INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

SINCE THE PIONEERING EXPERIMENTS of Haller, Galvani, and others more than 300 years ago, physiologists and biochemists have made extraordinary progress in understanding how muscles work (1, 12, 16, 31). Despite significant advances in this field, one problem that continues to challenge 21st century science is the etiology of muscle fatigue. The inability of a muscle to maintain a desired level of performance (or power output) is complex and involves a multiplicity of systems from the excitatory drive to the higher motor centers (e.g., motivation or effort) to the force-generating contractile apparatus itself (1, 12, 31). The relative contribution of these different sites to fatigue depends on the intensity, duration, and type of activity being performed (3).

In single muscle fibers and isolated whole muscles, fatigue has been associated with falls in ATP, phosphocreatine (PCr), and glycogen, increases in extracellular K⁺, intracellular Na⁺, cytosolic H⁺, lactate, and/or total P_i, and changes in free cytoplasmic [Ca²⁺] (1, 12, 31). In 1978, Fitts and Hollozy (13) observed a correlation between fatigue [a decline in twitch force (P_t) and tetanic tension (P_o)], increased lactate, and twitch broadening in isolated frog sartorius at 22°C stimulated at 30 Hz. A few years later, Sahlin et al. (26, 27) observed a similar phenomenon in isolated extensor digitorum longus of rats stimulated to contract isometrically at 2 Hz under CO₂-induced anaerobic conditions. Fatigue was associated with decreased ATP, increased lactate, and increased H⁺. On the basis of skinned fiber studies (10), Sahlin et al. concluded the prolonged relaxation times associated with fatigue were due to the pH-dependent sarcoplasmic reticulum (SR) Ca²⁺ reuptake process. Other candidates for the slowing of relaxation in isolated muscles have been a fall in the free energy of ATP hydrolysis (G'_ATP) and the accumulation of diprotonated phosphate (H₂PO). The latter proposal has received some support from skinned single fibers, rat gastrocnemius, and human muscle (22-24, 30).

One aspect of fatigue not widely studied is the role of the products of ATP hydrolysis (ATP, free [ADP], total free [P_i], free [Mg²⁺], and H⁺), glycogen, and lactate in vivo. The aim of the present study was to examine the effects of the products of ATP hydrolysis in rat gastrocnemius in vivo stimulated continuously at 2 Hz. This frequency was chosen because we previously showed it exceeds the apparent mitochondrial maximal velocity of aerobic respiration by a factor of 2.5 (4). During the early phase of constant stimulation, we report transient changes in H⁺, lactate, and H₂PO that were associated with transient increases in twitch broadening but not force decline. Force decline appears largely due to the fatigue of white fibers, not to the products of ATP hydrolysis inhibiting actomyosin activity. Other potential sites of fatigue include glycogen depletion and/or failure of nerve impulse transmission in these fibers.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animal preparation. Adult male Sprague-Dawley rats (300-400 g) were purchased from the Animal Resources Centre (Canningvale, Western Australia) and housed in the small animal facility at James Cook University. Rats were fed food and water ad libitum until the time of the experiment. Animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/ml) and placed on a thermostatically regulated heating pad (37°C). A tracheotomy was performed, and animals were ventilated on room air at a rate of 75 breaths/min and tidal volume of 25 ml to maintain arterial pH and blood gases in good physiological condition. The right carotid artery was cannulated with heparinized (100 U/ml) PE-50 tubing connected to a pressure transducer for continuous measurement of blood pressure, the output of which was displayed on a MacLab. Blood gas measurements were performed on a Ciba-Corning 865 analyzer. Anesthesia was maintained by passing 1.0% isoflurane at a rate of 0.5 ml/min via artificial ventilation. Although isoflurane and other inhaled anesthetics such as halothane, nitrous oxide, and barbiturate narcotics augment neuromuscular block by inhibiting motor end-plate transmission, they also exhibit muscle relaxant properties (21). Although the mechanisms remain unclear, the possibility exists that the use of isoflurane may impact on the results.

