9 26.8 ± 1.7 23.2 ± 1.8 27.4 ± 1.6 24.8 ± 1.3 20.7 ± 1.7
PCr 77.2 ± 4.9 22.0 ± 4.9 10.8 ± 2.8 83.3 ± 3.7 48.9 ± 3.9* 12.6 ± 4.5
Creatine 56.5 ± 3.5 111.1 ± 5.6 125.5 ± 5.6 53.7 ± 2.4 85.1 ± 6.8* 126.6 ± 6.3
Lactate 6.2 ± 0.7 38.2 ± 4.5 69.7 ± 9.2 2.8 ± 4.7* 25.5 ± 4.6* 76.9 ± 13.7
Acetylcarnitine 2.2 ± 0.7 4.9 ± 0.5 11.8 ± 0.9 12.7 ± 0.6* 15.0 ± 1.8* 13.9 ± 1.2
Carnitine 23.4 ± 1.5 20.7 ± 1.9 12.2 ± 1.4 11.5 ± 1.9* 8.9 ± 2.1* 7.9 ± 0.9
Citrate (C) 0.7 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.4 ± 0.1
Malate (M) 0.6 ± 0.2 0.9 ± 0.2 1.7 ± 0.5 0.4 ± 0.2 0.6 ± 0.1 1.0 ± 0.2
Fumarate (F) 0.07 ± 0.01 0.07 ± 0.02 0.1 ± 0.02 0.06 ± 0.01 0.08 ± 0.02 0.08 ± 0.02
ΣC + M + F 1.3 ± 0.2 1.49 ± 0.3 2.26 ± 0.5 1.0 ± 0.2 1.1 ± 0.1 1.4 ± 0.2
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Muscle metabolite concentrations in canine gracilis muscle at rest, after 1 min and 6 min of contraction with and without prior activation of pyruvate dehydrogenase complex (using DCA) with a tetanic stimulation protocol of 75 ms on and 925 ms off (12.5 V, 30 Hz, 0.1 ms). Values are means ± s.e.m., expressed as mmol (kg dry mass)−1. Muscles were stimulated via the obturator nerve for 6 min.

*

Significantly different from the control group (P < 0.05).

Discussion

It is widely accepted that for mammalian skeletal muscle to maintain force production for any great length of time (> 20 s) then oxidative phosphorylation is the principal route of ATP regeneration (see Greenhaff & Timmons, 1998). As a consequence the interaction between SLP and oxygen delivery has been studied using a variety of models (Hogan et al. 1994, 1998a,b; Grassi et al. 1996, 1998a,b, 2000, 2002; Timmons et al. 1996a, 1998b; Haseler et al. 1998). Our initial studies using DCA as a tool, utilized submaximal stimulation, where fatigue was induced by reducing muscle blood flow. In subsequent publications by Howlett et al. (1999b), Bangsbo et al. (2002) and Savasi et al. (2002) exercise models that yielded close to, or greater than, maximal oxygen uptake were used. For example, Bangsbo et al. (2002) used a workload that yielded 110% of thigh peak O2 uptake, and found no evidence for the acetyl group deficit. Savasi et al. (2002) used an exercise protocol at ∼90% peak whole-body O2 uptake for 90 s, where the rate of PCr degradation was 2–4 times more rapid than in our studies (Timmons et al. 1997, 1998a; Roberts et al. 2002). Indeed, in the studies that have failed to find any evidence for the acetyl group deficit (Howlett et al. 1999b; Grassi et al. 2002; Bangsbo et al. 2002) the average rate of PCr degradation over the first minute of exercise exceeded 1 mmol (kg dry mass)−1 s−1 (Table 3). Interestingly, in both the studies by Howlett et al. (1999a) and Bangsbo et al. (2002) an exercise protocol was used that involved the subjects pausing during the exercise protocol for a muscle biopsy, before undergoing a ‘second’ rest-to-work transition. We have previously explained (Timmons et al. 1998b) that this removes the treatment effect of DCA, since both ‘prior exercise’ and DCA activate PDC. Our present study combined with our meta-analysis confirms that when the initial rate of PCr exceeds a particular threshold, priming oxidative metabolism may not be readily measurable. Exercise protocols which do not rely on a large mitochondrial contribution to ATP regeneration, are unlikely to be influenced by acetyl group availability. Equally, this threshold or ‘break point’ will vary with the training status of the subjects or the intrinsic mitochondrial capacity. Thus, careful consideration must be given to both the exercise model, subject characteristics and the study design, before any reliable conclusions can be reached.

