INTRODUCTION

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THE ONSET OF CONTRACTILE WORK in skeletal muscle is characterized by increases in metabolic rate that are dependent on the work intensity. The demand for ATP is met by substrate-level phosphorylation (phosphocreatine hydrolysis and anaerobic glycolysis) and aerobic metabolism (oxidative phosphorylation), and the differential activation of each phosphorylating system during exercise depends on work intensity and the metabolic characteristics of the contracting muscle. At the onset of relatively high-intensity work, the imposed energy demand is primarily met by substrate-level phosphorylation, and as exercise proceeds, the contribution of ATP from oxidative phosphorylation increases (see Ref. 18). If work continues beyond a critical point (determined as a function of work intensity), muscle performance becomes compromised and fatigue ensues. The precise mechanisms relating the reduction of muscle performance during fatigue and the differential energy contribution from anaerobic to aerobic metabolism have not been well elucidated.

Previous experiments investigating the relative contribution of energy from each phosphorylating system have been confounded by muscle fiber type and oxygen delivery heterogeneity. To circumvent these problems, the isolated, single muscle fiber model has been used by various investigators to study metabolic processes related to muscle fatigue (see Refs. 4, 17, 18). In the single muscle fiber preparation, individual fibers are discriminated according to fiber type, and the single fiber is surrounded by a homogeneous medium. Three distinct contractile phases have been described in single Xenopus skeletal muscle fibers when the frequency (0.25, 0.33, 0.5, 1.0 Hz) of repetitive tetanic contractions is increased every 2 min until fatigue ensues (17). Phase 1 corresponds to an initial decrease in developed tension to ~85% initial maximal tension, phase 2 is characterized by a steady-state period of force production, and phase 3 describes a final decrease in developed tension. It has been suggested that the phases may correspond to the activation and failure of distinct energy-supplying pathways (15, 17). To investigate the relative contribution of the varied energy-supplying pathways, we compared the performance of isolated, single Xenopus fast-twitch fibers subjected to similar work protocols during oxygen-limited and -unlimited conditions. Calculated ATPase rates and O₂ consumption kinetic values were then used to examine the temporal correlation between the ATP generation from each system and the three previously described contractile phases in this model.

	METHOD

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Adult female Xenopus laevis were doubly pithed and decapitated. Lumbrical muscles were removed, and single, living, type-1 ("fast twitch") muscle fibers (n = 6) were microdissected from the muscle. Myocytes were fiber typed during microdissection according to twitch characteristics and appearance under dark-field illumination (13). Cross-sectional area was determined by averaging the three largest and three smallest diameters measured with an optical reticle. Platinum clips were attached to the tendons, and the fibers were mounted in a glass chamber and continually perfused with Ringer solution (in mM: 112 NaCl, 1.87 KCl, 0.82 CaCl₂, 2.38 NaHCO₃, and 0.07 NaH₂PO₄) at 20°C and 7.0 pH. Before each contraction period, the resting fiber was passively stretched until the force produced by a single tetanic contraction was maximal.

Tetanic contractions were induced by direct stimulation (50 impulses/s of 1-ms duration at 9 V, with a train duration of 200 ms) with platinum conducting electrodes on either side of the fiber, using a Grass (model S48, Quincy, MA) stimulator. Force development (P) was measured with a 5 g force transducer system (Aurora Scientific model 400A, Ontario, Canada). A Biopac Systems MP100WSW (Santa Barbara, CA) analog-to-digital converter was used to transform the analog signal, and the digital data were analyzed with AcqKnowledgeIII 3.2.6 software (Biopac Systems). The waveforms were also recorded on a flatbed chart recorder. Developed tension was analyzed at 10-s intervals using AcqKnowledgeIII software. Fatigue was determined by comparing individual peak tensions (P₀) to the highest peak tension within that run.

Experimental protocol. Each fiber (n = 6) had its rate of fatigue development measured during two separate work bouts (with a 45-min rest in between). Each fiber was subjected to both a high (159 mmHg) and a low PO₂ treatment (0 mmHg). To minimize possible order effects, three fibers were initially subjected to the high PO₂ condition, and three fibers were initially subjected to the low PO₂ condition, thereby incorporating both possible orders of oxygenation. During each work bout, fibers were stimulated at increasing frequencies (0.25, 0.33, and 0.5 Hz) in a sequential manner, with each stimulation frequency lasting 2 min until force fell to 30% of maximal. Low PO₂ of the Ringer solution was generated by 95% N₂-5% CO₂ aeration and maintained in the chamber with constant perfusion. The PO₂ of the Ringer solution in the chamber was monitored with a Clark-style electrode (Diamond General, Ann Arbor, MI) placed adjacent to the working fiber.