Muscle stimulation and force measurements. Muscles were prepared for sciatic nerve stimulation as previously described in Cieslar and Dobson (4). Briefly, the animal was placed in a custom-built Perspex cradle fitted with a 37°C water-heated pad, and the right hindleg was immobilized onto a mechanical ground in the cradle with brass pins. The tendon of the gastrocnemius was isolated and attached to a calibrated ultraprecision miniload cell (Transducer Techniques, MDB10) with noncompliant 2-0 silk string. The load cell was fixed onto a rack-and-pinion device that allowed adjustment of muscle length. The gastrocnemius was exposed by blunt dissection and covered with thin plastic film to prevent the exposed tissue from drying out. Isometric twitch contractions were induced by sciatic nerve stimulation using bipolar electrodes attached to a Harvard student stimulator. The length of the muscle was adjusted until maximum twitch force was obtained during a supramaximal contraction in response to a square-wave pulse (0.1 ms, 5-15 V). Isometric force development (in newtons) during stimulation was continuously displayed on a MacLab data-acquisition unit (Chart version 3.6).

Fatigue protocol. The animal was introduced into the bore of an Oxford 7.05-T horizontal NMR magnet. Muscles (n = 7) were stimulated to contract isometrically at 2 Hz (0.1-ms duration) for 20 min using a protocol described by Cieslar and Dobson (4). ³¹P NMR spectra were acquired in 4-min blocks (number of transitions = 128) while twitch force was continuously recorded and averaged every 2 min (4). These are difficult experiments to perform in the magnet, and shorter NMR acquisition times would have seriously compromised the quantitation of free Mg²⁺, pH, and phosphorus metabolites because of the lower signal to noise. As a result the 4-min time-averaged blocks (2-, 6-, 14-, and 18-min time average points) do not always correlate with the force measurements taken every 2 min. Preliminary experiments at 5, 10, and 50 Hz did not show the same patterns of twitch broadening and force decline observed at 2 Hz. The total twitch duration at 2 Hz was analyzed for the contraction time and relaxation time. The contraction time was defined as the time measured from the onset of isometric twitch development to the peak, whereas the time elapsed from the twitch peak to one-half tension was defined as the one-half relaxation time. Peak P_o was obtained in separate experiments (n = 4) by interrupting the constant-stimulation protocol of 2 Hz every 2 min with a 100-Hz stimulus (5 s).

NMR. ³¹P NMR experiments were performed at 121.47 MHz in the 110-mm bore of an Oxford 7.05-T superconducting magnet. A three-turn surface coil (14-mm OD) was placed at the center of the gastrocnemius muscle and served as transmitter and receiver. A small latex balloon filled with a solution of 10 mM phenylphosphonic acid (PPA) in saline was placed above the coil in the same geometric arrangement as the muscle below and served as an external standard. Magnetic field homogeneity was optimized by observing the off-resonance proton signal of muscle water. The surface coil was tuned and matched to resonate at 121.47 MHz. All NMR experiments were performed using a radio-frequency (RF) pulse of 10-µs duration that was transmitted at a near 90° flip angle by the surface coil. ³¹P NMR spectra were collected as 9,600 data points using a sweep width of 6,000 Hz. The free induction decays (FIDs) were acquired over 0.8 s with an interpulse delay time of 1 s. The FIDs were multiplied by a line-broadening factor equivalent to 20 Hz to improve signal-to-noise ratio. Resultant line widths for the in vivo muscle experiments were typically 40-60 Hz. The RF pulse was not synchronized with a nerve stimulation.

Analytical biochemical protocol. Parallel experiments were carried out on the laboratory bench, and muscles were freeze-clamped at rest and after 2, 6, 10, 14, and 18 min of 2-Hz stimulation using aluminum tongs precooled in liquid nitrogen. As mentioned above each of these time points on the bench corresponded with the midpoint of the ³¹P NMR spectra acquired in 4-min time-averaged blocks. The freeze-clamped muscles were ground to a fine powder under liquid nitrogen, the connective tissue was removed, and the powder was kept at 80°C until analysis of lactate, glycogen, pyruvate, PCr, and creatine (Cr). Conversion of total tissue contents (µmol/g wet wt) into intracellular concentrations (µmol/ml) was carried out using the published total tissue water contents (0.76 ml/g wet wt tissue) and intracellular space values (84% total tissue water) previously reported for rat gastrocnemius in vivo (5). Total tissue water (defined as g water/g wet wt) was determined by the difference in wet weight and dry weight after drying tissue for 48 h at 80°C.

