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Journal of Physiology (2002), 538.1, pp. 195-207
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.012984
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ABSTRACT |
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The aim of the present study was to determine whether the activation of the pyruvate dehydrogenase complex (PDC) by dichloroacetate (DCA) is associated with faster O2 uptake (O2) on-kinetics.
) was measured continuously in the popliteal vein; arterial and popliteal vein O2 contents were measured at rest and at 5-7 s intervals during the transition. Muscle
(Received 12 July 2001; accepted after revision 25 September 2001)
Corresponding author B. Grassi: Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Milano, LITA-Via Fratelli Cervi 93, I-20090 Segrate (MI), Italy. Email: bruno.grassi{at}unimi.it
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INTRODUCTION |
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Recent studies have demonstrated that, in the isolated dog gastrocnemius in situ, convective and diffusive O2 delivery do not represent the main limiting factors for oxygen uptake (O2) on-kinetics (Grassi et al. 1998a,b, 2000), thereby suggesting that such factors mainly reside in an inertia of skeletal muscle oxidative metabolism. The metabolic pathways of oxidative metabolism are complex, and several rate-limiting steps have been hypothesized. One of these steps could be represented by the pyruvate dehydrogenase complex (PDC), an enzyme localized within the inner mitochondrial membrane, which catalyses the decarboxylation of pyruvate to acetyl-CoA; this can subsequently be utilized in the tricarboxylic acid (TCA) cycle. More specifically, it has been hypothesized that an inherent lag in PDC activation at the onset of submaximal contraction could limit substrate delivery to the TCA cycle during this period, thereby limiting mitochondrial oxidative flux and accelerating non-oxidative energy provision (Timmons et al. 1996, 1997, 1998a,b; Howlett et al. 1999a).
PDC has two regulatory enzymes, a phosphatase and a kinase, which control an activation-inactivation cycle, and is under the influence of several allosteric effectors (Wieland, 1983). Dichloroacetate (DCA) activates PDC by inhibiting the kinase responsible for phosphorylating, and thus inactivating, the enzyme complex (Stacpoole, 1989). Different groups have recently demonstrated, both in isolated ischaemic dog gracilis muscle (Timmons et al. 1996, 1997) and in humans (Timmons et al. 1998a,b; Howlett et al. 1999a), that activation of PDC and stockpiling of acetyl groups at rest, by the administration of DCA, results in less phosphocreatine (PCr) degradation and lactate accumulation during rest-to-contraction or rest-to-submaximal exercise transitions. Such an attenuation in anaerobic energy provision could be attributed to a faster adjustment of skeletal muscle oxidative phosphorylation at the onset of contractions, as a consequence of reducing the proposed inertia in substrate supply to the TCA cycle (Timmons et al. 1996, 1997, 1998a,b; Howlett et al. 1999a). However, no study to date has assessed O2 on-kinetics following DCA administration. The aim of the present study, therefore, was to evaluate whether, in the isolated dog gastrocnemius preparation in situ, the activation of PDC by DCA results in faster O2 on-kinetics during the transition from rest to electrically stimulated submaximal contractions.
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METHODS |
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The study was conducted with approval of the Institutional Animal Care and Use Committee of Auburn University, Auburn, AL, USA, where the experiments were performed.
Six adult crossbred dogs of either sex (body mass 17.5 ± 2.0 kg, mean ± S.E.M.) were anaesthetized with pentobarbitone sodium (30 mg kg-1), with maintenance doses given as required to extinguish any reflex indicating a state other than a deep plane of anaesthesia appropriate for surgery. The dogs were intubated with an endotracheal tube and ventilated with a respirator (Harvard, model 613). The rectal temperature was maintained at ~37 °C with a heating pad and a heating lamp. After surgical preparation the animals were treated with heparin (2000 u kg-1). Ventilation was maintained at a level that produced normal arterial partial pressures of oxygen (PO2) and carbon dioxide (PCO2).
Surgical preparation
The gastrocnemius-plantaris-flexor digitorum superficialis muscle complex (for convenience referred to as 'gastrocnemius') was isolated as described previously (Stainsby & Welch, 1966). Briefly, a medial incision was made through the skin of the left hindlimb from mid-thigh to the ankle. The insertion tendons of the sartorius, gracilis, semitendinosus and semimembranosus muscles, which overlie the gastrocnemius, were cut with a hot blade to allow these muscles to be folded back, thus exposing the gastrocnemius. To isolate the venous outflow from the gastrocnemius, all the vessels draining into the popliteal vein, except those from the gastrocnemius, were ligated. The popliteal vein was cannulated and flow () was measured with a 3 mm cannulating-type electromagnetic flowmeter (Narco Biosystems, model RT-500). Venous outflow was returned to the animal via a reservoir attached to a cannula in the left jugular vein. The arterial circulation to the gastrocnemius was isolated by ligating all vessels from the femoral and popliteal arteries that did not enter the gastrocnemius. The right femoral artery was also cannulated and connected to a pressure transducer (Narco Biosystems, model RP-1500) in order to monitor blood pressure and to sample arterial blood.
