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Journal of Physiology (2002), 544.2, pp. 591-602
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2002.021097
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ABSTRACT |
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Considerable debate surrounds the identity of the precise cellular site(s) of inertia that limit the contribution of mitochondrial ATP resynthesis towards a step increase in workload at the onset of muscular contraction. By detailing the relationship between canine gracilis muscle energy metabolism and contractile function during constant-flow ischaemia, in the absence (control) and presence of pyruvate dehydrogenase complex activation by dichloroacetate, the present study examined whether there is a period at the onset of contraction when acetyl-coenzyme A (acetyl-CoA) availability limits mitochondrial ATP resynthesis, i.e. whether a limitation in mitochondrial acetyl group provision exists. Secondly, assuming it does exist, we also aimed to identify the mechanism by which dichloroacetate overcomes this 'acetyl group deficit'. No increase in pyruvate dehydrogenase complex activation or acetyl group availability occurred during the first 20 s of contraction in the control condition, with strong trends for both acetyl-CoA and acetylcarnitine to actually decline (indicating the existence of an acetyl group deficit). Dichloroacetate increased resting pyruvate dehydrogenase complex activation, acetyl-CoA and acetylcarnitine by ~20-fold (P < 0.01), ~3-fold (P < 0.01) and ~4-fold (P < 0.01), respectively, and overcame the acetyl group deficit at the onset of contraction. As a consequence, the reliance upon non-oxidative ATP resynthesis was reduced by ~40 % (P < 0.01) and tension development was increased by ~20 % (P < 0.05) following 5 min of contraction. The present study has demonstrated, for the first time, the existence of an acetyl group deficit at the onset of contraction and has confirmed the metabolic and functional benefits to be gained from overcoming this inertia.
(Resubmitted 25 March 2002; accepted after revision 7 August 2002; first published online 30 August 2002)
Corresponding author P. A. Roberts: E Floor, School of Biomedical Sciences, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK. Email: p.roberts{at}nottingham.ac.uk
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INTRODUCTION |
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The immediate increase in energy demand at the onset of muscular contraction must be matched by a rapid increase in ATP resynthesis to enable skeletal muscle contraction to continue for longer than a few seconds. The readjustment of mitochondrial ATP resynthesis to meet this demand is not immediate and follows an approximately exponential time course (for review see Tschakovsky & Hughson, 1999). During this period, the shortfall in ATP supply is met by ATP resynthesis from non-oxygen-dependent routes (i.e. ATP and phosphocreatine (PCr) hydrolysis and glycolysis), frequently termed substrate level phosphorylation (SLP; Lundgren et al. 1988). The activation of ATP production from SLP at the onset of exercise has classically been attributed to a lag in muscle blood flow and thereby oxygen delivery to contracting muscle (Margaria et al. 1933; Saltin, 1990), hence the genesis of the term the 'oxygen deficit'. However, more recently it has been proposed that oxygen availability may not be the sole determinant of the 'oxygen deficit' (Sahlin et al. 1988; Grassi et al. 1998), such that its true physiological genesis remains open to debate (for review see Tschakovsky & Hughson, 1999; Grassi, 2001; Hughson et al. 2001).
Recent work within our laboratory has investigated the functional role of the pyruvate dehydrogenase complex (PDC) during submaximal skeletal muscle contraction (Timmons et al. 1996a, b, 1997, 1998a, b, c). Of focal interest have been the metabolic events occurring under conditions of ischaemic exercise, where blood flow to the contracting muscle was maintained at its resting rate, thus preventing any increase in oxygen delivery at the onset of contraction (Timmons et al. 1996a). In a recent series of studies, dichloroacetate (DCA) was used to test the hypothesis that mitochondrial substrate availability, in the form of acetyl-coenzyme A (acetyl-CoA), rather than muscle blood flow alone, dictates the onset of mitochondrial ATP resynthesis (Timmons et al. 1996b, 1997, 1998a, b, c) and therefore the reliance upon ATP resynthesis from SLP at the onset of exercise. During 6 min of ischaemic contraction, a reduction in ATP resynthesis from SLP of ~45 % and an improvement in contractile function of ~30 % were observed from control muscle following DCA administration (Timmons et al. 1997). From this work we concluded that there must be a period, early in the rest-to-work transition in the control state, when acetyl-CoA supply does not meet the energy demand of contraction.
