INTRODUCTION

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THE REGULATORY EFFECT of O₂ concentration ([O₂]) on oxidative phosphorylation during exercise is an issue that presently remains unresolved due, in part, to differences associated with work intensity and the organ/system studied. Within isolated mitochondria, as PO₂ falls across a physiological range, cytochrome c becomes more reduced, whereas O₂ consumption (O₂) remains uniform to levels near 1 Torr (32, 33). In intact, exercising muscle, the microcirculatory PO₂ considered rate limiting at the level of the mitochondria is dependent, in part, on the capillary-to-myocyte interface, fiber type, and mitochondrial capacity. In addition to the complexity of examining the effect of [O₂] on steady-state O₂, Hogan and colleagues (12, 13) demonstrated that the effect of O₂ delivery (O₂) on maximal O₂ is further confounded by both convective and conductive issues associated with the interaction between PO₂ and O₂. Experiments designed to reduce O₂ availability, even at levels thought well above that considered rate-limiting, slow the O₂ kinetic response to a step-wise increase in metabolic rate (4, 17, 21). This is of physiological significance in that a slower O₂ on-kinetic response necessitates greater reliance on substrate-level phosphorylation to meet initial ATP requirements of the working muscle, which may result in an earlier onset of fatigue.

Proton magnetic resonance spectroscopy permits detection of myoglobin (Mb)-associated PO₂ in whole muscle of exercising humans. Controversy exists regarding the PO₂ response to graded exercise in that one study demonstrated that Mb desaturates rapidly to ~3 Torr with low exercise intensities and remains at that level at intensities near maximal (25). Contrasting work reported a step-wise reduction in Mb saturation with increasing work intensities (24). Recently, our laboratory (16) demonstrated a proportionately larger reduction in frog single myocyte intracellular PO₂ (PI_O2) with increasing contraction frequency, which is in agreement with that expected from muscle cells lacking Mb (10). Mathematical modeling of Groebe and Thews (9) predicts that capillary PO₂ of ~27 Torr is adequate for the maintenance of nonanoxic areas within myocytes of heavily working "red" muscle, which is consistent with in vivo findings (27, 31). For cells lacking Mb, the authors (9) calculate that capillary PO₂ would have to be approximately twofold higher (>55 Torr) to avoid intracellular areas of zero PO₂ under similar heavy-intensity exercise. To date, the PI_O2 in single muscle cells in vivo at which cell function becomes compromised during contractions remains undetermined.

The frog isolated single myocyte preparation in conjunction with phosphorescence quenching techniques for assessment of PI_O2 (11) represents an excellent tool to study the role of O₂ availability on metabolic function without complications associated with muscle O₂ diffusing capacity (i.e., capillarity, O₂ vs. PO₂, O₂/metabolic matching). For muscle cells lacking Mb such as in the Xenopus laevis, O₂ flux (i.e., O₂) is proportional to the fall in PI_O2, and it might be expected that at a higher extracellular PO₂ (PE_O2), initial rates of O₂ flux may be greater due to the higher driving gradient of the PE_O2. Thus the purpose of this investigation was to study the effect of varying PE_O2 on PI_O2 dynamics in single muscle cells from rest across a bout of high-intensity tetanic contractions. We tested two general hypotheses: 1) the initial rate of change of PI_O2 (calculated as net fall in PI_O2/, where  is the time constant) would be slower at a lower PE_O2 (low, ~20 Torr) compared with that at higher values [i.e., ~40 (moderate) or ~60 Torr (high)] and 2) the magnitude of the fall in PI_O2, corresponding to a proportional increase in O₂, would be progressively greater at each respectively higher PE_O2.

	METHODS

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Female adult African clawed frogs (X. laevis) were used in this investigation. All procedures were approved by the University of California-San Diego animal use and care committee and conform to National Institutes of Health standards.