A portion of the calcaneus, with the two tendons from the gastrocnemius attached, was cut away at the heel and clamped around a metal rod for connection to an isometric myograph via a load cell (Interface SM-250) and a universal joint coupler. The universal joint allowed the muscle to pull always directly in line with the load cell and thus prevented the application of torque to the load cell. The other end of the muscle was left attached to its origin; both the femur and the tibia were fixed to the base of the myograph by bone nails. An adjustable support rod was placed parallel to the muscle between the tibial bone nail and the arm of the myograph to minimize flexing of the myograph.
The sciatic nerve was exposed and isolated near the gastrocnemius. The distal stump of the nerve, ~1.5-3.0 cm in length, was pulled through a small epoxy electrode containing two wire loops for stimulation. The muscle was covered with saline-soaked gauze and a thin plastic sheet to prevent drying and cooling.
Experimental design
To evoke muscle contractions, the nerve was stimulated by supramaximal square pulses of 4-6 V amplitude and 0.2 ms duration (Grass S48 stimulator), isolated from ground by a stimulus isolator (Grass SIU8TB). Before each experiment, the muscle was set at optimal length (L0) by progressively lengthening the muscle as it was stimulated at a rate of 0.2 Hz, until a peak in developed tension (total tension minus resting tension) was obtained. For the experiments, isometric tetanic contractions were triggered by stimulation with trains of stimuli (4-6 V, 200 ms duration, 50 Hz frequency) at a rate of two contractions every 3 s for a 4 min period. Prior studies (Kelley et al. 1996; Ameredes et al. 1998) have shown that this stimulation pattern should elicit ~60-70 % of peak metabolic rate for this muscle. Each isometric tetanic contraction lasted 200 ms and was separated from the following by 1.3 s, during which the muscle was relaxing or quiescent.
For each muscle, the experiment consisted of two contraction periods of 4 min duration, preceded by a resting baseline. The contraction periods were separated by at least 45 min of rest. The resting baseline was chosen (vs. a baseline of lower metabolic intensity) in order to increase the gain of the metabolic transition, thus improving the signal-to-noise ratio of the investigated variables. The investigated metabolic transition was therefore a transition from rest to submaximal contractions. Two conditions were compared: (1) control (C; 45 ml of saline, given over ~45 min by infusion into the jugular reservoir before the trial); and (2) DCA infusion (300 mg (kg body mass)-1 of DCA [sodium dichloroacetate, Aldrich No. 34,779-5] in 45 ml saline, given over ~45 min by infusion into the jugular reservoir before the trial). This dosage of DCA should have completely transformed PDC into its active form (Stacpoole, 1989). Since DCA has a half-life in plasma between 0.5 and 2 h (Stacpoole, 1989), the order of treatments could not be randomized and C was always performed before DCA.
At the end of the experiment the dogs were killed with an overdose of pentobarbitone. The gastrocnemius was excised and weighed, and the weight was used to normalize variables per unit of muscle mass as appropriate.
Measurements
Output from the pressure transducer was recorded on a strip chart recorder while outputs from the load cell and flowmeter were fed through strain gauge and transducer couplers, respectively, into a computerized (PowerComputing PowerBase 240 Macintosh clone) data acquisition system (GW Instruments Inc., SuperScope II and instruNet Model 100B D-A input-output system). The load cell reaches 90 % of full response within 1 ms while the flowmeter has a pulsatile cut-off frequency of 30 Hz; both signals were sampled at a rate of 100 Hz by the computerized data acquisition system. The load cell was calibrated with known weights before each experiment. The flowmeter was calibrated with a graduated cylinder and clock during and after each experiment. Muscle blood flow () was averaged over every five contractions and then fitted to a smooth curve to allow precise calculation of values corresponding to the time of venous samples, as described below. Vascular resistance was calculated as muscle perfusion pressure (BPm) divided by .
Samples of arterial blood entering the muscle and of venous blood from the popliteal vein were drawn anaerobically in heparinized syringes. Since the arterial values are known to vary only slightly during this type of experiment, arterial samples were taken at rest, before the contractions and immediately after the contraction periods. A polyethylene tube (0.8 mm i.d., 37 cm long, 0.25 ml total volume including luer hub) was threaded into the popliteal vein cannula to the point where the vein exited the gastrocnemius. This allowed collection of venous blood immediately draining from the muscle. Venous samples were taken at rest (~10 s before the onset of contractions), every 5-7 s during the first 75 s of contractions, and every 30-45 s thereafter until the end of the contraction period. The precise time of each venous sample was recorded.
Blood samples were immediately stored in ice and analysed within 30 min of collection. Both arterial and venous blood samples were analysed at 37 °C for PO2, PCO2 and pH by a blood gas, pH analyser (Instrumentation Laboratories, IL 1304), and for haemoglobin concentration ([Hb]) and percentage saturation of Hb (Sa,O2, %) with a CO-Oximeter (Instrumentation Laboratories, IL 282), set for dog blood. These instruments were calibrated before and during each experiment. Blood O2 concentration was calculated by also taking into account the dissolved O2.
