INTRODUCTION

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DURING MODERATE-INTENSITY steady-state contractions, skeletal muscle cells derive most required ATP from the aerobic breakdown of substrates via the process of oxidative phosphorylation. For oxidative phosphorylation to occur, there must be a constant delivery of oxygen to the terminal oxygen acceptor of the respiring mitochondria, cytochrome aa₃. To ensure this constant flux of oxygen to the mitochondria, the working muscle cell maintains a gradient between the capillary or extracellular PO₂ and the intracellular PO₂ to create a driving force for oxygen delivery (14). To maintain this gradient, the intracellular PO₂ is thought to be regulated at a very low level. Indeed, in many models for calculating diffusion, it is often assumed that the PO₂ at the mitochondria approaches zero, especially at higher exercise work outputs (21).

Although an assumption of low PO₂ at high work intensities has been sufficient for many studies (10, 17), the exact relationship between intracellular PO₂ and muscle metabolism is both important and not well known. Unfortunately, it has long been problematic to measure intracellular PO₂ in intact, exercising muscle fibers. Several methods, most notably oxygen microelectrodes (27), cryospectroscopy (5), and near-infrared spectroscopy (2), among others (25), have been used to investigate cellular oxygen status in the past. Although these methods have produced interesting results, they have been known to suffer from inherent methodological problems (25).

One model for estimating intracellular PO₂ that has had recent success involves measuring myoglobin saturation from a section of whole muscle using magnetic resonance spectroscopy (12, 18, 19, 23). Using the deoxygenated myoglobin signal, it is possible, by ischemic calibration, to estimate the intracellular PO₂ of intact, working skeletal muscle. Recently, this method has been utilized by two separate groups to investigate intracellular PO₂ during different contraction paradigms. The results of these studies have been somewhat equivocal, however, as one group (19) has shown that PO₂ falls rapidly to a low level and does not change thereafter with changes in workload, whereas the other group (12) has shown that PO₂ continues to decline with increases in work output. This discrepancy, which may be due to methodological differences, has not been resolved (16).

One measurement system that has been developed and allows for the direct optical measurement of PO₂ utilizes oxygen-sensitive porphyrin compounds (26). This technique has previously been used extensively by those studying microcirculation (13). We have recently modified this technique to measure intracellular PO₂ in intact, single-skeletal muscle fibers (7). By using this technique on single fibers, it has been demonstrated that intracellular PO₂ falls with the onset of oxidative phosphorylation (7) and that the kinetics of this fall mirror the onset kinetics of oxygen uptake by the muscle (8). Whereas it is known that oxygen consumption (O₂) of single, isolated skeletal muscle fibers increases linearly with work rate (3, 24), no systematic investigation of the relationship between intracellular PO₂ and muscle work rate has been attempted with this model. The aim of the present study was to investigate whether intracellular PO₂ decreased in single skeletal muscle fibers as stimulation frequency, and thus energy demand, increased. Because these single amphibian fibers do not contain myoglobin and, therefore, behave as ideal cylinders for diffusion, our hypothesis was that PO₂ would be linearly related to stimulation frequency as predicted by the relationship between PO₂ and O₂ established by the Fick relationship.

	METHODS

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Animal care. Female adult Xenopus laevis were used for this study. They were doubly pithed and decapitated, the hindfeet were removed, and the lumbrical muscles (II-IV) were dissected free. All procedures were approved by the University of California at San Diego animal care and use committee and conform to National Institutes of Health guidelines.

Measurement systems. Single, living muscle fibers (n = 7) were micro-dissected with tendons intact in a chamber filled with Ringer solution (112 mM NaCl, 1.87 mM KCl, 0.82 mM CaCl₂, 2.38 mM NaHCO₃, 0.07 mM NaH₂PO₄, 0.1 mM EGTA; pH 7.0). Cells were microinjected with a solution of 0.5 mM palladium-meso-tetra(4-carboxyphenyl)porphine bound to bovine serum albumin (containing 10 mM fura 2 for visual monitoring of the injection using excitation light at 390 nm) by micropipette pressure injection (PV830 pneumatic picopump, World Precision Instruments, Sarasota, FL).

Experimental protocol. Platinum clips were attached to the tendons of the cells, and they were mounted in a chamber filled with Ringer solution. One end of the fiber was fixed, and the other free end was attached to an adjustable force transducer (model 400A, Aurora Scientific, Aurora, Ontario), allowing the muscle to be set at a length that produced maximal tetanic force (P_o). The analog signal from the force transducer was recorded via a chart recorder.