Table 3.

Approximate rates of PCr utilization, calculated over ∼1st minute of contraction, depending on available data

References Exercise mode and sample size PCr utilization (mmol (kg dry wt)−1 s−1)
Parolin et al. (2000)* Human, cycling (n = 6), 11% hypoxia 0.18
Timmons et al. (1998a)* Human, single leg knee extension (n = 9) 0.3
Gibala & Saltin (1999)† Human, single leg knee extension (n = 6) 0.3
Rossiter et al. (2003)* Human, knee extension (n = 6) ∼0.4
Timmons et al. (1997)* Canine gracilis, 3 Hz twitch stimulation with blood flow limited to 25% of normal (n = 6–8) 0.6
Roberts et al. (2002)* Canine gracilis, 3 Hz twitch stimulation, blood flow limited to 25% of normal (n = 6–8) 0.6
Present data‡ 75 ms (30 Hz) tetanic stimulation (n = 8) 0.9
Howlett et al. (1999a)* Human, aerobic cycling (n = 9) 1.07
Savasi et al. (2002) Human, high intensity aerobic cycling (n = 9) 1.2
Grassi et al. (2002)§ Canine gastrocnemius, 200 ms tetanic (50 Hz) stimulation, 2 per 3 s for 4 min (n = 6) ∼1.2
Bangsbo et al. (2002) Human, voluntary single leg knee extension (n = 4–6) ∼1.4
Howlett et al. (1999b) Maximal isokinetic cycling (n = 10) 5.4
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*

Demonstrated a reduced contribution from ‘anaerobic’ ATP regeneration or PCr sparing with dichloroacetate (DCA).

†

Gibala & Saltin demonstrated a 27% reduction in both PCr utilization and ‘substrate level phosphorylation’ but with 6 subjects this did not reach statistical significance.

‡

Reduced PCr degradation at 1 min by 35% but did not prevent PCr depletion at 6 min ([PCr] < 15 mmol (kg dry mass)−1).

§

Tetanic stimulation for 150 ms gives ∼1.2 mmol kg−1 s−1 PCr utilization (J.A. Timmons, unpublished observation). The original data demonstrated ∼0.5 mmol kg−1 s−1 but this PCr degradation rate appears to be exceptionally low for such a stimulation protocol or the reported rate of oxygen consumption.

Regulation of in vivo PDC activation

As with previous studies, DCA was the tool used in the present study to increase muscle acetyl group availability. DCA is a weak (IC50 > 200 μm) yet selective inhibitor of the kinase pyruvate dehydroxygenase kinase (PDHK) that inactivates PDC. The selectivity profile of DCA for PDHK (over unrelated kinases) is high since DCA is not an ATP analogue. Furthermore, the chronic effect of the di-chloro component of DCA structure has questionable relevance for the short-term administration to skeletal muscle or muscle bioenergetics. It is expected, however, that PDC activation, using DCA or any other PDC activator, will alter systemic pyruvate concentrations and thus influence other metabolites, such as the Krebs cycle intermediates. Indeed, we studied such an effect in the present study and no connection with DCA's effect on PCr sparing was found (Table 2). Such changes are not, however, additional pharmacological actions of DCA and hence when critiquing the usefulness of this experimental tool (Rossiter et al. 2003) greater care should be taken to fully appreciate what reflects the pharmacology of DCA and what simply reflects the biochemical consequences of PDC activation.

The physiological mechanisms responsible for PDC activation in human skeletal muscle are not well characterized. For example, maximal exercise results in a greater PDC activation than submaximal exercise (Constantin-Teodosiu et al. 1991; Howlett et al. 1999a). This probably reflects the greater muscle fibre recruitment, rather than a hypothetical (Spriet & Heigenhauser, 2002) increase in [Ca2+] within already contracting myocytes, as fibre recruitment rather than rate modulation of motor units is utilized by humans during exercise involving large muscle groups. Exemplification of this lack of necessity for a change in [Ca2+] can also be found in the observation that DCA fully activates PDC in resting skeletal muscle (Timmons et al. 1996a). Overall, it is not clear why PDC activation is suboptimal under physiological conditions, especially since full PDC activation does not cause acute hypoglycaemia in humans (Timmons et al. 1998b). It is plausible that at some point in the evolution of metabolic control, excessive PDC activation compromised glucose availability and hence a complex system of four inactivating kinases emerged to provide a survival benefit.