O2 of the gastrocnemius was calculated by the Fick principle as O2 = 
C(a-v)O2, where C(a-v)O2 is the difference in O2 concentration between arterial blood (Ca,O2) and venous blood (Cv,O2). O2 was calculated at discrete time intervals corresponding to the timing of the blood samples.
During both trials muscle biopsies were obtained by superficial excision of muscle pieces with a scalpel, at rest and during the last 15 s of the contraction period. Biopsy samples were immediately frozen in liquid nitrogen. Subsequently, samples were freeze-dried, dissected from visible connective tissue and blood, powdered, and extracted in 0.5 M perchloric acid containing 1 mM EDTA. After centrifugation, the supernatant was neutralized with 2.2 M KHCO3 and used for spectrophotometric determination of adenosine triphosphate (ATP), phosphocreatine (PCr), creatine and lactate concentrations (Harris et al. 1974). The extract was also used for the determination of free carnitine and acetylcarnitine concentrations by enzymatic assays with radioisotopic substrates, as described previously (Cederblad et al. 1990). Muscle metabolite concentrations are expressed in millimoles per kilogram of dry mass (DM). Substrate level phosphorylation (total 'anaerobic' ATP yield) was estimated (in mmol (kg DM)-1 of ATP) as: 
[PCr] + (1.5 [lactate]) + (2 [ATP]), in which indicates the difference between concentrations at rest and during contractions (for ATP and PCr) or between concentrations during contractions and at rest (for lactate) (Greenhaff et al. 1994).
Analysis of kinetics
In order to evaluate mathematically and compare the on-kinetics of physiological variables in the two experimental conditions, values obtained for each experiment during the contraction period (as well as the value corresponding to 'time 0', i.e. the resting value) were fitted by a function of the type:
y(t) = yBas + A(1 - e-(t - TD)/ ),
| (1) |
and parameter values (TD, ) were determined that yielded the lowest sum of squared residuals. In the above equation, yBas indicates the baseline value; A the amplitude between yBas and steady-state value during contractions, TD the time delay and the time constant of the function. To check for the presence of a 'slow component' (Gaesser & Poole, 1996) of the on-kinetics, data were also fitted by a function of the type:
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y(t) = yBas + Ap(1 - e-(t - TDp)/p) + As(1 - e-(t - TDs)/s).
| (2) |
In eqn (2) Ap indicates the amplitude between yBas and steady-state value during the primary component (yBas + Ap); As the amplitude between (yBas + Ap) and the steady-state value during the slow component; TDp and TDs the time delays and p and s the time constants of the functions during the two components of the response (see e.g. Grassi et al. 2000). The presence of a slow component was confirmed when the sum of squared residuals was lower for eqn (2) than for eqn (1).
To compare the overall O2 on-kinetics in the two experimental conditions, eqn (1) or eqn (2) was solved to calculate the time necessary to reach 50 % (t50%, corresponding to the half-time of the overall response) and 63 % (t63%, corresponding to the 'mean response time'; Hughson, 1990) of the differences between resting baselines and steady-state values obtained towards the end of contractions. The resulting times correspond to the points at which the O2 response passed through 50 and 63 % of the difference between the resting baseline and the steady-state value towards the end of contractions (Grassi et al. 1996, 1998a,b, 2000).
Statistical analysis
Values are expressed as means ± S.E.M. To determine the statistical significance of differences between two means, Student's paired t test (2-tailed) was performed. To determine the statistical significance of differences among more than two means, a repeated-measures analysis of variance was performed. Tukey's post hoc test was utilized to discriminate where significant differences occurred. The level of significance was set at P < 0.05. Data fitting by exponential or polynomial functions was performed by the squared residuals method. All statistical tests were conducted using a software package (InStat, version 3.0 for Windows, GraphPad Software).
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RESULTS |
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The mass of the gastrocnemius muscles was 74 ± 9 g.
Resting values of the main variables pertinent to O2 transport and utilization, acid-base status and haemodynamics are shown in Table 1 for the two experimental conditions. No significant differences were observed between C and DCA, indicating that the 'baseline' condition, as indicated by these variables, was the same, although the order of treatments could not be randomized (see Methods).
Steady-state values during contractions for the main variables pertinent to O2 transport and utilization, acid-base status and haemodynamics are shown in Table 2 for the two experimental conditions. No significant differences were observed between C and DCA, with the exception of pH, which was slightly higher in DCA.
Force production at the beginning of the contraction period (mean values calculated over 5 contractions) was 4.81 ± 0.49 N (100 g)-1 in C and 4.95 ± 0.49 N (100 g)-1 in DCA (n.s.). Mean (± S.E.M.) values of the fatigue index (FI, i.e. average values of force determined every 10 s/initial force) are shown in Fig. 1. FI was higher (i.e. the muscles showed less fatigue) in DCA than in C. The difference was statistically significant from 80 s of contractions onward. At the end of the contraction period FI was 0.73 ± 0.03 in C and 0.81 ± 0.05 in DCA (P = 0.02).