Statistics. All values are presented as means ± SE. To test for significance, one-way ANOVA was performed, and significance was tested with a Tukey least significant difference, post hoc test. The relationship between PO₂ and contraction frequency was also tested with a linear regression analysis, and a line of best fit was calculated for the data. The level of significance was set at P < 0.05 throughout.

	RESULTS

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Calibration. Figure 1 shows the phosphorescence decay curves when the intracellular PO₂ was ~30 (Fig. 1A) and 5 Torr (Fig. 1B). As PO₂ decreased in the environment around the porphyrin compound, the phosphorescence lifetime lengthened in a systematic manner.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Phosphorescence-decay curves from a single Xenopus laevis skeletal muscle fiber containing the oxygen-sensitive probe porphyrin before and during tetanic stimulation at 0.5 Hz when intracellular PO2 was 30 Torr (A) and 5 Torr (B).

Intracellular PO₂. Figure 2 shows the relationship between intracellular PO₂ and stimulation frequency for an individual single fiber. Previously, it has been shown that these fibers achieve steady-state O₂ by 2 min of stimulation (24). Intracellular PO₂ returned to resting levels within each 3-min rest period between stimulation periods.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Raw tracing of intracellular PO2 vs. time for a single representative skeletal muscle fiber. The order of the stimulation frequencies used for the stimulation protocol was randomized between all fibers. Note that intracellular PO2 rapidly returned to resting values during the rest periods between stimulation periods.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Relationship between intracellular PO2 and stimulation frequency in single Xenopus muscle fibers. Values are means ± SE. *Significantly different from rest; significantly different from 0.15 Hz; significantly different from 0.25 Hz (P < 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Regression line of best fit for the relationship between stimulation frequency and intracellular PO2 for all fibers in the experiment.

Force. Figure 5 demonstrates the force production for a given individual fiber during the five successive stimulation periods. For each individual fiber, maximal force production was not significantly different between each of the five stimulation periods of an experimental run, remaining at 75% P_o. Fatigue, defined by a fall in force production of >50% P_o, was evident in five fibers at 0.5 Hz and two fibers at 0.33 Hz.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Tracing of relative force for a single representative skeletal muscle fiber. Note that maximal force development during each of the five stimulation periods remained 75% of the initial tetanic force and was not significantly different between periods. Significant fatigue (i.e., <50% initial force) is evident only at 0.5 Hz.

	DISCUSSION

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These results clearly demonstrate that, under the conditions of the present study, intracellular PO₂ falls in a linear manner with increasing stimulation frequency in Xenopus single skeletal muscle fibers.

Based on Fick's law of diffusion, the relationship between O₂ and PO₂ for these single cells can be written by the equation

(1)

where O₂ is the constant of diffusion and extracellular PO₂ is the oxygen partial pressure in the chamber. Given that the extracellular PO₂ remains constant during the entire protocol in the present study and assuming that O₂ is also a constant, any increase in O₂ in these cells must be driven by a decrease in intracellular PO₂. Whereas O₂ and intracellular PO₂ have not been simultaneously measured in these cells to date, previous studies have demonstrated that O₂ in Xenopus single muscle cells does increase in a linear manner with increasing stimulation rate (3, 24). Therefore, it is very likely that the linear decrease in cellular PO₂ in this study was directly related to an increase in O₂ in the same fibers, as predicted by the above equation. O₂ should remain constant across stimulation frequencies, as the composition of the intra- and extracellular solutions, extracellular driving force (oxygen tension), and properties of the membrane (plasmalemma) do not change.

One interesting result of this study is that, although intracellular PO₂ did fall to fairly low levels during the most intense stimulation protocol, it did not go below ~5 Torr. Given that all cells demonstrated a fall in force production indicative of fatigue at this work intensity, it is likely that they were at or approaching maximal O₂ (3). Although this PO₂ value is somewhat higher than that seen in whole muscle from exercising humans (19, 20), it is still well above the oft-cited 0.5-1.0 Torr level that is required in vitro to maintain cytochrome turnover in isolated mitochondria (1). Currently, it is controversial as to whether the critical in vitro PO₂ necessary for mitochondrial function is different than the in vivo PO₂ that is required for normal cell bioenergetics (9, 28). It has been demonstrated in numerous studies that decreases in PO₂ can cause alterations in normal cell metabolism even at levels well above those thought to be limiting for oxidative metabolism (6, 9, 22). Therefore, it is likely that cellular respiration is not limited by intracellular PO₂ values in this range but requires a greater driving force to achieve a given O₂. This increased driving force is reflected in an increase in the magnitude of PCr breakdown and hydrogen ion accumulation as energy demand increases (12).