Metabolic control and muscle fatigue development

Hogan et al. (1994) concluded that the initial rate of fatigue development in the contracting gastrocnemius muscle directly related to reduced oxygen availability. This might appear at odds with our earlier study, where the development of muscle fatigue appeared largely due to the metabolic consequences of the acetyl group deficit (Timmons et al. 1997). However, studies by Hogan and colleagues altered oxygen delivery only after the muscle had reached a steady-state force production (Hogan et al. 1994, 1998a,b; Haseler et al. 1998). Thus, under these two conditions, different determinants of muscle fatigue are being studied. We feel that our model is more representative of every day physical activity patterns (Girard et al. 2001) but both models have provided important insight into metabolic control during muscle contraction. If during submaximal exercise, a significant proportion of the OD can be avoided, then this may have performance-enhancing implications for muscle function under low blood flow conditions. DCA is not an ideal long-term pharmacological agent as, for example, it demonstrates a poor pharmacokinetic profile. As newer generation PDC activators emerge (Mayers et al. 2003), with optimized pharmacological properties, then the time will come when the clinical utility of PDC activation for treating exercise intolerance in cardiovascular disease patients can be reasonably addressed.

Recently, Grassi et al. (2002) were able to demonstrate that DCA improved muscle function independently of altered metabolism. It occurred to us that aspects of metabolic control may differ during the rest-to-work transition period if tetanic stimulation was used (rather than twitch contractions). We used a tetanic stimulation protocol that achieved a steady-state oxygen consumption comparable with our previous twitch models (e.g. 3.4–4.2 ml min (100 g wet tissue)−1). Intramuscular lactate concentration and muscle lactate efflux (e.g. 15.3 versus 14.1 μmol min (100 g wet tissue)−1) were also similar to our earlier canine studies. However, despite a comparable steady-state oxidative flux, [PCr] was approximately half of that found during twitch contraction (Timmons et al. 1996b, 1997) indicating that metabolic control of respiration differed. In a previous study, steady-state [PCr] was achieved within ∼1 min of contraction, following DCA pretreatment (Timmons et al. 1997). In the present study DCA attenuated PCr utilization during the first minute of contraction; however, [PCr] continued to decline until the 6th minute. It is therefore apparent that during tetanic stimulation, the time taken to reach a steady-state [PCr] must be substantially longer than during a twitch protocol (Timmons et al. 1997) despite similar steady-state oxygen consumption. Nevertheless, it would seem clear that DCA pretreatment will not reduce ischaemic muscle fatigue when the final metabolic profile of the muscle is unaltered.

Determinants of SLP – the oxygen deficit versus the acetyl group deficit

Until recently it was believed that the major factor which limited the rate of increase in oxidative phosphorylation during the rest-to-work transition period was oxygen availability (see Hughson et al. 2001). During submaximal contraction, i.e. below peak V̇CO2, neither peripheral oxygen diffusion (Grassi et al. 1998a) nor oxygen delivery kinetics (Grassi et al. 1998b) limits oxygen consumption kinetics during the rest-to-work period. Unfortunately, none of these studies measured muscle [PCr] which would have directly demonstrated that muscle SLP was unaltered by enhanced oxygen delivery. Specific mention must also be given to the study by Linnarsson et al. (1974) since it is the most cited evidence that oxygen delivery limits mitochondrial ATP regeneration at the onset of exercise. In this study, hyperbaric hyperoxia did not significantly alter PCr utilization when compared with normoxia, despite extensive citation stating otherwise (Macdonald et al. 1997; Grassi et al. 1998b; Tschakovsky & Hughson, 1999; Parolin et al. 2000; Savasi et al. 2002). Furthermore, it is not possible to determine if the study by Linnarsson et al. (1974) used an appropriate randomized experimental design, where clearly such factors would impact on the lactate production (e.g. glycogen status or experimental duration) and PCr metabolism. It is also difficult to understand why the magnitude of the effect observed on muscle lactate was identical (in opposite directions of course) for both hyperbaric hyperoxia and hypobaric hypoxia – yet clearly hypobaric hypoxia would have a greater effect on blood oxygen content due to the characteristics of the oxyhaemoglobin curve. In more recent studies, hyperoxia reduced the ‘respiratory’ OD during the 3rd to 6th minute of the transition period (Macdonald et al. 1997) but had no effect on intramuscular PCr utilization (Savasi et al. 2002). This suggests that respiratory gas exchange during non-steady-state conditions may not reliably reflect the metabolic status of the contracting muscle tissue.