DCA is a specific pharmacological activator of the PDC that acts by allosterically inhibiting the kinase responsible for phosphorylating and thus deactivating the enzyme complex (Whitehouse et al. 1974; Pratt & Roche, 1979). The PDC catalyses the physiologically irreversible reaction that commits carbohydrates to their oxidative fate inside the mitochondria through the conversion of the glycolytic product pyruvate into mitochondrial acetyl-CoA. The resulting acetyl groups can subsequently be utilised by the tricarboxylic acid (TCA) cycle, or can alternatively be buffered towards carnitine when acetyl-CoA availability exceeds its rate of TCA cycle utilisation (Childress et al. 1966). The buffering of acetyl groups in this way has been said to maintain both a viable pool of free coenzyme A (CoASH) for the PDC reaction to continue and create a readily available reservoir of substrate, in the form of acetylcarnitine, for the TCA cycle (Lysiak et al. 1988). Pretreatment with DCA greatly enhances acetyl group availability to the TCA cycle at rest compared to control by near maximally activating the PDC and acetylating the carnitine pool (Timmons et al. 1996b, 1997, 1998b). During moderate-to-intense skeletal muscle contraction in untreated muscle, acetylcarnitine has been shown to accumulate almost linearly with time (Childress et al. 1966; Harris et al. 1987; Howlett et al. 1999), with this accumulation being greater under conditions of reduced blood flow (Timmons et al. 1996a, b, 1997), inferring that acetyl-CoA production is always in excess of TCA cycle requirements. However, studies to date have failed to look at the metabolic events occurring within the first seconds of contraction, or indeed at any time point during contraction prior to PDC activation.
In the present study we tested the hypothesis that early in the rest-to-work transition period there is a lag in mitochondrial ATP resynthesis, and that this lag must be in part due to inadequate supply of acetyl-CoA to meet the increased demand of the TCA cycle. Secondly, we assessed whether DCA conferred its beneficial biochemical and functional effects by overcoming this period of metabolic inertia at the onset of contraction. By taking multiple muscle biopsy samples during the first 60 s of contraction, we examined the time course of PDC activation, acetyl group availability and substrate utilisation during 5 min of electrically induced contraction in the ischaemic canine gracilis muscle (Timmons et al. 1996a).
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METHODS |
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Animals
All in vivo procedures were performed in full accordance with United Kingdom legislation and Home Office approval. A canine gracilis muscle perfusion model (Timmons et al. 1996a) was used because the gracilis muscle has a similar fibre type composition to human skeletal muscle (Maxwell et al. 1977) and the model enables repeated and rapid biopsy sampling to be performed at the onset of contraction. Following an overnight fast, 12 female beagle dogs (Animal Breeding Unit, AstraZeneca Pharmaceuticals, Alderley Park, Cheshire, UK; body mass 9.3 ± 0.3 kg) were premedicated with morphine sulphate (morphine, 10 mg, I.M.) 30 min prior to the induction and maintenance of anaesthesia with sodium pentobarbitone (pentobarbitone, Sagatal, Rhône Merieux, Harlow, UK). Anaesthesia was introduced as a bolus of pentobarbitone (45.9 ± 0.1 mg (kg body mass)-1) followed by a continuous infusion throughout each experiment (0.10 ± 0.01 mg (kg body mass)-1 min-1, I.V.). Once adequate anaesthesia was established, the trachea was intubated and the animals were artificially ventilated with room air (24 cycles min-1, tidal volume 13-15 ml (kg body mass)-1; Model 16/24, Palmer Bioscience, London, UK).
Surgical procedures
The right carotid artery was cannulated and mean arterial blood pressure recorded using a pressure transducer (PDCR 75, Druck, Barendrecht, The Netherlands) and an eight-channel chart recorder (Graphtec Linearcorder, mk8 WR3500, Nantwich, UK). The right brachial artery and antecubital vein were cannulated for the collection of arterial blood samples for the monitoring of blood pH, PCO2, PO2 (280 Blood Gas Systems, Ciba-Corning, Medfield, MA, USA) and for the I.V. infusion of heparin, physiologically buffered saline (PBS) and DCA. Both the left and right gracilis muscles were surgically and vascularly isolated leaving only the arterial and venous blood supply to and from them intact. The distal tendon of each gracilis muscle was severed and attached to an isometric force transducer (Grass FTC 10, Quincy, Medfield, MA, USA). The resting tension of the muscle was altered to ~300 g, in an attempt to re-establish the in situ appearance and fibre alignment of the gracilis muscle prior to the severing of the distal tendon. The popliteal artery was catheterised for the recording of gracilis muscle perfusion pressure. Following surgery and prior to the connection of the perfusion circuit, an infusion of heparin (Multiparin, 1 U (kg body mass)-1 min-1, I.V.) was commenced; this infusion was continued for the duration of the experiment. The femoral arteries supplying each gracilis muscle were cannulated proximally and distally and were attached sequentially to a perfusion pump (Miniplus 3, Gilson, Villiers-Le-Bel, France). The resting blood flow to each gracilis muscle was then fixed by setting the flow rate of the perfusion pump to ~6.5 ml min-1. This has been shown previously to equate to resting blood flow in the gracilis muscle within this animal model (Timmons et al. 1996a). Blood flow was maintained constant for the duration of the experiment and equated to ~25 % of the normal contraction-induced flow to the gracilis when stimulated using the present parameters (Timmons et al. 1996a). The fixing of blood flow at its resting level prevented exercise hyperaemia, and thereby any increase in oxygen delivery to the muscle during subsequent contraction. Each animal was infused with either 30 ml PBS (CON, n = 6) or 300 mg (kg body mass)-1 DCA (n = 6) in 30 ml PBS over a period of 30 min. The dose of DCA used in the present study maximally transforms PDC to its active 'a' form (PDCa) in vivo (Timmons et al. 1996b, 1997) and has a half life of ~20 h in dogs (Lukas et al. 1980; Stacpoole, 1989).