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Figure 1. Fatigue index Mean (± S.E.M.) values of fatigue index (average values of force determined every 10 s/initial force) as a function of time of contraction in the two experimental conditions, control (C) and dichloroacetate (DCA) administration. * Statistically significant difference. | ||
Muscle metabolite concentrations at rest and at the end of the contraction period for the two experimental conditions are shown in Table 3. There were no differences in [ATP] and [PCr] at rest between the two conditions. Whereas [ATP] was substantially unchanged (vs. rest) during contractions in both experimental conditions, [PCr] decreased ~30 % less during contractions in DCA than in C (although the difference did not reach statistical significance, P = 0.14). Resting [lactate] was significantly lower in DCA than in C. Since increases in [lactate] during contractions were not significantly different in the two conditions, [lactate] at the end of contractions showed a tendency to be higher in C than in DCA. The calculated substrate level phosphorylation amounted to 38.0 ± 7.0 mmol ATP (kg DM)-1 in C, and to 31.5 ± 2.9 mmol ATP (kg DM)-1 in DCA (n.s.). DCA induced 9-fold higher resting [acetylcarnitine] (P < 0.0001 vs. C) and a significantly lower [free carnitine] (P < 0.001 vs. C), indicating a significant PDC activation. At the end of contractions [acetylcarnitine] was still higher in DCA, despite a small decline (-1.9 ± 2.5 mmol (kg DM)-1) compared to resting values; this decline was significantly different (P < 0.01) from the accumulation observed in C (5.7 ± 2.0 mmol (kg DM)-1).
Mean (± S.E.M.) values of , C(a-v)O2 and O2 are shown for the two experimental conditions in Fig. 2. The on-kinetics of the three variables appear remarkably similar in the two conditions, although steady-state values during contractions were slightly higher (and C(a-v)O2 values were slightly lower) in C than in DCA.
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Figure 2. Mean (± S.E.M.) values of muscle blood flow, arterio-venous O2 concentration difference and muscle O2 uptake The vertical dashed lines indicate the onset of contraction. | ||
O2 values for individual animals are shown in Fig. 3, together with the curves obtained by fitting eqn (1) or (2). In one of the dogs (i.e. dog 6) eqn (2) provided a better fit of the data than eqn (1) (both in C and in DCA), indicating the presence of a slow component. In the same dog a slow component was also observed for .
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Figure 3. Individual values of muscle O2 uptake Points at time 0 indicate the resting baselines. The best-fitting curves (see Methods) are also shown. | ||
Mean (± S.E.M.) values of t50% and t63% for the O2 on-kinetics are shown in Fig. 4. For both parameters no significant differences were observed between DCA and C.
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Figure 4. Parameters of muscle O2 uptake on-kinetics Mean (± S.E.M.) values of calculated times (see Methods) to reach 50 % (t50%) and 63 % (t63%) of differences between resting baselines and steady-state values obtained towards the end of contraction, for the | ||
Kinetics parameters (time constant [], time delay [TD] + TD and amplitude of the response) for , C(a-v)O2 and O2 in the two experimental conditions are presented in Table 4. For dog 6, in which a slow component was observed for and O2, , TD, + TD and amplitude data for these variables refer to the 'primary' component of the kinetics (see Methods). The time constant for O2 on-kinetics was slightly but significantly higher (indicating a slower kinetics) in DCA than in C, whereas for + TD no significant difference between the two conditions was observed. In both conditions increased immediately at the onset of contractions (as indicated by the slightly negative TD), following an exponential time course characterized by a of ~15-20 s, whereas for C(a-v)O2 a very rapid exponential increase, characterized by a of ~8-11 s, was preceded by a ~6 s TD. Also for O2 an exponential increase with a of ~15-19 s was preceded by a TD of ~4-6 s. The sum of TD and was not different between and O2, whereas values for C(a-v)O2 were significantly lower than those for O2.
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DISCUSSION |
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Is PDC part of the metabolic inertia of oxidative metabolism at the onset of contractions?
The main result of the present study was that, in the isolated dog gastrocnemius preparation in situ, during transitions from rest to electrically stimulated contractions corresponding to ~60-70 % of the muscle peak O2, administration of DCA resulted in a significant activation of PDC as evidenced by a marked stockpiling of acetylcarnitine at rest, and was associated with less muscle fatigue, but it did not significantly affect 'anaerobic' energy provision and O2 on-kinetics. Thus, in the present experimental model, PDC activation status does not seem to be responsible for the 'metabolic inertia' in the adjustment of oxidative phosphorylation to sudden increases in the demand for ATP regeneration.