In contrast to these submaximal studies it was demonstrated that when a canine gastrocnemius preparation was stimulated at peak V̇CO2, a more rapid adjustment of blood flow during the rest-to-work transition period reduced the OD (calculated from gas exchange) and muscle fatigue (Grassi et al. 2000). However, there are some problems with the interpretation of this observation. Firstly, in the study by Grassi et al. (2000) the ‘high blood flow’ experimental period always followed the ‘normal blood flow’ period and was accompanied by intra-arterial adenosine infusion. It is also important to appreciate that a minimum recovery period between two successive contraction periods, that leaves acetyl group availability (or the rate of PDC activation) unaffected by the prior exercise, has not been established. It therefore still remains to be seen if oxygen delivery kinetics regulate adjustment during rest to peak oxidative flux conditions.

To date, little discussion has been given to the potential source of the ‘extra’ oxygen consumed to off-set the reduction in SLP following DCA administration. Assumptions that it can be measured at the level of respiratory gases and/or across the exercising limbs may be sanguine (Grassi et al. 2002; Rossiter et al. 2003; Koppo et al. 2004). The molar equivalent of molecular oxygen required to ‘compensate’ for the reduced SLP will be modest (due to the P : O ratio being estimated as high as ∼6). Calculations using the molar volume of oxygen (22.41 ml mmol−1) need also to consider that oxygen is partially dissolved and not exposed to standard temperature−pressure conditions. It is therefore unclear how to accurately translate a molar equivalent of oxygen into an accurate volume of oxygen and conclude that it is measurable under such conditions. The study by Rossiter et al. (2003) also highlights that DCA alters the characteristics of the V̇O2–workload relationship creating further doubt over the applicability of standard analysis methodologies. If PDC activation increases carbohydrate oxidation, then up to ∼12% less oxygen utilization is required for the same ATP regeneration when using lipid as the fuel. A recent study highlighted several problems associated with the OD calculation, derived from respiratory gas values (Özyener et al. 2003), further indicating that it is not a useful approach to studying the acetyl group deficit. One further recent study (Koppo et al. 2004) failed to design the study to ensure that PDC activation differed between control and DCA-treated conditions (they could not measure PDC status but plasma lactate concentrations during exercise supported the contention that no substantial ‘metabolic’ intervention took place) highlighting the importance of a rational experimental design when investigating the OD.

A recent study demonstrated that the decline in intramuscular PCO2 in a skinned muscle fibre preparation was accelerated during the rest-to-work period following DCA administration (Howlett & Hogan, 2003). Using the Xenopus single fibre preparation, devoid of myoglobin and therefore excluding any complication with this oxygen ‘store’, they were able to provide evidence for more rapid oxygen consumption following DCA administration. In humans, myoglobin represents an oxygen reservoir which is not necessarily in proportional equilibrium with venous saturation during submaximal exercise (Richardson et al. 2001). Given the modest amount of oxygen required to explain the reduction in SLP it is also possible that a small contribution from muscle oxygen stores further limits the possibility of detecting a difference using current in vivo measurement techniques (Grassi et al. 2002; Rossiter et al. 2003).

Conclusions

The present results support the idea that when the initial PCr degradation rate is ∼1 mmol (kg dry mass)−1 s−1 or less, acetyl group availability measurably impacts on SLP at the onset of exercise, when an early muscle biopsy is obtained. The present data suggest that if the stimulation intensity exceeds the maximum rate of increase in mitochondrial ATP regeneration, prior PDC activation will not prevent [PCr] tending towards zero, nor will it enhance ischaemic skeletal muscle function. Future studies aimed at measuring the net increase in oxygen consumption following PDC activation by DCA must appreciate that the measurement of oxygen mass balance across the muscle (or at the mouth) may not be sufficient. Overall, we would argue that careful examination of the published literature (Table 3), coupled with the present data set, demonstrates that acetyl group deficit is a widely applicable limitation to oxidative phosphorylation during the transition from rest to exercise. This also suggests that the acetyl group deficit may be an important physiological determinant of exercise capacity in health and disease.

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