Experimental protocol
Thirty minutes after the onset of CON or DCA infusion, the gracilis muscle was stimulated to contract, via electrical stimulation of the obturator nerve (Grass S88 stimulator, Quincy). Square-wave impulses of 0.1 ms duration, 3 Hz frequency, and 6 V submaximal voltage were applied for 5 min, resulting in complete muscle fibre recruitment. This stimulation protocol produces a workload of ~80 % maximal oxygen uptake (
O2,max) within the gracilis muscle with normal blood flow intact (Timmons et al. 1996a). Mean arterial blood pressure, hind-limb perfusion pressure and the isometric tension profile were recorded during electrical stimulation. The duration of stimulation was based on a previous study using this animal model where a steady-state force output was observed following 6 min of contraction (Timmons et al. 1997). Once completed, the stimulation procedure was repeated in the animal's contralateral gracilis muscle. Following the completion of both gracilis experiments (left and right muscles) each animal was humanely killed, whilst still under anaesthesia, by the infusion of pentobarbitone and the I.V. infusion of saturated potassium chloride.
Muscle sampling and analyses
Immediately prior to contraction, a resting muscle biopsy sample was taken by superficial excision of tissue from the distal end of the left gracilis muscle, using a scalpel blade and forceps. On all occasions the left gracilis muscle was the first muscle to be investigated, but muscle-sampling times varied (protocols 1 and 2) and are depicted in Fig. 1. Varying the tissue sampling times between limbs precluded any potential bias between the left and right gracilis muscle experiments. In protocol 1, biopsy samples were taken at rest, and after 20, 60 and 300 s of contraction. In protocol 2, biopsy samples were taken at rest, and after 10, 40 and 180 s of contraction. In protocol 2, stimulation was maintained for a further 2 min after the final biopsy sampling to generate comparable isometric force data between the two protocols. All excised muscle biopsy samples were frozen immediately (i.e. within 2 s) by submersion in liquid nitrogen. A scalpel blade and forceps were used here in preference to the Bergström needle technique (Bergström, 1975) due to the quicker sampling they allow, which is obviously essential for short time course studies.
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Figure 1. Experimental overview In protocol 1, biopsy samples were taken at rest, and after 20, 60 and 300 s of contraction. In protocol 2, biopsy samples were taken at rest, and after 10, 40 and 180 s of contraction. In protocol 2, stimulation was maintained for a further 2 min after the final biopsy sampling to generate comparable isometric force data between the two protocols. PBS, physiologically buffered saline; DCA, dichloroacetate. | ||
All biopsy samples were divided into two equal portions under liquid nitrogen. Subsequently, one portion was freeze-dried, dissected free from visible blood and connective tissue and powdered. Following extraction in 0.5 M perchloric acid containing 1 mM EDTA, the supernatant was neutralised with 2.2 M KHCO3 and used for the spectrophotometric determination of ATP, PCr, creatine and lactate (Harris et al. 1974). The extract was also used for the determination of free carnitine, acetylcarnitine, free CoASH and acetyl-CoA using radioisotopic substrates, as described previously (Cederblad et al. 1990). Freeze-dried muscle powder was also used for the determination of muscle glycogen levels (Harris et al. 1974). The remaining portion of frozen, wet muscle was used to assess the activation status of the PDC (Constantin-Teodosiu et al. 1991b).
Calculations and statistics
All data are reported as means ± S.E.M. Comparisons between treatments for both absolute concentrations and changes from rest were carried out using two-way analysis of variance (ANOVA) with repeated measures. When a significant F-value was obtained (P < 0.05), a 'least significant difference' post hoc test was used to locate any differences (SPSS Base 8.0). Significance was accepted at the 5 % level, unless stated otherwise in the text. With the exception of lactate, the content of all muscle metabolites was adjusted to the mean total creatine concentration within each individual animal. By this means it was possible to compensate for any admixture of connective tissue and other non-muscular elements within each muscle biopsy sample (Gilbert et al. 1971; Harris, 1981).
The amount of ATP produced from SLP was calculated from changes in the concentration of muscle PCr, lactate and ATP using the following equation (eqn (1), Spriet et al. 1987; the symbol 
represents the difference in concentration between two consecutive sampling time points for the stated muscle metabolite):
SLP = PCr + (1.5 
lactate) + (2 ATP). (1)
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RESULTS |
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Blood gas status and haemodynamics
There was no effect of DCA on arterial blood PO2 (CON = 107.1 ± 2.3 mmHg vs. DCA = 107.5 ± 2.2 mmHg), PCO2 (CON = 42.5 ± 0.7 mmHg vs. DCA = 43.0 ± 0.7 mmHg) and pH (CON = 7.36 ± 0.01 vs. DCA = 7.35 ± 0.01) throughout the experiments. There was no effect of DCA on mean arterial blood pressure (CON = 114.1 ± 2.8 mmHg vs. DCA = 110.1 ± 1.7 mmHg), heart rate (CON = 154 ± 3 beats min-1 vs. DCA = 148 ± 2 beats min-1) or hind-limb perfusion pressure (CON = 61.4 ± 3.1 mmHg vs. DCA = 58.9 ± 4.3 mmHg).