Previous studies conducted using the same preparation as in the present study showed that during transitions from rest to electrically stimulated contractions corresponding to ~60 % of the muscle peak O2, neither convective nor diffusive O2 transport to muscle fibres was a limiting factor for the O2 on-kinetics (Grassi et al. 1998a,b). Further experiments, conducted for transitions from rest to contractions corresponding to the muscle peak O2 demonstrated that, in those conditions, the well-known exponential adjustment of convective O2 delivery could account for only a relatively minor portion (~) of the O2 deficit (Grassi et al. 2000). These results were mainly in agreement with the hypothesis (see e.g. Cerretelli et al. 1980; Whipp & Mahler, 1980) that the main limiting factor for O2 on-kinetics lies in an inertia of oxidative metabolism to adjust to sudden increases in the needs for ATP regeneration. Experiments conducted in ischaemic dog muscles (Timmons et al. 1996, 1997) and in humans during transitions from rest to submaximal exercise (Timmons et al. 1998a,b; Howlett et al. 1999a) pointed to PDC as one of the sites where this metabolic inertia may occur. These authors demonstrated, following activation of PDC and stockpiling of acetyl groups at rest by DCA, less muscle PCr degradation, less lactate accumulation and less fatigue during submaximal contraction. They hypothesized that the attenuation of anaerobic energy production during the on-transition could be explained by a faster adjustment of oxidative phosphorylation. In none of these studies, however, was O2 on-kinetics determined.
PDC is localized within the inner mitochondrial membrane and catalyses the decarboxylation of pyruvate to acetyl-CoA. This reaction is essentially irreversible, committing pyruvate to entry into the TCA cycle and to oxidation. PDC has two regulatory enzymes, a phosphatase and a kinase, which regulate an activation-inactivation cycle, and is under the influence of several allosteric factors. Activation status is regulated via changes in calcium and pyruvate availability and by the ratios of ATP : ADP, acetyl-CoA : CoA-SH and NAD+ : NADH (see e.g. Wieland, 1983). The complexity of the enzymatic regulation confirms the key role of PDC in oxidative metabolism of carbohydrates. DCA activates PDC by inhibiting the kinase responsible for phosphorylating, and thus inactivating, the enzyme complex (Stacpoole, 1989). Infusion of DCA also results in the acetylation of most of the intramuscular carnitine pool at rest (Timmons et al. 1996, 1997, 1998a,b). The latter provides a readily available pool of oxidizable substrates via the acetylcarnitine transferase reaction, which transforms acetylcarnitine to acetyl-CoA; this can then enter the TCA cycle (Lysiak et al. 1988). In the present study we did not specifically measure PDC activity. However, the finding of a 9-fold higher resting [acetylcarnitine] with DCA (vs. C) demonstrates significant activation of the enzyme at rest and is in line with previous reports in which PDC activation status following DCA administration has been determined (Timmons et al. 1996, 1997; 1998a,b; Howlett et al. 1999a). Another indication of significant PDC activation was the finding of a markedly lower muscle [lactate] at rest following DCA, also confirming previous observations by others (Timmons et al. 1996, 1997, Howlett et al. 1999a).
PDC activation during contractions, in physiological conditions, seems to be achieved mainly through increases in mitochondrial [Ca2+] (Hansford, 1994) and [pyruvate] (Putman et al. 1995), and therefore the extent of PDC activation is highly dependent on power output (Constantin-Teodosiu et al. 1991; Howlett et al. 1998). In physiological conditions, PDC activation following sudden (but submaximal) increases in metabolic demand is delayed. For example, Timmons et al. (1996) observed, in ischaemic dog gracilis muscle after 1 min of contractions, a level of PDC activation corresponding to ~65 % of that observed after 6 min of contractions. Howlett et al. (1999a) described in humans, during constant-load cycling at ~65 % of O2,max, that PDC activation after 30 s of exercise was ~70 % of that observed after 2 and 10 min of exercise. After DCA administration these authors (Timmons et al. 1996; Howlett et al. 1999a) observed significantly higher levels of PDC activation (vs. control) during the critical phase (i.e. the first 1-2 min) of the transition. Conversely, PDC activation is known to be very rapid during near maximal intensity exercise (Putman et al. 1995; Howlett et al. 1999b).
The mechanism(s) by which the activation of PDC at rest might influence the O2 on-kinetics can be summarized as follows. At the onset of exercise the delivery of substrates to the TCA cycle must be rapidly increased and the rate of activation of PDC (which is low at rest) might not be sufficient to meet the increased demand for substrate flux. However, following PDC activation at rest by DCA, acetyl-CoA will accumulate and most of the carnitine pool becomes acetylated (due to the low TCA cycle flux and the increased flux through PDC), i.e. acetyl group production is in excess of demand. This stockpile of acetyl groups (acetyl-CoA and acetylcarnitine) could then be utilized immediately when contractions start, thereby eliminating a possible limitation in the rate of adjustment of oxidative phosphorylation (Timmons et al. 1996, 1997, 1998a,b). In addition, acetylation of intramuscular carnitine should also reduce the proposed competition for acetyl-CoA between the carnitine acetyltransferase reaction and the TCA cycle at the onset of contraction (Timmons et al. 1997). In line with previous studies (Timmons et al. 1996, 1997, 1998a,b), in the present study muscle [acetylcarnitine] declined slightly during contractions following DCA administration, whereas it increased in C. This suggests that, during contractions following DCA administration, acetyl groups were mainly directed towards the TCA cycle and not to acetylcarnitine accumulation, whereas in C the increased flux of substrates was also directed towards acetylcarnitine accumulation. According to other authors (Howlett et al. 1999a) the effects of DCA are simply related to an increased flux of substrates through PDC after exercise begins and not to the increased resting muscle [acetylcarnitine], which, in those experiments (Howlett et al. 1999a), accumulated early in exercise.