Biochemical assays. All chemicals used in the metabolic assays were purchased from either Sigma Chemical or Boehringer Mannheim and were of the highest grade. Frozen tissue (~100 mg) was homogenized in equal volumes of ice-cold 0.1 M HCl in methanol and 3.6% perchloric acid (19) using glass beads in a high-speed Biospec mini-beadbeater. The homogenate was centrifuged (9,000 g; 2 min), and a volume of acid extract was removed and mixed with an aliquot of KHCO₃ (0.3 M and 0.2 M stocks) to neutralization (pH 6-7). The supernatant was immediately measured either spectrophotometrically or fluorometrically for pyruvate, lactate, PCr, and Cr according to the methods of Lowry and Passonneau (19). Total tissue glycogen was measured separately according to the method of Passonneau (25). Briefly, 1 ml of 0.5 M NaOH was added to frozen tissue (~50 mg) and heated in boiling water for 20-30 min to remove tissue free glucose. The suspension was made acidic with a known volume of 12 N HCl (pH <3.0). An aliquot of acid extract (~50 µl) was removed and added to 950 µl of 200 mM acetate buffer (pH 4.7) containing amylo--1,4--1,6-glucosidase to digest tissue glycogen. After a 2-h incubation, the acid extract was fluorometrically assayed for glucose (25).

Quantitation of NMR spectra. ³¹P NMR spectral intensities for the phosphorus-containing compounds were determined by computer integration using the VNMRX software. Saturation correction factors were determined from fully relaxed and control spectra by taking the ratio of the area under a given peak with a 20-s relaxation delay to the area of the spectra with a 1-s relaxation delay (4). The mean correction factors for the 10 mM PPA external standard, P_i, PCr, and -ATP were 2.20 ± 0.05, 1.80 ± 0.06, 1.56 ± 0.02, and 1.22 ± 0.02 (means ± SE, n = 18), respectively. Intracellular concentrations of the -ATP, PCr, and P_i were calculated by equating the spectral intensities of the saturation-corrected phosphorus metabolites to the spectral intensity of the saturation-corrected external standard (10 mM)


Intracellular pH (pH_i) was calculated from the chemical shift [, in parts per million (ppm)] of P_i relative to PCr in the ³¹P spectra using the NMR version of the Henderson-Hasselbalch equation

(1)

Intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was calculated from the observed chemical shift difference (, in ppm) between -P and -P resonances of ATP in the ³¹P spectra using the modified form of the London equation (14)

(2)

where  = [H⁺]/K_H,  = (K_D/K'_D), K_H is the dissociation constant for the H⁺/ATP⁴ equilibrium, K_D is the dissociation constant for the ATP⁴/Mg²⁺ equilibrium, and K'_D is the dissociation constant for the ATPH³/Mg²⁺ equilibrium. The parameters ₁, ₂, ₃, and ₄ were assigned published values of 10.600, 11.660, 8.165, and 8.52 ppm, respectively, K_D was 9.0 × 10⁵ M, K_H was 3.4 × 10⁷ M, and K'_D was 7.2 × 10⁴ M (14).

(3)

(4)

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1

(5)