Muscle contractile function
Resting gracilis tension did not differ between the two groups (CON = 936 ± 158 vs. DCA = 913 ± 98 g tension (100 g wet muscle)-1). Peak isometric tension was observed after 40 s of contraction within each animal. No difference in peak isometric tension existed between groups (CON = 4539 ± 361 vs. DCA = 3830 ± 266 g tension (100 g wet muscle)-1). Following 5 min of contraction, isometric tension had declined by 57 % from peak (2051 ± 410 g tension (100 g wet muscle)-1) in CON with the corresponding value in the DCA group being 40 % at 5 min (2324 ± 386 g tension (100 g wet muscle)-1; P < 0.05, Fig. 2).
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Figure 2. Effect of pretreatment with DCA on isometric tension development in the canine gracilis muscle Isometric tension development (percentage of peak) during 5 min of ischaemic muscle contraction following pretreatment with saline (CON, | ||
PDC activation
There was no significant increase in PDC activation during the first 20 s of contraction in CON (Fig. 3). However, PDC was activated between the 20 and 60 s time points (CON at 20 s = 0.53 ± 0.07 vs. CON 60 s = 1.59 ± 0.30 mmol acetyl-CoA min-1 (kg wet muscle)-1, P < 0.01), with this degree of activation remaining for the duration of contraction.
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Figure 3. Effect of pretreatment with DCA on levels of the active form of the pyruvate dehydrogenase complex (PDCa) during ischaemic contractions Levels of PDCa at rest and during 5 min of ischaemic contraction following pretreatment with saline (CON, | ||
DCA infusion resulted in a > 20-fold difference in the amount of PDC existing in its active form (PDCa) at rest compared with CON (CON = 0.13 ± 0.05 vs. DCA = 2.86 ± 0.51 mmol acetyl-CoA min-1 (kg wet muscle)-1, P < 0.01). This difference between groups remained throughout the first minute of contraction (Fig. 3). However, no difference in PDCa was seen at subsequent time points, as PDCa increased and declined from its resting value in the CON and DCA trials, respectively (Fig. 3).
Carnitine, CoASH and their acetylated forms
There was no increase in acetylcarnitine concentration from rest during the first minute of contraction in CON (Fig. 4A). In fact, during the first 20 s of stimulation there was a strong trend for acetylcarnitine to decline (CON rest = 5.9 ± 0.9 vs. CON 20 s = 4.2 ± 0.6 mmol (kg dry muscle)-1, P = 0.08). Acetylcarnitine then increased at the 40 s time point (CON 20 s = 4.2 ± 0.6 vs. CON 40 s = 6.6 ± 0.6 mmol (kg dry muscle)-1, P < 0.05), after which there was an almost linear increase in its concentration (r 2 = 0.96, P < 0.05, Fig. 4A).
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Figure 4.Effect of pretreatment with DCA on concentrations of acetylcarnitine and free carnitine in the canine gracilis muscle during ischaemic contractions Acetylcarnitine (A) and free carnitine (B) concentration at rest and during 5 min of ischaemic contraction following pretreatment with saline (CON, | ||
DCA infusion substantially elevated acetylcarnitine concentration at rest from that seen in CON (CON = 5.9 ± 0.9 vs. DCA = 21.6 ± 0.8 mmol (kg dry muscle)-1, P < 0.01), with this degree of acetylation of the carnitine pool being maintained and remaining higher than CON at all time points during contraction following DCA (P < 0.01, Fig. 4A).
Changes in acetylcarnitine concentration were mirrored by changes in the concentration of carnitine (Fig. 4B), with no differences existing in the concentration of the total carnitine (the sum of acetylcarnitine and free carnitine) pool between groups (CON = 24.0 ± 0.4 vs. DCA = 24.6 ± 0.2 mmol (kg dry muscle)-1).
There was no increase in acetyl-CoA from its resting concentration at any time point during contraction in CON (Fig. 5A). However, during the first 10 s of contraction there was a strong trend for acetyl-CoA to decline from rest (CON rest = 12.2 ± 1.7 vs. CON 10 s = 5.7 ± 1.1 µmol (kg dry muscle)-1, P = 0.06), before reverting to its resting concentration at 1 min (Fig. 5A).
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Figure 5. Effect of pretreatment with DCA on concentrations of acetyl-CoA and free CoASH in the gracilis muscle during ischaemic contractions Acetyl-CoA (A) and free CoASH (B) concentrations at rest and during 5 min of ischaemic contraction following pretreatment with saline (CON, | ||
Acetyl-CoA was significantly elevated above CON at rest following DCA administration (CON = 12.2 ± 1.7 vs. DCA = 30.5 ± 2.7 µmol (kg dry muscle)-1, P < 0.01). The concentration of acetyl-CoA in DCA remained significantly higher than CON throughout the first 3 min of contraction (P < 0.05, Fig. 5A). Then, at 3 min, it started falling from its resting concentration (DCA rest = 30.5 ± 2.7 vs. DCA 180 s = 18.4 ± 1.9 µmol (kg dry muscle)-1, P < 0.01), such that no differences in the concentration of acetyl-CoA existed between groups at the end of contraction (Fig. 5A).