In any case, we were unable to confirm the working hypothesis of the present study because O2 on-kinetics was not faster in DCA than in C. This suggests that, in the present experimental model, despite activation of PDC and the presence of excess acetyl groups, substrate availability for the TCA cycle did not appear to limit the rate of adjustment of oxidative phosphorylation. The question might then arise regarding the locus/mechanism responsible for the inertia of skeletal muscle oxidative metabolism. The interested reader is referred to some recent reviews on the topic (Balaban, 1990; Meyer & Foley, 1996; Tschakovsky & Hughson, 1999; Grassi, 2001; Hughson et al. 2001). Besides the role of products of high-energy phosphates splitting, as discussed below, a regulatory role can be hypothesized for mitochondrial Ca2+ (see e.g. Hansford, 1994), redox and phosphorylation potential (see e.g. Tschakovsky & Hughson, 1999), other rate-limiting enzymes or (for high-intensity exercise or contractions) O2 availability (see e.g. Grassi, 2001).
Effects of PDC activation on 'anaerobic' energy provision
The above conclusion should viewed with some caution, since it might be specific to the present experimental model. In the present study the substantially unchanged O2 on-kinetics after DCA administration was associated with non-significant changes in energy provision from 'anaerobic' energy sources (i.e. PCr splitting and lactate accumulation; see estimates of substrate level phosphorylation in the Results), although a trend toward less PCr splitting and less reliance on substrate level phosphorylation with DCA was described. In previous studies, conducted on different experimental models (Timmons et al. 1996, 1997, 1998a,b; Howlett et al. 1999a) anaerobic energy yield was significantly reduced following DCA.
The trend towards lower PCr degradation (PCr 'sparing') with DCA ([PCr] 19.7 mmol (kg DM)-1 in DCA vs. 27.9 mmol (kg DM)-1 in C, corresponding to ~30 % PCr 'sparing') was less pronounced than that observed after DCA administration during previous studies conducted in humans (Timmons et al. 1998a,b; Howlett et al. 1999a) or in ischaemic dog gracilis muscle (Timmons et al. 1996, 1997) and it did not reach statistical significance (P = 0.14). No clear-cut explanation can be offered for this discrepancy from the previous studies. As for the human studies (Timmons et al. 1998a,b; Howlett et al. 1999a), the difference could relate to the particular contraction pattern (electrically stimulated isometric tetanic contractions) in our preparation, as well as to the fact that the dog gastrocnemius is a highly 'aerobic' muscle, predominantly made up of slow oxidative (type I) or fast oxidative-glycolytic (type II a) fibres (Maxwell et al. 1977). This aspect could influence direct comparisons between the different experimental approaches. It must also be pointed out that a recent study conducted on humans cycling at 55 % O2,max in acute hypoxia did not find any difference in PCr degradation after 1 min of exercise, after DCA administration vs. control (Parolin et al. 2000). As for previous studies on dog gracilis muscle (Timmons et al. 1996, 1997), they evaluated contractions in ischaemic (~20 % of normal blood flow) conditions, which could alter the balance between energy sources compared to conditions of unrestricted blood flow.
In the present study muscle lactate accumulation during the contraction period was not different in the two conditions. This observation is at variance with the lower muscle lactate accumulation observed after DCA by Howlett et al. (1999a) (humans cycling at 65 % O2,max), but appears in agreement with the unchanged muscle lactate accumulation after DCA described by Timmons et al. (1998a) in humans during low-intensity knee-extensor exercise. Muscle lactate concentrations observed during contractions in the ischaemic dog gracilis muscle (Timmons et al. 1996, 1997) or in hypoxic (Parolin et al. 2000) or ischaemic (Timmons et al. 1998b) humans were two to five times higher than those of the present study, making direct comparisons more difficult.
Is PCr an energy buffer or the 'driving force' for muscle respiration?
Kinetics parameters presented in Table 4 show that, after a 5-6 s time delay, the time constant of O2 on-kinetics was significantly higher (indicating a slower kinetics) in DCA than in C. Values of + TD (as well as t50% and t63%, see Fig. 4) were slightly (although not significantly) higher in DCA than in C. The observation of a slightly slower O2 on-kinetics with DCA appears intriguing, and it might be of some interest in terms of the mechanism(s) regulating oxidative phosphorylation in skeletal muscle. PCr degradation, indeed, may be considered not just as an ATP buffer, but it could represent (through changes in [Cr], or of other variables related to this) one of the main controllers of oxidative phosphorylation (see e.g. di Prampero & Margaria, 1968; Whipp & Mahler, 1980; Cerretelli & di Prampero, 1987; Binzoni & Cerretelli, 1994; Meyer & Foley, 1996). Indirect evidence in favour of this concept is provided by the substantially identical kinetics of phase II pulmonary O2 increase and PCr degradation recently described by Rossiter at al. (1999) in humans performing constant-load quadriceps exercise. Thus, if muscle O2 kinetics is somehow coupled to PCr degradation, in the presence of less PCr degradation then O2 should be lower. Lower O2 values (in DCA vs. C) at different times during the transition, and the same steady-state O2 in the two conditions, would yield a slower O2 on-kinetics in DCA, which is the trend that we actually observed (see Table 4). In our study, differences in O2 on-kinetics between the two conditions were rather small; however, among the various parameters used to evaluate the kinetics we observed a significantly greater for the primary component in DCA vs. C.
