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Journal of Physiology (2001), 536.3, pp. 905-915
© Copyright 2001 The Physiological Society
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ABSTRACT |
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 TopAbstract IntroductionMethodsResultsDiscussionAppendixReferences |
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INTRODUCTION |
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The ATP demand in working muscle is tightly coupled to mechanical output (Hochachka & McClelland, 1997; Hogan et al. 1998; Stary & Hogan, 2000) and depends on numerous variables such as the type of contractile activity, i.e. dynamic or isometric (Ryschon et al. 1997), the intensity and duration of contractions (Crow & Kushmerick, 1982; Bergstrom & Hultman, 1988; Hogan et al. 1998) and the fibre type composition of the muscle (Crow & Kushmerick, 1982; Newham et al. 1995; Ratkevicius et al. 1998). For these reasons, the rate of ATP utilization shows considerable variability among the different forms of exercise (Fitts, 1994). The assessment of the ATP cost of contraction provides a good tool to compare the bioenergetics of these different forms of exercise, by normalizing the ATP utilization to force output.
It is well established that ATP cost of contraction is reduced throughout fatiguing exercise. Chemical analysis of isolated mouse muscle (Crow & Kushmerick, 1982) and of rat muscle stimulated in situ (de Haan et al. 1986) has shown that the ATP cost of a single tetanic contraction inducing fatigue decreases progressively with the increased duration of the contraction. Similar observations have been reported in human muscle; 31P-magnetic resonance spectroscopy (31P-MRS) studies have shown that the ATP cost of contraction declines during a single 90 s maximal voluntary contraction (MVC) (Boska, 1994; Newcomer et al. 1999) and also throughout a 15 min protocol consisting of brief repeated MVCs (Newcomer & Boska, 1997). Likewise, the ATP cost of a brief MVC (5 s duration) has been demonstrated to be lower during recovery from a 4-5 min exhaustive dynamic exercise than during the pre-exercise period (Smith et al. 1999). It is of interest to stress that all these previous studies have investigated the changes in ATP cost of contraction throughout a given fatiguing protocol. However, the relationship between the level of fatigue and the reduction in the ATP cost of contraction has to our knowledge never been addressed.
The purpose of the present study was to test in vivo whether the reduction in ATP cost of contraction during rat gastrocnemius muscle stimulation was related to fatigue level. Muscles were electrically stimulated to perform 6 min isometric contractions at different frequencies; one non-fatiguing protocol (0.8 Hz) and five fatiguing protocols (2, 3.2, 4, 5.2 or 7.6 Hz) were used. We have quantified non-invasively using 31P-MRS the rates of ATP turnover from oxidative and anaerobic pathways (Kemp & Radda, 1994; Conley et al. 1998; Newcomer et al. 1999; Walter et al. 1999) in order to calculate the ATP cost of contraction. A preliminary account of this work has been presented previously (Giannesini et al. 2000).
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METHODS |
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Animal care and feeding
Forty-four male Wistar rats (CERJ, Le Genest St Isle, France) weighing 350-375 g were used for these experiments, following the guidelines of the National Research Council Guide for the care and use of laboratory animals, and the French Law on the Protection of Animals. Rats were housed in an environmentally controlled facility (12 h light-12 h dark cycle, 22 °C), and received water and standard rat food ad libitum until the time of experiment. At the end of experiments, animals were immediately killed by an intracardiac injection of sodium pentobarbitone.