Changes in acetyl-CoA were mirrored by changes in the concentration of free CoASH (Fig. 5B), with no difference existing in the concentration of total CoASH (sum of acetyl-CoA and free CoASH) pool existing between groups.
Muscle metabolites at rest and during contraction
ATP concentrations were maintained during the majority of the period of contraction in both groups. However, after 5 min of contraction, ATP concentration in the DCA-treated group was significantly higher than that in the CON group, which had fallen from its resting concentration (P < 0.05, Table 1).
Resting PCr concentration was similar between groups (Table 1). After 20 s of contraction, PCr had fallen from its resting concentration in both groups (P < 0.01, Table 1). Furthermore, the rate of PCr hydrolysis was similar in both groups over the initial 40 s of contraction. However, from 1 min onwards, PCr concentrations were better maintained following DCA pretreatment, and at 5 min PCr was 56 % of its resting concentration, compared to 27 % in the CON group (P < 0.01, Table 1). Changes in the concentration of PCr mirrored changes in creatine concentration (Table 1), with no difference in the concentration of the total creatine (the sum of PCr and creatine) pool existing within and between groups (CON = 124.6 ± 0.2 vs. DCA = 124.2 ± 0.1 mmol (kg dry muscle)-1.
Resting muscle glycogen concentration was no different between groups (Table 1). During the first 40 s of contraction the rate of glycogenolysis was similar in both groups (Table 1). However, from 60 s onwards the rate of glycogen degradation in the DCA group was significantly lower than that of CON (P < 0.05, Table 1). Overall, by the end of contraction muscle glycogen degradation was twice as high in CON as in the DCA-treated group (CON = -74 ± 18 vs. DCA = -31 ± 6 mmol (kg dry muscle)-1, P < 0.01).
Muscle lactate concentration was lower than CON at rest and throughout contraction following DCA administration (P < 0.05, Table 1). Lactate concentration increased from rest to 40 s of contraction in both groups (P < 0.05, Table 1). Following 5 min of contraction, lactate accumulation from rest was 43 mmol (kg dry muscle)-1 in CON, with the corresponding value in the DCA group being ~45 % lower at 23 mmol (kg dry muscle)-1 (P < 0.01, Table 1).
The calculated contribution from SLP to ATP resynthesis over the entire 5 min of contraction was ~40 % lower following DCA pretreatment than in the CON group (CON = 122 ± 9 vs. DCA = 73 ± 6 mmol ATP (kg dry muscle)-1, P < 0.01). During the first minute of contraction, the calculated rate of ATP production from SLP was significantly lower following DCA (CON = 60 ± 2 vs. DCA = 43 ± 5 mmol ATP min-1 (kg dry muscle)-1, P < 0.05, Fig. 6), and this was also the case at 1-3 min of contraction (CON = 20 ± 4 vs. DCA = 9 ± 2 mmol ATP min-1 (kg dry muscle)-1, P < 0.05). However, no difference in SLP-derived ATP production was observed between groups during the final 2 min of contraction (CON = 13 ± 2 vs. DCA = 8 ± 2 mmol ATP min-1 (kg dry muscle)-1, Fig. 6).
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Figure 6. Effect of pretreatment with DCA on the rate of ATP production from substrate-level phosphorylation (SLP) at various times during ischaemic muscle contraction Rate of ATP production from SLP between rest and 1 min, 1 min and 3 min, and 3 min and 5 min of ischaemic skeletal muscle contraction following pretreatment with saline (CON, | ||
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DISCUSSION |
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The present study examined in detail the time course of PDC activation, acetyl group availability and substrate utilisation during 5 min of submaximal ischaemic contraction in canine skeletal muscle. A significant finding of the present work was the lag in acetyl group provision (in the form of acetyl-CoA and acetylcarnitine) during the first seconds of contraction, which resulted from, and was mirrored by, a lag in PDC activation. This is the first study to demonstrate clearly the existence of this 'acetyl group deficit' in contracting skeletal muscle. Although the present findings were obtained in profoundly ischaemic canine skeletal muscle, previous work by our group using this experimental model (Timmons et al. 1996a, b, 1997) has yielded results that have subsequently been validated by numerous research groups within ischaemic (Timmons et al. 1998b) and non-ischaemic human skeletal muscle (Timmons et al. 1998a; Howlett et al. 1999; Parolin et al. 1999, 2000) at a variety of exercise workloads.
In accordance with previous studies, the infusion of DCA prior to the onset of contraction resulted in near-maximal activation of the PDC to PDCa and acetylation of the free CoASH and carnitine pools to levels reported following maximal exercise (Harris et al. 1987; Putman et al. 1995; Timmons et al. 1997). The increased availability of substrate for the TCA cycle was associated with a reduced reliance upon SLP and an improvement in contractile function during the subsequent rest-to-work transition period, thereby implying that a greater contribution from mitochondrial-derived ATP resynthesis must have occurred to meet the ATP demand of contraction. It is important to recognise that a second significant finding of the present work was that no difference in ATP resynthesis from SLP existed between groups during the final 2 min of contraction. Based upon our data, we suggest that this was due to the concentration of acetyl-CoA falling from its elevated resting concentration during this period following DCA administration. Collectively, these findings point to the availability of acetyl-CoA as being a key limitation towards mitochondrial ATP delivery, at least at the onset of contraction at submaximal workloads.