Myocyte preparation. Single muscle cells (n = 12; 80.5 ± 16.0 µg) were isolated and prepared as described previously (11). Briefly, frogs were doubly pithed, and the lumbrical muscles (II-IV) were removed from the hindfeet. Single myocytes were dissected with tendons intact in a chamber of physiological Ringer solution. Myocyte fiber type was assessed during dissection according to twitch characteristics and appearance under dark-field illumination (30). Cells were microinjected with a solution consisting of 0.5 mM Pd-meso-tetra (4-carboxyphenyl)porphine bound to bovine serum albumin (for phosphorescence quenching as described in Assessment of PI_O2) and 10 mM fura 2 (Molecular Probes, Eugene, OR) by micropipette pressure injection (PV830 pneumatic picopump, World Precision Instruments, Sarasota, FL). The fura 2 was injected for direct visual confirmation of cell injection at an excitation light of 390 nm. After microinjection, cells were given a minimum of 1 h of recovery.

Experimental protocol. Platinum clips were attached to the tendons of each myocyte to facilitate fiber positioning within the Ringer's solution-filled chamber. One end of the tendon was fixed, whereas the other end was attached to an adjustable force transducer (model 400A, Aurora Scientific, Aurora, Ontario, Canada), allowing the muscle to be set at optimum muscle length (i.e., length at which maximal tetanic force is produced). The analog signal from the force transducer was recorded via a data acquisition system (AcqKnowledge, Biopac Systems, Santa Barbara, CA) for subsequent analysis. Fibers were perfused throughout the experiment with Ringer's solution previously equilibrated with 5% CO₂ and 3, 5, or 8% O₂ in N₂ balance. Constant perfusion was maintained throughout the protocol to maintain the experimental PE_O2 [i.e., ~20 (low), 40 (moderate), or 60 Torr (high)] and to reduce the possible occurrence of an appreciable unstirred layer surrounding the cell. Tetanic contractions were elicited by using direct (8-10 V) stimulation of the muscle (model S48, Grass Instruments, Warwick, RI). The stimulation protocol consisted of 200-ms trains of 70-Hz impulses of 1-ms duration. Myocytes were subjected to trials of ~2 min at a stimulation frequency of 3 contractions every 2 s with a 5-min recovery period at each of the three PE_O2. The order of PE_O2 treatments was such that, of the 12 cells in which data are reported, 2 cells each were subjected to one of the six possible order combinations. After the contraction protocol, the fibers were mounted at a constant muscle length, and fiber width (widest and narrowest at 2 locations) and length measurements were taken in duplicate. Volume (V) was calculated by assuming an ellipse as V =  · r(1) · r(2) · length, where r is the cell radius (2) and was converted to a mass (1.10 g/cm³).

Assessment of PI_O2. The myocyte was observed with a Nikon ×40 fluor objective (0.70 numerical aperture). The phosphorescence quenching of the porphyrin compound within the myocyte was measured via a system consisting of a flash lamp (Oxygen Enterprises, Philadelphia, PA), a 425-nm band-pass excitation filter, a 630-nm cut-on emission filter, and a photomultiplier tube for collection of the phosphorescence signal. To calculate phosphorescence lifetimes from the intracellular O₂ probe, the phosphorescent decay curves from a series of 10 flashes (15 Hz) were averaged, and a monoexponential function was fit to the subsequent best-fit decay curve (analysis software from Medical Systems, Greenvale, NY). The O₂ dependence of phosphorescence quenching is described by the Stern-Volmer equation, where
Thus PO₂ = (_o/  1)/(k_qo), where _o and  are the phosphorescence lifetimes at anoxia and a given PO₂, respectively, and k_q is the quenching constant (in Torr/s), is a second-order rate constant that is related to the frequency of collisions between O₂ and the excited triplet state of the porphyrin and the probability of energy transfer when collisions occur. The constants for the compound used, Pd-meso-tetra(4-carboxyphenyl)porphine bound to albumin in solution, have been well characterized (22), and for this preparation, k_q and _o were set at 690 Torr/s and 100 µs, respectively (11). Phosphorescent decay curves were recorded every 4 s from each cell throughout the experimental period.