Statistical analysis. All values are shown as means ± SE. Unless otherwise indicated, a one-way ANOVA was applied for statistical comparison of the means followed by a Newman-Keuls post hoc test. Linear regression analysis was used to evaluate the relationship between variables. Statistical significance was established at P < 0.05. The software package used for this analysis was SuperANOVA version 1.1 (Abacus Concepts, 1989).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isometric twitch contractile properties and characterization of fatigue in vivo. Time-dependent changes in peak P_t, peak P_o, contraction time, and relaxation time during continuous isometric contractions at 2 Hz are presented in Fig. 1. All mechanical data are expressed as a percentage of the initial value. P_t increased to values higher than the initial tension in the first 4 min and was accompanied by a marked broadening of the twitch duration (phase 1). In contrast peak P_o did not change significantly in phase 1. Prolonged twitch duration in phase 1 was associated with increases in the one-half relaxation time (20%) and the contraction time (10%) shown in Fig. 1B. After 4 min, the increased twitch duration was reversed and accompanied the rapid decline in P_t to 40% of the initial value at the end of stimulation (phase 2). By contrast, peak P_o decreased steadily between 4 and 10 min, then plateaued at 80% of the initial value (Fig. 1A). In our study, therefore, phase 1 was characterized by twitch force development and increased twitch duration, and phase 2 was characterized by the decline of twitch and tetanic force and decrease in twitch duration. The next stage of our study was to examine the relationship between muscle metabolism and the differences in isometric contractile properties in phases 1 and 2.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Isometric twitch contractile properties [tension (A) and twitch duration (B)] of rat gastrocnemius in vivo during 2-Hz constant stimulation. Phase 1 was characterized by an increase in peak twitch force (Pt) and twitch duration, which corresponds to an increase in the one-half relaxation time (RT) and contraction time (CT). Phase 2 was characterized by a decline in Pt and peak tetanic force (Po) and a decrease in twitch duration. Bars represent SEs from the mean.

Changes in free P_i, PCr, free Mg²+, cytosolic pH, and calculation of free ADP, H₂PO, and G'_ATP during constant stimulation of 2 Hz. ³¹P NMR-determined concentrations of PCr, ATP, free [Mg²⁺], total free [P_i], pH_i, and [H₂PO] and G'_ATP were determined at rest and after 2, 6, 10, 14, and 18 min (Table 1). Prestimulation or control value for the ratio PCr/ATP was 4.3, which is in accord with other NMR studies on rat gastrocnemius (20, 23, 28). Values in Table 1 represent the time-averaged mean of the NMR spectra acquired in 4-min blocks, keeping in mind that the isometric contractile properties were taken every 2 min. In the 0- to 4-min period (time averaged to 2 min corresponding to phase 1), PCr decreased by 60% (P < 0.05) with only a small change in ATP (4% fall). This major decrease in PCr was accompanied by a significant decrease in pH_i (7.21 to 6.91), a 10-fold increase in [H₂PO] (0.73 to 7.56 mM), 20% increase in free [Mg²⁺] (0.58 to 0.70 mM), a 5-fold increase in free [ADP] (18.3 to 91.2 µM), and a 6-fold increase in total free [P_i] (2.8 to 17.9 mM) (Table 1). The cytosolic G'_ATP fell dramatically from 65 to 55 kJ/mol (P < 0.05), corresponding to a fall of the cytosolic phosphorylation ratio from 219,000 to 3,000 M¹ (Table 1).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Time-dependent changes in H+ (A) and diprotonated inorganic phosphate (H2PO; B) during 2-Hz constant stimulation in rat gastrocnemius in vivo. Points represent time-averaged midpoints of the NMR spectra acquired in 4-min blocks. Bars represent SEs from the mean.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Recovery of H+ (A) and diprotonated inorganic phosphate (B) as a function of the recovery of prolonged relaxation in phase 2 (6, 10, 14, and 18 min) of our constant-stimulation protocol. The linear regression equation for H+ was [H+] = 0.21x  19.12 with r = 0.99 (P < 0.05) and for H2PO was [H2PO] = 0.72x  63.87 with r = 0.94 (P < 0.05), where x is the one-half relaxation time expressed as percentage of the initial value. Bars represent SEs from the mean, and the n values are found in Table 1.