Hindlimb surgical preparation
General anaesthesia was induced with an intraperitoneal injection of sodium pentobarbitone (50 mg (kg body weight)-1), and was maintained during the experiment by repeated administrations of anaesthetic (10 mg (kg body weight)-1, every 30 min) through an intraperitoneal catheter. The left hindlimb was surgically prepared for in situ sciatic nerve stimulation of the gastrocnemius muscle. The Achilles' tendon was exposed and removed from the foot at the site of calcaneus bone attachment, leaving intact the neurovascular supply of the muscle. The distal part of the gastrocnemius tendon was attached to a home-built force transducer via a silk thread. Through a small incision made at the hip level, the left sciatic nerve was exposed in its gluteal course between the quadratus femoris and the caudofemoralis muscles, and was carefully cleared of connective tissue. A home-built bipolar electrode connected to an electrical stimulator (SI-10, NARCO, USA) was placed around the sciatic nerve, which was cut proximally to the electrode attachment. The part of the sciatic nerve connected to the bipolar electrode was put back in place at the hip level and the incision was surgically closed. The rat was placed backwards in a custom-made Perspex cradle integrating a warm-water heating pad. Body temperature (typically 35-36 °C) was monitored throughout the experiment using a rectal probe. The left leg was firmly immobilized by securing the foot with straps and by inserting a non-magnetic brass pin into the tibia head. In that position, the belly of the gastrocnemius muscle was located above the 31P-MRS surface coil. At rest, muscle was passively loaded (typically 1.3-1.5 N) by adjusting the position of the force transducer in order to give maximum isometric twitch tension in response to supramaximal square wave pulses (1-10 V, 1 ms duration) delivered to the sciatic nerve. At this stage, the cradle was inserted into the magnet.
Stimulation protocol
The stimulation protocol consisted of 6 min of isometric contractions, which were electrically induced via the sciatic nerve with supramaximal square-wave pulses (1-10 V, 0.2 ms duration). Animals were randomly divided to perform six different stimulation protocols: one non-fatiguing protocol at 0.8 Hz (n = 7) and five fatiguing protocols at 2 Hz (n = 7), 3.2 Hz (n = 9), 4 Hz (n = 9), 5.2 Hz (n = 6) and 7.6 Hz (n = 6). Isometric contractions were synchronized to MRS data acquisition.
Force measurements
Force measurements were conducted with a home-built force transducer, which was linear from 0 to 10 N. During muscle stimulation, the electrical signal coming from the force transducer was amplified (reference: 13-4515-50, Gould, USA), converted to a digital signal and processed on a personal computer using ATS software (SYSMA, France). Isometric force (in N s) was calculated every 15 s of stimulation by integrating isometric tension (in N) relative to time (in s) and was expressed as tension-time integral. Isometric force per twitch (N s twitch-1) was calculated by scaling isometric force to the stimulation frequency. Muscle relaxation was characterized as the extent of the return to rest tension between consecutive twitches.
Magnetic resonance spectroscopy and data processing
31P-MRS investigations were performed in a horizontal superconducting magnet (Brüker 47/30 Biospec system, Karlsruhe, Germany) operating at 4.7 T. Magnetic resonance (MR) data were collected with a home-built 31P-MRS surface coil (10 
14 mm). Magnetic field homogeneity was optimized by monitoring the water signal until the width of the water resonance at half height was less than 0.25 p.p.m. 31P-MR signal was acquired at 81 MHz following 20 µs radiofrequency pulses applied with a repetition time of 2.4 s. MR data acquisition was gated to stimulation in order to record signals between consecutive contractions and then reduce motion artifacts due to contraction. Free-induction decays (FIDs; 12 scans, 4000 Hz sweep width, 512 data points collected) were continuously recorded in 30 s blocks throughout the experimental protocol: during the 6 min before stimulation (rest), during stimulation (6 min) and during the 30 min after stimulation (recovery). FIDs were transferred to an IBM RISC 6000 workstation and processed using NMR1 spectroscopy processing software (New Methods Research, USA). After deconvolution to a line broadening of 15 Hz and application of zero filling (2 K), FIDs were Fourier-transformed into spectra and baseline correction was performed as previously described (Mazzeo & Levy, 1991). Signal areas corresponding to PCr, Pi and 
-ATP were measured by curve fitting of the spectrum signals to a Lorentzian shape function (Mazzeo & Levy, 1991), and were corrected for magnetic saturation effects using fully relaxed spectra collected at rest with a TR of 20 s. Absolute concentrations of PCr and Pi were expressed relative to a resting -ATP concentration of 5.8 mM reported from fluorimetrical measurements in the same muscle from the same strain of rat (Kemp et al. 1996). Intracellular pH (pHi) was calculated from the chemical shift of Pi relative to PCr (-2.45 p.p.m.) as previously described (Arnold et al. 1984). Time points for the time course of pHi and phosphorylated metabolite concentrations were assigned to the midpoint of the acquisition interval as previously described (Kemp et al. 1994, 1996).