The acetyl group deficit
Studies that have investigated acetyl group accumulation during moderate-to-intense skeletal muscle contraction have reported a near-linear increase in acetylcarnitine with time (Childress et al. 1966; Harris et al. 1987; Timmons et al. 1997; Howlett et al. 1999), implying that PDC activation, and hence acetyl-CoA availability, are always in excess of the demands of the TCA cycle during contraction. However, studies to date have failed to investigate the metabolic events occurring within the first seconds of contraction, or at any contraction time point prior to PDC activation. Therefore, at present it is unknown whether PDC activation, and thereby acetyl-CoA availability to the TCA cycle, is limiting towards mitochondrial ATP resynthesis at the onset of skeletal muscle contraction. One recent study (Bangsbo et al. 2002), however, has reported an increase in PDC activation following just 5 s of high-intensity exercise in human skeletal muscle. This observation could be taken to indicate that acetyl-CoA availability may not be limiting towards TCA cycle demand at the onset of contraction, at least at near-maximal workloads (Bangsbo et al. 2002). However, interpretation of the data presented by Bangsbo et al. (2002) should be viewed with some caution. The authors reported PDC activation status at rest to be ~50 % lower in both saline- and DCA-treated subjects when compared to similar studies reported in the literature (Timmons et al. 1998c; Howlett et al. 1999). Furthermore, given that muscle acetyl-CoA and acetylcarnitine values were not presented by Bangsbo et al. (2002), it is not possible to determine whether acetyl group availability was markedly increased as a result of DCA administration. A second interpretation could be that the rate of PDC activation at the onset of maximal-intensity exercise is sufficiently rapid that no effect of DCA could be expected (Bangsbo et al. 2002), a point we shall return to later in the text.
In the present study, a lag in the accumulation of acetylcarnitine (Fig. 4A) and acetyl-CoA (Fig. 5A) was seen within the first seconds of contraction in the CON group, whilst PDC was inactive (Fig. 3). Indeed, there was a trend for both acetyl-CoA (P = 0.06) and acetylcarnitine (P = 0.08) to decline during the first 20 s of contraction. Acetylcarnitine (Fig. 4A) and acetyl-CoA (Fig. 5A) tended to increase from 20 to 40 s of contraction and this was paralleled by further transformation of the PDC to PDCa (Fig. 3). It would appear, therefore, that activation and flux through the PDC limits substrate delivery towards the TCA cycle during the first 20 s of contraction. However, subsequent to this, the rate of acetyl group formation was greater than its rate of entry into the TCA cycle as PDC activation increased markedly from rest (Fig. 3). This is exemplified by the almost linear rise in the concentration of acetylcarnitine from 40 s onwards, reflecting the sequestering of excess acetyl groups by carnitine, via the carnitine acetyltransferase equilibrium reaction (Fig. 4A). In a recent study by Howlett et al. (1999), no lag in acetylcarnitine accumulation was seen in human skeletal muscle during 30 s of contraction at 65 % of O2,max, which is in contrast to the findings presented here. One explanation for this discrepancy is that the work intensity employed in the Howlett et al. (1999) study was lower than that employed in the present experiment. Thus, the activation of PDC and demand for acetyl-CoA during exercise would have been lower than in the present study. In other words, there would have been better matching between rates of acetyl-CoA supply and demand during the rest-to-work transition, thereby reducing the magnitude of the acetyl group deficit.
The idea that exercise intensity may influence the magnitude of any acetyl group deficit at the onset of exercise is supported by recent papers by Grassi et al. (2002) and Savasi et al. (2002) in canine and human skeletal muscle, respectively. Here the authors demonstrated, contrary to several earlier papers (Timmons et al. 1996b, 1997, 1998b; Howlett et al. 1999), that DCA administration and the resultant stockpiling of acetyl groups had no effect on oxygen consumption kinetics (Grassi et al. 2002), PCr degradation or lactate accumulation (Grassi et al. 2002; Savasi et al. 2002) at the onset of relatively high-intensity contraction. Whilst it is difficult to reconcile the biochemical data of Grassi and co-workers (2002) with work published at a similar workload in the literature (Timmons et al. 1996b, 1997; Howlett et al. 1999), the paper of Savasi et al. (2002), taken together with the findings of Bangsbo et al. (2002), would suggest that there is an upper workload intensity above which acetyl group delivery is no longer limiting towards TCA cycle demand, due to the rapid activation of PDC at this exercise intensity (Howlett et al. 1998). Therefore, at workloads above this intensity, pretreatment with DCA is unlikely to be associated with a reduction in ATP resynthesis from SLP and an improvement in contractile function. Similarly, based on the findings of Howlett et al. (1999) outlined above and the more recent findings of Evans et al. (2001), we would propose that there will also be a lower exercise intensity below which acetyl group delivery via the PDC reaction is not limiting towards TCA cycle demands (i.e. where PDC flux and/or fat-mediated acetyl group delivery is sufficient to match the energy demands of contraction). Presumably this exercise intensity equates to one where no increase or decline in acetylcarnitine concentration would be seen upon the initiation of exercise. In this respect, the study by Evans et al. (2001) investigated changes in acetyl group availability during 2 min of moderate-intensity (65 % O2,max) exercise in human volunteers following near maximal acetylation of the muscle carnitine and CoASH pools by sodium acetate infusion. In line with our proposal, no changes in acetyl-CoA or acetylcarnitine concentrations were observed following 30 s of exercise in either saline- (control) or sodium acetate-treated subjects, despite the PDC activation that occurred in both groups. It would appear, therefore, that an acetyl group deficit will exist over a predictable range of exercise intensities (between ~65 and 90 % O2,max), above and below which increasing resting acetyl group availability will be ineffective at reducing SLP and improving contractile function.