By incidence, analysis of TD, and + TD for O2, and C(a-v)O2 obtained in the C condition of the present study shows interesting similarities with homologous data obtained by Grassi et al. (1996) for exercising legs in humans. In both of these studies increased immediately at the onset of contractions, following a substantially exponential time course, whereas C(a-v)O2 showed a 'biphasic' time course, in which a TD of several seconds was followed by a very rapid exponential increase to the new steady state. Moreover, in both studies a faster on-kinetics compared to O2 on-kinetics was observed, providing further (although indirect) evidence that, during transitions to contractions of submaximal metabolic intensity, convective O2 delivery is not limiting O2 on-kinetics.
Effects of DCA on the metabolic efficiency of muscle contraction
Analysis of O2 on-kinetics in the present study could also be somehow confounded by the fact that the metabolic efficiency (force produced/metabolic output) of contractions was somehow different in the two conditions. The force of contractions, substantially the same in the two conditions at the onset of contractions (see Results), was higher during the remaining contraction period in DCA, as shown by the higher values of the fatigue index, demonstrating less muscle fatigue (Fig. 1) and confirming previous observations (Timmons et al. 1996, 1997, 1998a). It must also be noted, however, that FI in the two conditions was very similar during the first ~30 s of contractions (i.e. during the most critical portion of the transition, as far as the O2 on-kinetics are concerned), and that differences reached statistical significance only from 80 s of contractions onward. A comparison of the relationship between force and metabolic output is easier at the end of the contraction period, when O2 has reached a steady state. In those conditions, for the same O2, force was significantly higher in DCA than in C. The presence of a slightly lower PCr degradation in DCA, and of similar increases in muscle [lactate] in the two conditions (see Table 3), suggests a higher metabolic efficiency with DCA. This could be explained by a preferential utilization of carbohydrate energy sources induced by the drug. It is well known that the energy made available at the muscle level per unit of O2 consumed is slightly higher when pyruvate is oxidized than when free fatty acids are utilized. Thus, for the same O2, more ATP can be generated and a higher force can be sustained when carbohydrates are preferentially utilized as energy fuels (vs. fats). This, in fact, may be one of the principal effects of DCA administration. DCA exerts a direct inotropic effect on the heart, presumably by facilitating oxidative metabolism of carbohydrates over fats (see e.g. Stacpoole, 1989). DCA has been utilized to support myocardial contractility during heart failure (Berzin et al. 1994). Since myocardial O2 and coronary blood flow were not affected by DCA (Berzin et al. 1994), the decreased O2 requirements to oxidize pyruvate compared to fatty acids provides a reasonable mechanism by which the drug could cause the observed increase of myocardial efficiency. A recent study demonstrated that DCA increases cardiac efficiency during reperfusion of ischaemic hearts by increasing the efficiency of ATP utilization (Taniguchi et al. 2001). To the best of our knowledge, no previous studies have specifically investigated the effects of DCA administration on skeletal muscle efficiency.
Another factor that could have contributed to the reduced fatigue after DCA administration is represented by the lower lactate levels observed, both at rest and during contraction (although, in the latter case, the difference vs. control did not reach statistical significance), suggesting less disturbance of muscle acid-base homeostasis with the drug.
Methodological considerations
The question could be raised whether the relatively small PCr sparing observed in the present study could yield a detectable signal in terms of O2 on-kinetics. Let us assume, as proposed by the previously mentioned authors (Timmons et al. 1996, 1997, 1998a,b; Howlett et al. 1999a), that PCr sparing would be associated with a faster O2 on-kinetics. Could we detect a difference in the latter, with our level of confidence in parameter determination? This issue can be examined by performing some calculations on our data. We observed 8.2 mmol (kg DM)-1 less PCr degradation with DCA, vs. C, corresponding to ~2.1 mmol per kilogram of 'wet' muscle (WM). At a P : O2 ratio of 6, this would correspond to ~0.343 mmol O2 (kg WM)-1. For 22.4 ml O2 (mmol O2)-1, this would correspond to ~0.77 ml O2 (100 g WM)-1 to be consumed 'in excess' to account for the observed PCr sparing, according to the hypothesis by the previously mentioned authors. Assuming a half-time for O2 on-kinetics of ~16 s (as observed in the present study; see the t50% value in Fig. 4), half of this 'extra' O2 should have occurred during the first 16 s of contractions, corresponding to an average 'extra O2' of ~1.46 ml (100 g)-1 min-1. Similar calculations were performed for the remaining periods of contractions. After adding this 'extra O2' to the measured O2 values (averages for all subjects), we came up with a hypothetical O2 vs. time curve, which was compared with the real O2 vs. time curve described in Fig. 2 (C condition). The two curves were fitted by eqn (1), and we obtained + TD values which were 2.92 s lower with the hypothetical O2 vs. time curve compared to the real O2 vs. time curve. Could we detect a 2.92 s difference in + TD, with six dogs and the observed variance, by using Student's t test (paired analysis, two-tailed), at an 
value of 0.05 and a 
value of 0.2? The answer is positive: six muscles would be enough. That is to say, if the observed PCr sparing could be explained in terms of faster O2 on-kinetics, we would have been able to detect a significant difference in + TD for this kinetics. Recent, although still controversial, evidence of a P : O2 ratio lower than 6 (see e.g. Salway, 1999) would lend further support to this notion.