Calculations
The ATP turnover rates (mM s-1) by PCr hydrolysis (D), oxidative phosphorylation (Q) and glycolysis (L) were calculated as previously described (Kemp & Radda, 1994; Kemp et al. 1996; Ratkevicius et al. 1998; Walter et al. 1999) and are detailed in the Appendix. Total ATP turnover rate was calculated: (i) at the onset of the stimulation period from the sum of anaerobic ATP contributions (D + L), considering oxidative contribution (Q) as negligible at this stage of stimulation (Kemp et al. 1994); (ii) at the end of the stimulation period from the sum of anaerobic and oxidative ATP contributions (D + L + Q). Total ATP turnover per twitch (mM twitch-1) was calculated by scaling the total ATP turnover rate to the stimulation frequency. ATP cost of contraction (mM (N s)-1) was calculated as the ratio between total ATP turnover per twitch and isometric force per twitch.
Statistics
All results are presented as means ± S.E.M. Statistical difference was tested within each protocol using Student's two-tailed t test for paired observations, and among protocols using one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test for multiple comparisons. The level of significance was set at P < 0.05.
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RESULTS |
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Time-dependent changes in [PCr] and pHi
Typical 31P-MRS spectra from a single rat gastrocnemius muscle are presented in Fig. 1. [PCr] and pHi were not significantly different among resting muscles, averaging 20.7 ± 0.3 mM and 7.04 ± 0.02, respectively, over the six groups (Table 1). Throughout the stimulation period, [PCr] and pHi decreased to reach an end-of-stimulation value, which was lower at higher stimulation frequencies (Table 1). Time-dependent changes in [PCr] and pHi are illustrated in Fig. 2A and B.
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Figure 1. Typical 31P-MRS spectra from a single rat gastrocnemius muscle at rest (A) and at the end of 6 min repeated isometric contractions produced at 2 Hz (B) MR data acquisition was synchronized to stimulation in order to record 31P signals between consecutive twitches. Abbreviations for peak assignment (in p.p.m.) are: PME (phosphomonoester), Pi (inorganic phosphate), PCr (phosphocreatine), and | ||
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Figure 2. Typical time-dependent changes in [PCr] (A), pHi (B) and isometric force (C) during 6 min repeated isometric contractions produced at 3.2 Hz Values are means ± S.E.M. (n = 9). For the time-dependent changes in [PCr] and pHi, the first point indicates the resting value. | ||
Time-dependent changes in isometric force
Samples of isometric tension traces are presented in Fig. 3. At the onset of muscle stimulation (t = 0.25 min), no sign of fatigue was observed within each protocol (Fig. 4A). At this time, isometric force per twitch did not differ significantly (P = 0.45) among the six protocols (Fig. 4A), averaging 0.124 ± 0.004 N s twitch-1 over the six protocols. Throughout muscle stimulation, isometric force per twitch was not significantly altered (104 ± 7 % of initial value) for the non-fatiguing protocol (at 0.8 Hz) but decreased significantly for the fatiguing protocols (Fig. 4A). Time-dependent changes in isometric force are illustrated in Fig. 2C. At the end of the fatiguing protocols (t = 5.75 min), the level of fatigue increased proportionally with stimulation frequency (Fig. 4A): isometric force per twitch reached 0.105 ± 0.006 N s twitch-1 at 2 Hz (82 ± 4 % of initial value), 0.067 ± 0.006 N s twitch-1 at 3.2 Hz (60 ± 7 % of initial value), 0.054 ± 0.006 N s twitch-1 at 4 Hz (51 ± 8 % of initial value), 0.044 ± 0.006 N s twitch-1 at 5.2 Hz (35 ± 4 % of initial value) and 0.024 ± 0.004 N s twitch-1 at 7.6 Hz (19 ± 4 % of initial value). Differences were statistically significant (P < 0.01) among protocols at 0.8, 2 and 3.2 Hz and between protocols at 4 and 7.6 Hz, but were not statistically (P = 0.10) significant between protocols at 5.2 and 7.6 Hz (Fig. 4A).