In the present study, DCA pretreatment resulted in 4- and 3-fold increases in acetylcarnitine and acetyl-CoA concentrations, respectively (Fig. 4A and Fig. 5A), which was coupled to a greater than 20-fold increase in the activation status of the PDC (Fig. 3). Over the course of the subsequent 5 min of submaximal ischaemic contraction, DCA pretreatment overcame the acetyl group deficit, reduced SLP by ~40 % and improved contractile function by ~20 % compared with CON (Fig. 2). This reduction in ATP resynthesis from SLP following DCA occurred predominantly within the first 3 min of contraction (Fig. 6). Indeed, no differences in SLP existed between groups from 3 min and onwards (Fig. 6). Given that work output was the same between treatments during the initial 3 min of contraction (Fig. 2), it is of interest to calculate whether the deficit in energy delivery as a result of the reduction in SLP following DCA can be accounted for.
The resting concentration of acetyl-CoA in CON (~10 µmol (kg dry muscle)-1, Fig. 5A) was of the same magnitude as that found in previous studies (Constantin-Teodosiu et al. 1991a; Howlett et al. 1999). DCA increased resting acetyl-CoA levels by nearly three-fold, these levels remaining significantly higher than CON throughout the first 3 min of contraction (Fig. 5A). According to the in vitro-assessed Michaelis-Menton constant (Km) of citrate synthase for acetyl-CoA (up to 500 µmol l-1; Newsholme & Leech, 1983), the increased availability of acetyl-CoA would promote its entry into the TCA cycle via citrate synthase at the onset of contraction compared to CON. However, when we consider that the decline in SLP following DCA administration during the first minute of contraction was ~15 mmol (kg dry muscle)-1 of ATP equivalents (Fig. 6), it becomes apparent that the ~20 µmol (kg dry muscle)-1 increase in resting acetyl-CoA availability following DCA could not have solely overcome this energy deficit, accounting for just 1.2 % of the decline in SLP. DCA infusion near-maximally acetylated the muscle carnitine pool at rest (Fig. 4A) to a concentration similar to that seen during maximal exercise and to the same extent as previous DCA studies by our group (Timmons et al. 1996b, 1997). Using the assumption that 1 mmol of acetylcarnitine will produce 12 mmol of ATP equivalents through the TCA cycle, a net provision of 1.3 mmol acetylcarnitine (kg dry muscle)-1 would be required to account for the ~15 mmol ATP equivalents (kg dry muscle)-1 difference in SLP between treatments during the first minute of contraction. As the concentration of acetylcarnitine fell by 1.5 mmol (kg dry muscle)-1 during the first 20 s of contraction in CON (Fig. 4A), the results appear to indicate that the increased concentration of acetylcarnitine following DCA, with a modest contribution from acetyl-CoA, overcame the acetyl group deficit and resulted in the reduction in SLP during the first minute of contraction in DCA.
Following the first minute of contraction, PDC activation had increased substantially and acetyl group availability was no longer limited in the CON group, as reflected by the near-linear accumulation of acetylcarnitine (Fig. 4A, r 2 = 0.96) and the reversion of acetyl-CoA to its resting concentration (Fig. 5A). However, despite similarities in PDCa between the CON and DCA groups following 1 min of contraction, an ~20 mmol (kg dry muscle)-1 reduction in ATP resynthesis from SLP was observed between 1 and 3 min of contraction following DCA administration (CON = 19.8 ± 4.2 vs. DCA = 8.5 ± 1.9 mmol ATP min-1 (kg dry muscle)-1, P < 0.05, Fig. 6). During this period, no differences in contractile function existed between treatments, so we can assume a similar rate of ATP turnover in both groups (Fig. 2). Therefore, in order to meet the ATP demand of contraction, this ~20 mmol (kg dry muscle)-1 reduction in SLP following DCA must have been accompanied by a quantitatively similar increase in mitochondrial ATP resynthesis, and thereby increased acetyl-CoA entry into the TCA cycle. Between 1 and 3 min of contraction, both acetylcarnitine and acetyl-CoA had a tendency to decline in the DCA-treated group, with the concentrations of each metabolite increasing in the CON group (Fig. 4A and Fig. 5A). This reduction in acetylcarnitine ( 1.6 mmol (kg dry muscle)-1) and acetyl-CoA ( 8.9 µmol (kg dry muscle)-1) at 1 and 3 min of contraction following DCA equates to ~1.6 mmol (kg dry muscle)-1 of acetyl groups being sequestered by the TCA cycle during this period. As before, using the assumption that a 1 mmol (kg dry muscle)-1 decline in acetyl group availability equates to ~12 mmol (kg dry muscle)-1 of ATP equivalents, we can see this closely matches (19 mmol (kg dry muscle)-1 ATP equivalents) the ~20 mmol (kg dry muscle)-1 reduction in SLP between 1 and 3 min of contraction following DCA treatment (Fig. 6).