As mentioned above, PDC activation is highly dependent on power output (Constantin-Teodosiu et al. 1991; Howlett et al. 1998). Experiments conducted in humans during short supramaximal exercises (Howlett et al. 1999b) found that PCr degradation was the same after DCA administration vs. control, despite a greater activation of PDC (both at rest and after the exercise) obtained by the drug. This observation led the authors to conclude that, during very intense exercise, there might be an inability to utilize the extra oxidative substrate (from either stored acetylcarnitine or PDC activation), possibly because of O2 (Hughson, 1990; Tschakovsky & Hughson, 1999; Hughson et al. 2001) or other metabolic limitations. Another explanation is that during exercise at an intensity that exceeds the O2,max of the muscle, the rate of ADP accumulation, in the presence or abscence of DCA, is so marked that oxidative phosphorylation is maximally activated. In the present study the investigated transition was from rest to contractions at a metabolic level that should correspond, in the present preparation (muscle self perfused, i.e. not pump perfused) to ~60-70 % of peak O2 (Kelley et al. 1996; Ameredes et al. 1998). At this metabolic level, we have previously demonstrated that neither convective nor diffusive O2 delivery to muscle is a limiting factor for the O2 on-kinetics (Grassi et al. 1998a,b). Thus, the unchanged O2 on-kinetics observed in the present study after DCA administration could not be explained in terms of an O2 limitation, which, in theory, could counterbalance the effects on oxidative phosphorylation which represented the working hypothesis of the present study.
In the present study the order of treatments could not be randomized, i.e. DCA was always performed after C. However, some of our previous studies, conducted using the same experimental model as the present study, did not show any significant residual effect on a subsequent period of contractions deriving from a previous contraction period (interval between contraction periods of at least 45 min) (Grassi et al. 1998a,b).
Advantages and limitations of the present preparation (isolated dog gastrocnemius in situ) for the study of O2 on-kinetics were discussed at length in our previous studies (Grassi et al. 1998a,b, 2000). In short, the main disadvantages are represented by the particular contraction pattern (synchronous tetanic contractions), by the intrinsic invasiveness of the preparation and by the problem of extrapolating the results to exercising humans. While caution is warranted in this respect, it must be noted that: (1) basic mechanisms of regulation of oxidative phosphorylation appear the same across mammalian species (Balaban, 1990); and (2) the patterns of and O2 increase at contraction onset appear remarkably similar in canine (as shown by the present and by previous studies; see e.g. Piiper et al. 1968; Grassi et al. 1998a, 2000) and in human muscles (Grassi et al. 1996), the only difference being faster kinetics in dogs, presumably as a consequence of the higher percentage of oxidative fibres (Maxwell et al. 1977). In the present study (as well as in previous papers; see e.g. Grassi et al. 1998a, 2000) resting to muscle was higher and O2 extraction was lower than the values usually observed in human skeletal muscle (see e.g. Laughlin et al. 1996). The elevated resting is probably attributable to some loss of vascular tone related to the surgical denervation of the muscle (Laughlin et al. 1996). It must be noted, however, that the proposed mechanisms determining and regulating the increase in muscle at the onset of contractions (muscle pump, 'myogenic' control, 'propagated vasodilatation', endothelium-derived vasodilatation and increased concentration of metabolites in the interstitium; Laughlin et al. 1996) would be unaffected by the surgical denervation of the muscle. DCA did not have any significant haemodynamic effect, as demonstrated by the similar on-kinetics in the two conditions, as well as by the similar , BPm and vascular resistance values at rest and at steady state during contractions.
Conclusions
In the isolated dog gastrocnemius preparation in situ, during transitions from rest to electrically stimulated contractions corresponding to ~60-70 % of the muscle peak O2, DCA infusion determined a significant activation of PDC, a marked stockpiling of acetylcarnitine at rest and less muscle fatigue, but it did not significantly affect 'anaerobic' energy provision and O2 on-kinetics. In the present experimental model, PDC activation status is not responsible for the 'metabolic inertia' in the adjustment of oxidative phosphorylation to sudden increases in the needs for ATP regeneration. The increased metabolic efficiency with DCA might be explained by a preferential utilization of carbohydrates during contraction.
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Acknowledgements
This study was supported by NIH Grants 1RO1AR-40342 and AR-40155; by NATO Collaborative Research Grant no. 972111; and by Telethon-Italy Grant no. 1161C.
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