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Figure 3. Typical isometric tension traces from three rat gastrocnemius muscles electrically stimulated at 0.8, 4 or 7.6 Hz For each trace, isometric tension (in N) is shown at the onset (left panels) and at the end (right panels) of 6 min repeated isometric contractions. The dashed horizontal lines indicate the rest tension. Muscle stimulation produced no fatigue at 0.8 Hz (A), an intermediate level of fatigue at 4 Hz (B) and a high level of fatigue at 7.6 Hz (C). Note that muscle relaxation was not fully achieved (65 % relaxation) at the end of muscle stimulation at 7.6 Hz when muscle tension did not revert to rest values between consecutive twitches. | ||
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Figure 4. Isometric force (A), total ATP turnover (B) and ATP cost of contraction (C) at the onset and at the end of 6 min repeated isometric contractions produced during non-fatiguing (0.8 Hz) and fatiguing (2, 3.2, 4, 5.2 and 7.6 Hz) stimulation protocols Values are means ± S.E.M. * Significant difference (P < 0.05) between the onset (t = 0.25 min) and the end (t = 5.75 min) of each protocol. At the end of the stimulation period, statistical difference (P < 0.05) was tested among stimulation protocols using one-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls test for multiple comparisons: a significantly different from protocol at 0.8 Hz; b significantly different from protocol at 2 Hz; c significantly different from protocol at 3.2 Hz; d significantly different from protocol at 4 Hz; e significantly different from protocol at 5.2 Hz; f significantly different from protocol at 7.6 Hz. | ||
Muscle relaxation
For the six protocols, muscle relaxation was fully achieved (100 %) at the onset of the stimulation period when isometric tension returned to rest values between consecutive twitches (Table 2). At the end of the stimulation period, muscle relaxation was also fully achieved for protocols ranging from 0.8 to 4 Hz, but not for protocols at 5.2 and 7.6 Hz when muscle tension did not return to rest values between consecutive twitches (91 ± 5 and 75 ± 10 % relaxation at 5.2 and 7.6 Hz, respectively) (Table 2). This observation is illustrated in Fig. 3.
ATP turnover rate
Total ATP turnover per twitch did not differ significantly (P = 0.29) among the six protocols at the onset of the stimulation period (Fig. 4B), averaging 0.208 ± 0.012 mM twitch-1 over the six protocols. At this time, relative contributions of PCr hydrolysis (D) and glycolysis (L) to the total ATP turnover per twitch were affected by increasing stimulation frequencies: D declined with increasing stimulation frequency, from 0.167 ± 0.012 mM twitch-1 at 0.8 Hz (73 ± 7 % of the total ATP turnover) to 0.107 ± 0.009 mM twitch-1 at 7.6 Hz (43 ± 3 % of the total ATP turnover) (Table 3); on the other hand, L increased by a factor of 2 between 0.8 Hz (0.066 ± 0.019 mM twitch-1) and 7.6 Hz (0.150 ± 0.023 mM twitch-1) (Table 3).
At the end of the stimulation period, the total ATP turnover per twitch was significantly reduced in the six protocols (Fig. 4B). The extent of this reduction increased proportionally as the stimulation frequency increased (Fig. 4B): total ATP turnover per twitch reached 0.140 ± 0.021 mM twitch-1 (68 ± 7 % of initial value) at 0.8 Hz, 0.066 ± 0.009 mM twitch-1 (38 ± 4 % of initial value) at 2 Hz, 0.042 ± 0.007 mM twitch-1 (25 ± 4 % of initial value) at 3.2 Hz, 0.029 ± 0.004 mM twitch-1 (14 ± 2 % of initial value) at 4 Hz, 0.017 ± 0.003 mM twitch-1 (11 ± 3 % of initial value) at 5.2 Hz and 0.011 ± 0.002 mM twitch-1 (4 ± 1 % of initial value) at 7.6 Hz. Differences were statistically significant (P < 0.04) among protocols at 0.8, 2 and 3.2 Hz, but not among protocols at 4, 5.2 and 7.6 Hz (Fig. 4B).