Calculations. It has been demonstrated in Xenopus type-1 fibers that during moderate fatigue (P/P₀ > 0.5) the fall in ATP hydrolysis is correlated with the fall in tension (12), and this relationship was used to calculate the change in ATP hydrolysis rates. A previously described (3) maximum rate of ATP hydrolysis (20 nmol ATP · s¹ · mg dry wt¹) for Xenopus type-1 fibers similar to those used in the present study was applied, and the values were normalized. Dry weight was estimated from volume using a previously described (3) transform coefficient (0.23 mg/ml). Relative rates of ATP generation from oxidative phosphorylation were calculated from the difference in ATP hydrolysis rates between the oxygen-unlimited condition (ATP supplied by substrate phosphorylation + oxidative phosphorylation) and the oxygen-limited condition (ATP supplied by substrate-level phosphorylation only).

The energy needed to sustain maximal contractions available from phosphocreatine (PCr) hydrolysis alone was calculated from the maximal rate of ATP hydrolysis (3) and previously measured (12) intracellular concentrations of PCr in Xenopus type-1 fibers at rest. ATP available for contractions from PCr hydrolysis was calculated from the creatine kinase reaction
Statistics. Force development was compared between treatments at consecutive 10-s periods for all fibers and analyzed for statistical differences. Two-way repeated-measures ANOVA was used for the statistical analysis. In all analyses, the 0.05 level of significance was used.

	RESULTS

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The mean fiber diameter was 76 ± 4 µm. The PO₂ was maintained at 0 mmHg during the duration of the low PO₂ treatment and 159 mmHg during the high PO₂ treatment. No significant difference was found in the initial peak tensions between the high (334 ± 57 kPa) and the low (325 ± 41 kPa) PO₂ condition. Figure 1 compares the developed tension between the high and the low PO₂ condition for the first 90 s, analyzed at 10-s intervals. No significant difference was found between the peak tensions until the 60 s time point. At the 60 s time point, developed tension for the high PO₂ treatment (P/P₀ = 0.96 ± 0.02) was significantly higher (P < 0.05) than the low PO₂ treatment (P/P₀ = 0.92 ± 0.02).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Relative developed tension (P/P0) of single type-1 (fast twitch) fibers (n = 6) during first 90 s after onset of tetanic contractions (0.25 Hz) during a high (159 mmHg) and a low PO2 treatment (0 mmHg). * Significant difference (P < 0.05) between treatments.

Figure 2 compares the developed tensions between treatments for the entire work protocol. After 60 s, developed tension during the low PO₂ condition continued to decline in a somewhat linear fashion, whereas developed tension during the high PO₂ condition remained at a steady-state until stimulation frequency was increased. At 180 s, developed tension during the high PO₂ condition began to decline. After 180 s, the rate of tension decline (slope of the curve in Fig. 2) during the high PO₂ condition gradually increased until 240 s, at which point the developed tension began to fall in a linear fashion, at a rate similar to the low PO₂ condition at the same time point. Time to fatigue (P/P₀ = 0.3) was reached significantly sooner during the low (250 ± 16 s) than the high PO₂ condition (367 ± 28 s).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Relative P/P0 of single type-1 fibers (n = 6) during a fatiguing (until P/P0 < 0.3) work protocol during high and low PO2 treatment. Mean time to fatigue (P/P0 = 0.3) is indicated by  for low PO2 condition and  for high PO2 condition. * Significant difference (P < 0.05) between treatments.

Figure 3 illustrates the aerobic ATP production calculated from the difference in ATP hydrolysis rates between the two treatments. After 240 s after the onset of work, the slope of the disparity in ATP hydrolysis rates approached zero, indicating that the decrease in ATP hydrolysis rates was now similar between treatments. The decrease in ATP hydrolysis remained similar between treatments until stimulation was terminated.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Aerobic ATP production calculated from difference in ATP hydrolysis rates between high and low PO2 treatments. Slope of curve therefore is an indirect measurement of oxygen consumption (O2). A slope of 0 corresponds to no further increase in O2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHOD RESULTS DISCUSSION REFERENCES

The results of this study demonstrate that in Xenopus single, fast-twitch muscle fibers 1) during the first 50 s after the onset of 0.25-Hz tetanic contractions, no difference in force production was observed between conditions of unlimited- and limited-oxygen availability, suggesting an ability of substrate-level phosphorylation to compensate for the absence of oxidative phosphorylation; 2) a significant difference in force production was observed between treatments after 60 s of work, with force continuing to decrease during the low-oxygen condition (likely due to the greater disruption of metabolic homeostasis) while being maintained in the high-oxygen condition; 3) developed tension began to decline at 240 s during the high-oxygen condition at a rate similar to that observed during fatigue in the oxygen-limited condition; and 4) a temporal relationship was observed between the oxygen-dependent and -independent phases and previously described phases (17) of muscle contractility in these single fibers.