Previously, two separate laboratories (12, 19), both using magnetic resonance spectroscopy to quantify intracellular PO₂ based on saturation of myoglobin within sections of whole muscle, have demonstrated that PO₂ in human skeletal muscle does fall rapidly at the onset of exercise. However, one group found that PO₂ fell linearly over a range of increasing work intensities (12), whereas the other found that PO₂ fell to a low level at lower intensities and then did not change as work intensity increased to maximal (19). The authors of the latter paper have suggested that possible reasons for this discrepancy are the mode of exercise (knee extensor vs. plantarflexion), signal overlap from nearby muscles (i.e., soleus), and/or the method of increasing work intensity (increasing workload vs. increasing rate of contractions) (16). It is unclear which of these factors could affect the measurement of PO₂ in these studies, but the apparent discrepancy in results would suggest that some difference(s) between the two protocols resulted in either differences in PO₂ or differences associated with measurement.

It is interesting to note that a recent paper has suggested that the use of myoglobin saturation for estimating intracellular PO₂, as practiced by these two laboratories, may itself lead to a large underestimation of the true intracellular PO₂ (11). However, whereas these authors are correct in their interpretation of the nonlinearity of the myoglobin oxygen saturation curve, their criticism of previous nuclear magnetic resonance (NMR) work may not be valid. First, it is unlikely that any fibers would have an intracellular PO₂ over 70 Torr as used in the example, especially during exercise, given that mean capillary PO₂ is usually less than 40 Torr and that the amount of PCr degradation in the working muscle fibers during this type of work is significant. Second, the example uses the average of two numbers at extreme ends of the spectrum. In reality, two adjacent fibers would not have such a large difference in intracellular PO₂; many fibers would make up the measurement even with the very good resolution possible with NMR today, and the continuum of myoglobin saturation values in these fibers would average out to a representative whole muscle value. Finally, the degree of nonlinearity is much greater at the high end of the curve and not at the lower oxygen tensions found in the previous studies.

Because of the above limitations in the study of whole muscle PO₂, there are obvious advantages of the single-fiber preparation in studying changes in intracellular PO₂ as it affects and is affected by skeletal muscle metabolism. First, the extracellular milieu can be completely controlled, negating any potential effects of differences in blood flow and/or oxygen transport found in vivo. Blood flow to different regions of a whole muscle or to different muscle fiber types is not homogeneous. Also, during whole muscle contractions, intramuscular pressure can become large enough to restrict blood flow, potentially causing regions of ischemia (16). The cells in the present study are constantly perfused with a controlled PO₂ Ringer solution to maintain a constant extracellular oxygen. Second, there are no potential fiber-type or muscle-recruitment issues that are commonly seen as a methodological problem in other models. During submaximal workloads in a whole muscle, not all muscle fibers in a given area are active. Because skeletal muscle fiber types have different metabolic profiles, including a range of mitochondrial densities and myoglobin contents, measurement in a given area is an average of many individual muscle fibers, all of which are in a different metabolic state. Because only one fiber is being tested in the present study, there are no ambiguities regarding which fibers are actually active. Third, there are no spatial issues pertaining to the area of measurement that is seen with NMR and/or near-infrared spectroscopy. Like the recruitment issues, it is possible for the size of the area of interest to contain active and inactive muscle fibers, making it difficult to interpret results that are more global in nature. Finally, the system used in the present study is a direct measurement of intracellular PO₂ and is not dependent on myoglobin saturation.

Although the results of the present study agree with previous data that skeletal muscle intracellular PO₂ is linearly related to muscle work rate, some care still must be taken in making any comparisons between the isolated single fiber and in situ working human muscle. First, these single Xenopus muscle cells lack myoglobin. Without myoglobin, these fibers likely behave as ideal Krogh cylinders with a uniform oxygen gradient radiating from the core. Myoglobin has long been thought to play a role in the regulation of intracellular PO₂ and/or facilitating transport of oxygen from the blood to the mitochondria, but that role has now come under scrutiny (4, 11). A recent paper (11) suggests that myoglobin plays a very minor role in the transport of oxygen in the cell. Second, it is difficult to compare the work rates between contracting whole muscle and single fibers. Based on the previous work, it is apparent that the higher contraction frequencies will elicit O_2 max in these fibers (3, 24), and, as significant fatigue is seen at the highest contraction frequencies in these single fibers, it is likely that these cells reached or at least approached their maximum O_2 max in the present study. Finally, these cells are surrounded by a homogeneous field of oxygen, whereas, in working fibers in whole muscle, the surface area of a single fiber that is in contact with the microcirculation is less.

In summary, in isolated Xenopus single muscle fibers, the intracellular PO₂ decreases linearly with increases in contraction frequency. This suggests that intracellular PO₂ is linearly related to O₂ in these cells, as predicted by the Fick relationship.