ATP cost of contraction
ATP cost of contraction did not differ significantly (P = 0.45) among the six protocols at the onset of the stimulation period (Fig. 4C), with a mean value of 1.76 ± 0.11 mM (N s)-1 over the six protocols. At the end of the stimulation period, ATP cost of contraction was significantly reduced in the six protocols (Fig. 4C), reaching 1.08 ± 0.13 mM (N s)-1 (67 ± 9 % of initial value) at 0.8 Hz, 0.63 ± 0.07 mM (N s)-1 (48 ± 7 % of initial value) at 2 Hz, 0.64 ± 0.15 mM (N s)-1 (41 ± 8 % of initial value) at 3.2 Hz, 0.56 ± 0.07 mM (N s)-1 (29 ± 4 % of initial value) at 4 Hz, 0.41 ± 0.07 mM (N s)-1 (31 ± 6 % of initial value) at 5.2 Hz and 0.54 ± 0.17 mM (N s)-1 (26 ± 6 % of initial value) at 7.6 Hz; differences were statistically significant (P < 0.006) between the non-fatiguing protocol and the five fatiguing protocols (Fig. 4C). However, ATP cost of contraction did not differ significantly among the five fatiguing protocols and averaged 0.57 ± 0.05 mM (N s)-1 (35 ± 3 % of initial value).
Relationship between isometric force and ATP cost of contraction
Regression analysis performed on data pooled across the five fatiguing protocols did not show any significant correlation (P = 0.87, r 2 = 0.01) between the isometric force per twitch and the ATP cost of contraction at the end of the stimulation period (Fig. 5).
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Figure 5. Relationship between isometric force and ATP cost of contraction at the end of 6 min repeated isometric contractions produced during fatiguing stimulation protocols at 2, 3.2, 4, 5.2 and 7.6 Hz Regression analysis (continuous line) performed on pooled data from the fatiguing protocols (2, 3.2, 4, 5.2 and 7.6 Hz) did not show any significant correlation (P = 0.87, r 2 = 0.01). | ||
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DISCUSSION |
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This work represents the first attempt to investigate the relationship between the reduction in ATP cost of contraction during muscle exercise and the level of fatigue. We mainly report three novel findings: (i) at the onset of the stimulation period, no signs of fatigue were observed in the six protocols and ATP cost of contraction calculated from the total anaerobic ATP turnover (PCr hydrolysis plus glycolysis) remained independent of the stimulation frequency; (ii) at the end of the fatiguing protocols, ATP cost of contraction was reduced to the same extent (35 ± 3 % of initial value) whatever the level of fatigue; (iii) at the end of the non-fatiguing protocol, ATP cost of contraction was also reduced but to a lesser extent (67 ± 9 % reduction) when compared with the fatiguing protocols.
Effect of stimulation frequency on ATP cost of contraction before fatigue development
At the onset of the stimulation period, no signs of fatigue were observed in the six protocols and ATP cost of contraction did not differ significantly among protocols in spite of increasing stimulation frequency. These findings are in accordance with several previous experiments showing that the ratio of energetic demand to force output remained independent of the stimulation intensity during short duration exercises performed without any fall in force output. For instance, a thermodynamic study in isolated fibres from Xenopus laevis reports that muscle efficiency (defined as the ratio of mechanical power to total energy output) of 1.5 s repeated isometric contractions was not altered when stimulation frequency increased from 10 to 40 Hz (Buschman et al. 1995). Furthermore, 31P-MRS studies have demonstrated in vivo that ATP cost of contraction remained unaltered at the onset of a series of repeated isometric contractions whatever the stimulation frequency, ranging from 0.5 to 2 Hz in flexor digitorum superficialis of human forearm (Blei et al. 1993), from 1 to 8 Hz in rat gastrocnemius muscle (Foley & Meyer, 1993) and over 60 Hz in rattlesnake tail muscle (Conley & Lindstedt, 1996).