Metabolism and fatigue. Skeletal muscle has the extraordinary capacity to adapt to an immense and immediate increase in ATP demand. Although ATP turnover rates can increase several hundred-fold from resting values at the onset of intense exercise (8), intracellular ATP concentrations remain relatively unchanged (2, 11). The source of energy for rephosphorylation of ATP during relatively high-intensity work is determined by the time after the onset of work and the metabolic profile of the muscle being studied. In general, the initial onset of force production in skeletal muscle during high-intensity exercise is associated with intracellular changes indicating that ADP phosphorylation is predominantly supplied by substrate-level phosphorylation (anaerobic glycolysis and PCr hydrolysis). PCr stores begin to decline, intracellular P_i and ADP concentrations increase, and oxygen consumption is minimal (see Refs. 7, 18). As high-intensity work continues, oxidative phosphorylation increases progressively until a maximal oxidative capacity is reached (o_{2 max}) (see Ref. 1).

It was proposed (14) that when the demand for ATP exceeds the phosphorylating capacity of these systems, an imbalance between supply and demand develops and performance becomes compromised, indicated by an inability to generate force similar to the nonfatigued state. This may be a result of substrate limitation to oxidative phosphorylation (NADH, etc.), inadequate mitochondrial density, or insufficient oxygen availability as the terminal electron acceptor in oxidative phosphorylation. In the present study, the greater reliance on substrate-level phosphorylation during the low PO₂ condition likely resulted in an earlier disruption of metabolic homeostasis leading to a more rapid decline in developed tension.

Force production in single muscle fibers. In previous experiments, we have demonstrated an oxygen dependence on force production in working whole skeletal muscle (9-11). The dependence on oxygen availability at the cellular level is difficult to quantify in whole muscle, due to muscle fiber type and blood flow heterogeneity and oxygen delivery kinetics. In the present experiments, the working, isolated single muscle fiber model has been used to avoid some of these confounding factors. Extracellular oxygenation was homogenous and easily determined, and because the fibers were constantly perfused, the possibility of unstirred layers was minimized. Therefore oxygen availability was immediate, and the onset kinetics of oxidative phosphorylation were determined by intracellular metabolic changes and the diffusive properties of oxygen, independent of hemodynamics. In addition, a single muscle fiber type was used to minimize differences in mitochondrial density and, therefore, oxidative capacity.

Fatigue development in isolated single fibers during repetitive tetanic contractions has been characterized by three distinct phases of tension development (see Ref. 17) that occur as stimulation frequency is increased. The first phase entails a decline in developed tension to ~80-90% of initial tetanic contractions, lasting ~14-20 contractions, and is similar among all fiber types. Phase 2 is characterized as a period of steady-state force production, the duration of which is dependent on the oxidative capacity of the fiber investigated. Phase 3 occurs with a period of rapid force decline as stimulation frequency is increased. Using single fibers from a mouse and a similar work protocol, Westerblad and Allen (15, 16) temporally correlated the three phases of force production with measurements of free intracellular calcium concentrations ([Ca²⁺]_i). They noted that phase 1 was associated with an increase in free [Ca²⁺]_i, and the decline in developed tension was therefore likely due to an inhibition of force production by an accumulation of intracellular metabolites (P_i and ADP). The steady-state contractions defining phase 2 were associated with a stable level of free [Ca²⁺]_i. Phase 3 was correlated with both a decline in [Ca²⁺]_i and a decreased calcium sensitivity.

The results of the present study, in which oxygen delivery to the mitochondria was limited in fast-twitch muscle fibers, provide strong evidence that during the initial contractions substrate-level phosphorylation has the capacity to compensate for a reduction in oxidative phosphorylation (Fig. 1). Similar observations were made by Westerblad and Allen (15) in working mouse fibers (n = 3) exposed to anoxia and cyanide. This suggests that substrate-level phosphorylation provides the major source of ADP phosphorylation at the onset of work.