Kinetic modeling. For kinetic fitting of PI_O2 dynamics from resting baseline to PI_O2 steady state during contractions, kaliedagraph data analysis software (Synergy Software, Reading, PA) was used. A monoexponential model was used as follows
where t is elapsed time after contractions onset, b is resting baseline, A is asymptotic amplitude for the exponential curve, TD is time delay, and  the time constant.

Statistical analysis. Data are presented as means ± SE. Differences in mean values among the different PE_O2 were tested with a one-way ANOVA with repeated measures. When F values were significant, post hoc analysis of all pairwise multiple comparisons was performed via the Student-Neuman-Keuls test. Data were regressed linearly by using standard least-squares techniques. Statistical significance was accepted at P < 0.05.

	RESULTS

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Neither beginning nor ending relative force production differed significantly (P > 0.05) across the range of residing PE_O2 (Fig. 1).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Normalized initial and end force production (means ± SE) for single myocytes (n = 12) at 3 extracellular PO2 treatments. Although force production declined significantly (all P < 0.05) for each trial from beginning to end, there were no differences (P > 0.05) among trials.

Mean PI_O2 data for all 12 myocytes from rest and in response to electrical stimulation at the varying PE_O2 are shown in Fig. 2. The steady-state PI_O2 during contractions was not significantly different (P > 0.05) at the low and moderate PE_O2 (i.e., ~3 and 6 Torr, respectively); however, it was elevated significantly (P < 0.05) at the high (~17 Torr) compared with both the low and moderate PE_O2 (Fig. 3A). There was a significant increase (all P < 0.05) in the fall of PI_O2 at each sequentially greater PE_O2 (Fig. 3B).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Mean (±SE) intracellular PO2 profiles for all 12 single myocytes at rest (first 20 s) and in response to electrically induced tetanic contractions at the 3 extracellular PO2 treatments.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   A: mean (±SE) intracellular PO2 for single skeletal myocytes (n = 12) at rest and during steady-state contractions. B: net fall (means ± SE) in intracellular PO2 from rest to steady-state levels. * P < 0.05 vs. low. # P < 0.05 vs. moderate.

The PI_O2 kinetic parameters for the rest-to-contraction transitions, including the TD before the fall in PI_O2, the  for the fall in PI_O2, and the mean response time (MRT; TD + ), are shown in Fig. 4. No significant differences in TD existed (P > 0.05) at the three PE_O2 (Fig. 4). The  was significantly faster at the low compared with high PE_O2 (P < 0.05; Fig. 4). In addition, the MRT slowed significantly at high (61.8 ± 9.4 s) compared with both low (35.2 ± 5.1 s; P < 0.05 vs. high) and moderate (43.3 ± 6.4 s; P < 0.05 vs. high) PE_O2 (Fig. 4). MRT was correlated positively (r = 0.965) with the net fall in PI_O2 (Fig. 5). Because of the linear relationship between the net fall in PI_O2 (i.e., A) and the speed of that reduction (i.e., ), the initial rate of change in PI_O2, calculated as A/, was not different (P > 0.05) between PE_O2 trials (low, 1.3 ± 0.2; moderate, 1.6 ± 0.3; high, 1.3 ± 0.2 Torr/s; Fig. 5).

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Kinetic parameters (means ± SE) for fall in intracellular PO2 in response to electrically induced tetanic contractions at the 3 extracellular PO2 treatments. * P < 0.05 vs. high.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   A: mean response time (mean data for 12 myocytes at each of the 3 extracellular PO2 treatments) for the fall in intracellular PO2 in response to tetanic contractions linearly correlated (dashed line) to the net fall in intracellular PO2. B: initial metabolic response, calculated as [amplitude (A)/time constant ()], was not significantly different (P >0.05) among extracellular PO2 trials.