It is of interest to stress that ATP cost of contraction was calculated in these previous 31P-MRS studies at the very start of muscle stimulation from the initial burst of PCr breakdown, which was assumed to be the only pathway contributing to the total ATP turnover at this stage of stimulation; in the present experiments, our ability to measure the glycolytic ATP turnover throughout the stimulation period allowed us to demonstrate that the ATP cost of contraction also remained independent of the stimulation frequency in the latter stage of the stimulation period, after 0.25 min of repeated isometric contractions, when no signs of fatigue were observed.
The independence of ATP cost of contraction from stimulation frequency at the onset of the stimulation period may be explained by the combination of two processes. Firstly, the isometric force per twitch did not differ significantly between the six protocols. Indeed, for each experiment, stimulation parameters and muscle length were optimized at rest in order to produce maximal isometric force during stimulation (see Methods). Secondly, total ATP turnover per twitch calculated from the sum of anaerobic contributions (PCr breakdown plus glycolysis) also remains unaffected by the increase of stimulation frequency, which roughly coincides with the view that anaerobic metabolism is considered to provide enough energy to cope with a high ATP demand at the onset of exercise, including brief intense exercise (Foley & Meyer, 1993; Rico-Sanz et al. 1998; Sahlin et al. 1998; Walter et al. 1999). Our data showing that the decrease in the relative contribution from net PCr breakdown was compensated by the increase in the glycolytic contribution illustrates that the high precision of ATP production is regulated to meet ATP utilization throughout muscle exercise (Hochachka & McClelland, 1997; Sahlin et al. 1998; Stary & Hogan, 2000).
An interesting observation from the present study is that the total ATP turnover per twitch averaged 0.21 ± 0.01 mM twitch-1 over the six protocols at the onset of the stimulation period, which is strongly similar to previous data showing that the rate of ATP hydrolysed per twitch measured at the onset of muscle stimulation was 0.25 ± 0.01 mM twitch-1 in rat gastrocnemius muscle (Foley & Meyer, 1993) and 0.21 ± 0.01 mM twitch-1 in cat biceps (predominantly composed of fast-twitch fibres) (Harkema et al. 1997).
Reduction in ATP cost of contraction associated with muscle stimulation
In the six protocols, ATP cost of contraction was reduced between the onset and the end of 6 min repeated isometric contraction. In other words, muscle paradoxically needed less ATP to produce the same amount of isometric force at the end of the stimulation period compared with the onset.
Alteration of activation and relaxation processes have been proposed to play a role in the reduction in ATP cost of contraction throughout muscle exercise (Hogan et al. 1998; Smith et al. 1999). During repeated contractions, ATP is used for both contractile and non-contractile processes, the latter process being related to ion transport associated with the activation-relaxation cycle of muscle contraction. Therefore, any reduction of one of these processes throughout muscle activity would explain the reduction in ATP cost of contraction. This assumption is supported by the fact that, for a given duration of contraction, ATP cost of contraction is lower for a single sustained isometric contraction when compared with repeated isometric contractions (de Haan et al. 1986; Chasiotis et al. 1987; Bergstrom & Hultman, 1988; Spriet, 1989; Newham et al. 1995; Hogan et al. 1998): during a single sustained isometric contraction, ATP is mainly hydrolysed to maintain tension, whereas additional ATP consumption is required for ion transport between contractions during repeated isometric contractions; it has been reported previously that 20-50 % of the ATP utilized during contraction would be used for Ca2+ pumping across the sarcoplasmic reticulum (SR) membrane (Bergstrom & Hultman, 1988; Lou et al. 1997; Hogan et al. 1998). In the present study, muscle relaxation was not fully achieved between consecutive twitches produced during the fatiguing protocols at 5.2 and 7.6 Hz, hence suggesting an alteration in Ca2+ fluxes across the SR membrane. Consequently, at least for these levels of fatigue, the reduction in ATP cost of contraction throughout the stimulation period could be due to the reduction in the relative contributions of non-contractile processes.