Changes in glycogen, lactate, and pyruvate during constant stimulation of 2 Hz. Enzymatically determined concentrations of glycogen, lactate, and pyruvate are shown in Table 2. Tissues were frozen at times corresponding to the midpoint of the time-averaged NMR spectra. Glycogen decreased by ~17% (P < 0.05) in the first 2 min (phase 1), and lactate and pyruvate increased fivefold (P < 0.05) with no significant change in the lactate-to-pyruvate ratio. In phase 2, glycogen fell from 16.4 to 9.3 at a rate of 1.8 µmol · g¹ · min¹. Glycogen continued to decrease and the lactate-to-pyruvate ratio increased (P < 0.05), indicating a progressive reduction (decreased NAD/NADH) in the cytosolic environment. Between 10 and 14 min, the rate of glycogen utilization decreased to 0.59 µmol · g¹ · min¹ and was essentially zero after 14 min. The relationship between muscle glycogen and the decline in twitch force in phase 2 is shown in Fig. 4. A highly significant linear relation was found between 6 and 20 min where [glycogen] = 0.23x  2.0 with r = 0.99, where x is the decline in twitch force expressed as a percentage of initial value. By contrast, no correlations were found with total phosphate, free [ADP], free [Mg²⁺], or G'_ATP with either twitch broadening or force decline.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Glycogen as a function of twitch force decline in phase 2 (6, 10, 14, and 18 min) during 2-Hz constant stimulation. The linear regression equation for glycogen is [glycogen] = 0.23x  2.0 with r = 0.99 (P < 0.05), where x is the decline in twitch force expressed as a percentage of initial value. Bars represent SEs from the mean, and n values are found in Table 2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The primary goal of this study was to investigate the effects of the products of ATP hydrolysis and other kinetic and thermodynamic variables on the fatigue process in rat skeletal muscle in vivo. Muscles stimulated continuously at 2 Hz developed force in the first 4 min of stimulation accompanied by a 20% increase in relaxation time (phase 1). The prolonged twitch duration was associated with a decrease in cytosolic pH (6.91 to 6.58) and increases in lactate (8.41 to 12.6 µmol/g) and H₂PO (7.56 to 14 mM). Interestingly, changes in pH and H₂PO were reversed after 6 min (phase 2) despite an inhibition of force development between 6 and 20 min. This uncoupling of force decline from metabolic changes in H⁺, lactate, and H₂PO is an original finding and implicates other factors in the fatigue process.

Recovery of H+ and twitch broadening. As mentioned earlier, the prolonged relaxation is a common feature of muscle fatigue (1, 8, 9, 13, 27). Sahlin et al. (27) associated twitch broadening with muscle pH because muscles poisoned with iodoacetic acid (blocking of glycolysis and lactate production) showed a fall in tension but no change in pH or broadening. The transient change in H⁺ and twitch broadening in our study strongly implicates this monovalent ion in vivo (Figs. 1B and 2A). Two possible sites for slowing of relaxation include H⁺ inhibition of the cross-bridge cycle and/or the SR ATPase pump. The 20% decrease in peak tetanic tension (5-s stimulation at 100 Hz) measured at different times over the course of our experiment provides some support for a decreased number of active cross bridges (Fig. 3). However, the fall in peak tetanic tension was not transient with twitch broadening implicating other factors (Fig. 1A).

Diprotonated phosphate and twitch broadening. Over the past 20 years, interest has shifted from the effect of total [P_i] to the involvement of the diprotonated species of phosphate (H₂PO) in muscle fatigue. Our study shows that NMR-derived total free [P_i] increased six- to eightfold after the first 2 min but remained around 19-20 mM for the remainder of the stimulation protocol (Table 1). Total [P_i] was therefore not considered a likely candidate for twitch broadening in our fatigue protocol. In contrast to total [P_i], our results show a correlation between transient twofold rise in H₂PO (7.56 to 14 mM) and transient twitch broadening (Figs. 1B and 2B). However, because H₂PO is calculated from the pH dependency of the mono- and dibasic phosphate equilibrium, it is exceedingly difficult to separate their reported effects from those of H⁺.

Force decline in phase 2. The recovery of H⁺ and H₂PO toward near-control values after twitch force decline suggests that these metabolites do not inhibit force production in phase 2, nor is the depression of force likely linked to total [P_i], free cytosolic [ADP], or cellular G'_ATP and/or phosphorylation ratios (Table 1). No relationship was observed between these metabolites or thermodynamic parameters and the decline in twitch force (or peak tetanic force) during ongoing fatiguing stimulation (Table 1). Other in vivo studies have shown incremental increases in total P_i that were concomitant with a decrease in force at different stimulation frequencies (22, 23, 30). We argue that such correlation may reflect a closely coupled metabolic system and not be causative of the fatigue process per se. Although we are not totally discounting the contribution of each metabolite given the complexity of the in vivo system, no relationship was found between any of these parameters and force decline in our fatigue protocol.

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Perspectives