The increasing concentration of acetylcarnitine between 1 and 3 min in the CON group, during a period when the TCA cycle is capable of a greater flux, is surprising (Fig. 4A). We believe this observation indicates that the carnitine acetyltransferase reaction competes more favourably than the TCA cycle for acetyl groups at this time. This view is supported by evidence from in vitro studies showing that when the concentration of TCA cycle intermediates is low, as observed at the onset of contraction (Gibala et al. 1998), carnitine reduces the rate of acetyl-CoA oxidation by the TCA cycle (Childress et al. 1966; Sacktor & Wromser-Shavit, 1966; Gibala et al. 1998). It has been suggested that this is because carnitine acetyl transferase (Km 40 µM) competes more successfully for acetyl-CoA than citrate synthase (Km up to 500 µM; Newsholme & Leech, 1983) when the concentration of oxaloacetate is low (Gibala et al. 1998). In the present study, DCA near-maximally acetylated the carnitine pool at rest, such that there was no further increase in acetylcarnitine over the 5 min of contraction (Fig. 4A). This raises the possibility that the normally high concentration of free carnitine (~18 mmol (kg dry muscle)-1) at the onset of contraction in untreated muscle may limit the supply of acetyl-CoA to the TCA cycle, thereby limiting mitochondrial ATP delivery. Thus, the effects of DCA on mitochondrial ATP production and SLP could feasibly be the direct consequence of improving metabolic efficiency by reducing free-carnitine availability, rather than the 'stockpiling' of acetyl groups that is currently favoured (Timmons et al. 1996b, 1997, 1998a; Howlett et al. 1999). Indeed, the importance of the free carnitine pool in controlling the rate of carbohydrate vs. fat oxidation cannot be discounted. Carnitine serves to transport fats from their sites of storage in the cytosol to their place of oxidation inside the mitochondria (Fritz & Yue, 1963; Fritz & Marquis, 1965). Given the near-maximal acetylation of the cellular carnitine pool at rest following DCA, it is likely that very little carnitine-mediated lipid translocation into the mitochondria occurs during subsequent contraction. Therefore, DCA could be viewed as preferentially switching the muscle towards the oxidation of carbohydrates with the added benefit of an increased ATP yield per unit of oxygen.
During the final 2 min of contraction, no differences in PDCa (Fig. 3), acetyl group availability (Fig. 4A and Fig. 5A), SLP (Fig. 6) or glycogen utilisation (Table 1) were observed between treatments. Indeed, the only significant difference in metabolism between groups over this period was a fall in ATP concentration in CON (Table 1, P < 0.05). Despite the biochemical similarities between groups following 3 min of stimulation, tension development was ~20 % greater at 5 min following DCA (Fig. 2). Presumably the reduction in inorganic phosphate, lactate and proton accumulation during the initial 3 min of contraction following DCA administration, together with the preservation of ATP concentrations throughout contraction, reduced the inhibitory effects of metabolite accumulation on muscle cross-bridge cycling (Fitts, 1994) resulting in the observed improvement in contractile function.
In conclusion, the present study has documented, for the first time, the existence of metabolic inertia, in the form of acetyl-CoA availability, at the immediate onset of skeletal muscle contraction. The increased provision of acetyl groups at rest following DCA administration overcame this acetyl group deficit and reduced the requirement for SLP throughout the first 3 min of contraction, under conditions of identical force output and oxygen delivery. The envisaged faster onset of mitochondrial ATP resynthesis throughout the rest-to-work transition following DCA resulted in the ~20 % improvement in contractile function at 5 min. These results indicate that the supply of acetyl groups to the TCA cycle is limited at the onset of contraction, resulting in the activation of SLP and premature fatigue development.
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REFERENCES |
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Acknowledgements
The present work was supported by the Medical Research Council (UK).
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; n = 6) or DCA (
; n = 6). Results are expressed as means ± S.E.M. No difference in peak tension (which always occurred at 40 s) existed between groups. * Significantly different from the corresponding CON value (P < 0.05).
; n = 6) or DCA (
; n = 6). Results are expressed as means ± S.E.M. with units of mmol ATP equivalents min-1 (kg dry muscle)-1. *Significantly different from the corresponding CON value (P < 0.05).