This oxygen-independent time period corresponds with the time domain previously defined as phase 1 (17). At the onset of intense contractions in whole muscle, it has been demonstrated that the supply of energy is predominantly derived from PCr hydrolysis (7). Unfortunately, the differential energy contribution of each anaerobic pathway during the onset of contractions in single fibers is difficult to determine. However, it is possible to estimate the ADP phosphorylating capacity of the intracellular PCr reserve in single fibers, independent of glycolysis. This would provide a minimum temporal estimate of an oxygen-independent phase, because the energy contribution from anaerobic glycolysis during this period certainly provides some high-energy phosphates. Using published values of maximal ATP hydrolysis rates (3) and resting PCr concentrations in single, type-1 Xenopus lumbrical fibers (12), we calculated (equation 1) that 20 ± 1 maximal contractions worth of ATP are available from PCr alone, corresponding to ~80 s (at 0.25 Hz stimulation rate) after the onset of work. It is interesting that a reduction in developed tension is observed (60 s after the onset of work) before this minimum estimated time point at which PCr stores are exhausted. This suggests that force was attenuated before the absolute depletion of immediate energy stores as an energy-conserving mechanism, supporting the notion of fatigue as a protective mechanism (20).

Despite the characterization of the amphibian type-1 fibers used in the present experiment as "nonoxidative," only the initial ~15 contractions (first 60 s) were capable of being oxygen independent. This is similar to a recent finding (19) in exerising humans in which it was demonstrated that the initial 60 s of high-intensity work was unaffected by reductions in oxidative capacity. In the present study, after phase 1 (60 s after the onset of work), a significant difference in developed tension was observed between the high and the low PO₂ condition (Fig. 1). Developed tension continued to decline with increasing contraction frequency in a somewhat linear fashion (Fig. 2) for each subsequent measurement during the oxygen-limited condition, whereas force was maintained until 180 s after the onset of work during the high PO₂ condition. Maintenance of steady-state contractions during the high PO₂ condition only began to decline significantly after 180 s. The rate of fatigue progressively increased during the high PO₂ condition until the 240 s time point. Beyond 240 s, developed tension fell at a rate similar to the oxygen-limited condition, corresponding to the end of phase 2 and the onset of phase 3.

The difference in calculated ATP hydrolysis rates between the two PO₂ treatments is shown in Fig. 3. Because the difference in ATP hydrolysis rates between the conditions was the result of an imposed limitation to oxidative phosphorylation, the slope of the disparity in ATP hydrolysis rates is a correlate of oxygen use, and therefore provides an estimate of oxygen uptake (O₂) in these single fibers. The onset kinetics of the estimated O₂ are similar to previous kinetics measurements in single muscle fibers (12). Phase 2, in which a period of steady-state force production occurs, corresponds with that period in which oxidative metabolism has increased to meet the demand for ATP. The disparity in ATP hydrolysis rates (see Fig. 3) continues to increase until a plateau (slope ~0) is reached. At this point, ATP hydrolysis declines at a similar rate during both treatments. In the high PO₂ condition, with increasing stimulation frequency, this point initiates phase 3. This period corresponds to the work level at which ATP turnover exceeds aerobic capacity, and tension declines. The extension can be made that this point correlates with the o_{2 max}. An imposed ATP demand surpassing the oxidative capacity of the cell would then lead to an imbalance in the ATP supply/demand ratio. The significant increase in intracellular metabolites (P_i, ADP, and H⁺) associated with severe fatigue (phase 3) has been shown to directly inhibit the release of Ca²⁺ from the sarcoplasmic reticulum (see Ref. 17) and to inhibit the contractile apparatus (5, 6). This is in agreement with the decreases in [Ca²⁺]_i and Ca²⁺ sensitivity previously observed during stage 3 (15, 16).

In summary, the results of this study provide evidence for an oxygen dependency in "nonoxidative" (Xenopus type-1) fibers during relatively high-intensity contractions and a temporal relationship between the differential activation of the phosphorylating pathways and the three previously described contractile phases in these single muscle fibers. Specifically, phase 1 corresponds to an oxygen-independent phase where the imposed demand for ATP is primarily met by substrate-level phosphorylation and lasts ~60 s when the contraction frequency is 0.25 Hz. This is followed by an oxygen-dependent period, corresponding to phase 2, in which tension is maintained at a steady-state level, during which oxidative phosphorylation increases until a maximal level is reached with increasing stimulation frequency. Phase 3, characterized by an accelerated decline in developed tension, corresponds to the point at which the calculated maximal oxidative capacity of the working fiber is reached and ATP demand exceeds production.

Perspectives