	DISCUSSION

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The critical in vivo PE_O2 at which oxidative phosphorylation becomes compromised is uncertain. This present investigation provides data regarding the effects of PE_O2 on the oxidative metabolic capacity in intact, single skeletal muscle cells lacking Mb. Our results suggest that Mb-deficient cells depend critically on O₂ at levels significantly higher than that expected to be seen physiologically in vivo to attain peak metabolic rates. Furthermore, our findings that the initial rate of change in PI_O2 (i.e., A/) was not different among PE_O2 trials (across the range of 20-60 Torr) demonstrates that [O₂] does not play an integral role in setting the initial metabolic response to a heavy bout of fatiguing contractions in these single frog myocytes.

Extracellular-to-intracellular O₂ gradient. The O₂ gradient between the myocyte and extracellular fluid, as reported in the present investigation, was 50 Torr in some cells. To assess whether this is physiologically tenable or whether a large unstirred layer surrounding the myocyte was necessary to account for such a large intra- to extracellular O₂ gradient, the critical PO₂ at which anoxia will occur was calculated as described previously (3) as

where O₂ is O₂ diffusion.

3

1

1

Effects of PE_O2 on peak metabolism. The relationship between O₂ and PO₂ for single myocytes lacking Mb is described by Fick's law of diffusion as
where O₂m is the muscle O₂ constant and PO₂mito is mitochondrial PO₂. With the assumption that there was little or no gradient between cytosolic and mitochondrial PO₂, the difference between PE_O2 and PI_O2 is proportional to the net increase in O₂. The data reported in the present investigation demonstrate that, compared with the low PE_O2, O₂ was ~50% greater at the moderate and nearly twofold greater at the high PE_O2. Thus these data suggest that diffusive O₂ movement into the cell represents a major limitation to high rates of O₂ in myocytes lacking Mb. Because the oxidative phosphorylation response differed significantly among the three PE_O2 trials (Fig. 3), the unchanged force profile (Fig. 1) was likely maintained by substrate-level phosphorylation for the 2-min contractile period.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Intracellular PO2 dynamics (dashed line) and modeled fit (solid line) in response to tetanic contractions for an oxidative (A) and glycolytic (B) myocyte.

Effects of PE_O2 on the kinetic response to an elevated metabolic demand. At the transition to moderate-intensity exercise, the speed of the O₂ response does not depend critically on O₂ availability, whereas during supralactate threshold exercise, O₂ kinetics are dependent, in part, on O₂-related mechanisms (8, 28). To date, the specific PO₂ within the microvasculature that may alter the initial metabolic response from rest to high-intensity work is not known. Wilson and Rumsey (33) demonstrated that a PE_O2 of 30 Torr requires compensatory changes in phosphorylation and redox potential to maintain mitochondrial respiration in cell suspension that is minor until the PO₂ drops to ~10 Torr (32). Previous investigations (e.g., Refs. 4, 17, 21) have demonstrated that "hypoxia" slows the O₂ kinetic response at the transition to greater cycling work. However, these findings are confounded by the likelihood that blood flow to the working muscle was elevated during hypoxia to counteract the reduced hemoglobin saturation such that O₂ remained unchanged (14, 26).

Functional role of Mb in intramyocyte O₂. Classically, it has been thought that Mb facilitates O₂ transport within exercising myocytes and that this is important in the maintenance of a relatively homogeneous intramyocyte PO₂ distribution even during maximal aerobic work (15, 18, 34). Indeed, mathematical models of intramyocyte O₂ movement predict an important role for Mb-facilitated diffusion (5, 9). Garry et al. (6) demonstrated recently that Mb-deficient mice tolerate low and/or moderate levels of treadmill exercise. However, these Mb-knockout mice appear to adapt compensatory strategies, such as elevations in basal blood flow, capillary density, and hematocrit all acting to augment O₂ movement from capillary to sarcolemma (7), and thus the precise importance of Mb has not been defined. Data in the present investigation support the postulate of Groebe and Thews (9) in that isolated muscle cells lacking Mb appear to be O₂ limited during high-intensity tetanic contractions at PE_O2 nearing 60 Torr and possibly higher for the more oxidative cells. Our results suggest that contracting myocytes lacking Mb require a significantly greater PE_O2 compared with cells containing Mb to achieve peak rates of O₂.