Another attractive possibility to explain the reduction in the ATP cost of contraction might lie in the shift of the pattern of fibre type recruitment throughout the fatiguing protocol. Rat gastrocnemius muscle is a mixed muscle composed of 93 % of fast-twitch fibres and 7 % of slow-twitch fibres (Armstrong & Phelps, 1984). At the onset of electrical stimulation, all the fast- and slow-twitch fibres are recruited to produce muscle contraction; with fatigue development, because fast-twitch fibres are less fatigue resistant than slow-twitch fibres (Chasiotis et al. 1987), the proportion of fast-twitch fibre recruitment could, however, decrease compared with the proportion of recruited slow-twitch fibres. Given that slow-twitch fibres are known to contract more economically compared with fast-twitch fibres (Sawka et al. 1981; Crow & Kushmerick, 1982; Harkema et al. 1997), the recruitment of a higher proportion of slow-twitch fibres could therefore account for the reduction in ATP cost of contraction during the fatiguing protocols.
Reduction in ATP cost of contraction during non-fatiguing protocols
The reduction in ATP cost of contraction during the non-fatiguing protocols indicates that this phenomenon is not always associated with fatigue development, and further suggests that at least one additional mechanism could intervene. To our knowledge, potent alteration in ATP cost of contraction in the later stages of the non-fatiguing protocols has never been reported. The muscle-tendon complex is composed of contractile and series elastic (non-contractile) elements. During contraction, a muscle uses energy to shorten against series elastic elements (SEE), which produce a mechanical work external to contractile elements (Newham et al. 1995). This external work has been demonstrated to affect muscle energetics during stretch-shorten cycles (Ettema, 1996). To a lesser extent, it has been suggested that even under isometric conditions some energy utilized for contraction was wasted to overcome SEE (de Haan et al. 1986; Newham et al. 1995; Newcomer et al. 1999). The observation that during a single contraction, external work occurs only at the onset of contraction but not during subsequent maintenance of force, could explain why ATP cost of contraction has been found to be greater at the onset of a single contraction (Newham et al. 1995; Newcomer et al. 1999). Consequently, it is possible that in the present study, although muscle was stretched to its optimal length before stimulation, external work could affect ATP cost of contraction at the onset of stimulation. Therefore, external work could be attenuated with successive contractions, thereby accounting for the lower ATP cost of contraction measured by the end of muscle stimulation whatever the stimulation frequency. It is important to emphasize that this mechanism could also participate in the reduction in ATP cost of contraction during the fatiguing protocols.
Relationship between fatigue level and ATP cost of contraction
We can now ask why the reduction in ATP cost of contraction measured at the end of the five fatiguing protocols is not related to the level of fatigue? According to the view that limitation in energy availability is a classical hypothesis to explain muscle fatigue (Fitts, 1994; Sahlin et al. 1998; Ward et al. 1998), it is legitimate to consider that muscle needs to use ATP sparingly to avoid or at least attenuate fatigue development. Reduction in ATP cost of contraction throughout muscle activity might represent a way to optimize ATP utilization to produce force. Our hypothesis is supported by the observations that the reduction in ATP cost of contraction (i) was larger during the fatiguing protocols when compared with the non-fatiguing protocol and (ii) reached the same extent whatever the level of fatigue. These results might mean that ATP utilization is systematically optimized throughout muscle activity up to a threefold level, which could be associated with fatigue development. Further investigation is still required to understand the cause-to-effect relationship between fatigue development and reduction in ATP cost of contraction.
In conclusion, this study reports for the first time the effects of increasing fatigue levels on ATP cost of contraction. We have demonstrated that ATP cost of contraction was reduced throughout non-fatiguing and fatiguing protocols. During the non-fatiguing protocol, the reduction in ATP cost of contraction may be attributed to ATP consumption by non-contractile elements. During the fatiguing protocols, the larger reduction in ATP cost of contraction may be due to additional mechanisms such as shift in fibre type recruitment and alteration of activation-relaxation processes. The fact that the reduction in ATP cost of contraction during in vivo stimulation of rat gastrocnemius muscle is not related to the fatigue level leads us to propose that fatigue development could be associated with an optimization in ATP utilization.
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APPENDIX |
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The ATP turnover rates (in mM s-1) by PCr hydrolysis, oxidative phosphorylation and glycolysis, were calculated from the time-dependent changes in [PCr] and pHi (Kemp & Radda, 1994; Kemp et al. 1996; Ratkevicius et al. 1998; Walter et al. 1999).
The ATP turnover rate by net PCr hydrolysis (D) was directly calculated from the time-dependent changes in PCr during muscle stimulation:
| D = -dPCr/dt. | (A1) |
The oxidative ATP turnover rate (Q) was calculated at the end of muscle stimulation using the initial rate of PCr recovery (Kemp et al. 1994):
| Q = k PCrcons, | (A2) |
where PCrcons is the PCr concentration at the cessation of muscle stimulation. The pseudo-first order rate constant of PCr recovery (k) was estimated by fitting the PCr recovery profile to a monoexponential function (PCrt = PCrrest + PCrcons e-kt, where PCrrest is the resting PCr concentration).
The glycolytic ATP turnover rate (L) was inferred from the number of moles of protons (P) generated by lactate formation, considering that glycolytic ATP production is related to lactate synthesis with a stoichiometry of 1.5 moles of ATP per mole of lactate (Hochachka & Mommsen, 1983) (L = 1.5P). P was calculated from the observed changes in pHi taking into account the number of moles of protons (i) associated with PCr hydrolysis (H+PCr), (ii) passively buffered in the cytosol (H+ ), (iii) leaving the cell (rate of net proton efflux, H+efflux), and (iv) produced by oxidative phosphorylation (H+ox) (Kemp & Radda, 1994; Walter et al. 1999):
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P = H+PCr + H+ + H+efflux - H+ox.
| (A3) |
H+PCr (mM s-1) was calculated from the time-dependent changes in [PCr] and from the stoichiometric coefficient 
= 1/(1 + 10 (pHi-6.75) ), which represents the number of protons associated with PCr hydrolysis (Wolfe et al. 1988):
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H+PCr = -D.
| (A4) |
H+ was calculated from the apparent buffering capacity total (in slykes, millimoles acid added per unit change in pHi) and from the variations in pHi (
pHi = pHobserved - pHrest):
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H+ = -total pHi.
| (A5) |
total takes into account the buffering capacity of Pi (Pi ) and the buffering capacity of tissue tissue (total = Pi + tissue). Pi was calculated from the concentration of Pi with a pKa of 6.75 (Wolfe et al. 1988):
| (A6) |
tissue has been demonstrated to vary linearly between pH 7 (16 slykes) and pH 6 (37 slykes) (Adams et al. 1990); according to these data, tissue was calculated as follows:
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tissue = -21pHi + 163.
| (A7) |
H+efflux (mM s-1) was calculated during muscle stimulation using the proportionality constant 
relating proton efflux rate to pHi (Kemp & Radda, 1994; Newcomer et al. 1999):
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H+efflux = -pHi. (A8)
| (A8) |
(in mM s-1 (pH unit)-1) was determined during the initial recovery period using eqn (A6) and the rate of net proton efflux calculated at this stage of stimulation:
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H+efflux = D + total dpHi/dt.
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H+ox (mM s-1) was calculated from Q (eqn (A2)) and from the stoichiometric coefficient m = 0.16/(1 + 10 (6.1-pHi)), which represents the number of moles of protons produced in association with oxidative phosphorylation (Kemp & Radda, 1994):
| H+ox = m Q. | (A10) |
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REFERENCES |
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Acknowledgements
We would like to thank Ferdinand Tagliarini for his technical assistance. This work was supported by grants from CNRS (UMR 6612), ADEREM (Association pour le Développement des Recherches Biologiques et Médicales au CHR de Marseille) and Ministère de la Santé (PHRC 1997).
Corresponding author
D. Bendahan: Centre de Résonance Magnétique Biologique et Médicale (CRMBM), UMR CNRS 6612, Faculté de Médecine de Marseille, 27 Boulevard Jean Moulin, 13005 Marseille, France.
Email: david.bendahan{at}medecine.univ-mrs.fr
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