Intracellular PO₂ and bioenergetic measurements in skeletal muscle: the role of exercise paradigm

The following is the abstract of the article discussed in the subsequent letter:

	ABSTRACT

Molé, Paul A., Youngran Chung, Tuan Khanh Tran, Napapon Sailasuta, Ralph Hurd, and Thomas Jue. Intracellular PO₂ and bioenergetic measurements in skeletal muscle: the role of exercise paradigm. Am J Physiol Regulatory Integrative Comp Physiol 277: R173-R180, 1999.The present study evaluated whether intracellular partial pressure of O₂ (PO₂) modulates the muscle O₂ uptake (O₂) as exercise intensity increased. Indirect calorimetry followed O₂, whereas nuclear magnetic resonance (NMR) monitored the high-energy phosphate levels, intracellular pH, and intracellular PO₂ in the gastrocnemius muscle of four untrained subjects at rest, during plantar flexion exercise with a constant load at a repetition rate of 0.75, 0.92, and 1.17 Hz, and during postexercise recovery. O₂ increased linearly with exercise intensity and peaked at 1.17 Hz (15.1 ± 0.37 watts), when the subjects could maintain the exercise for only 3 min. O₂ reached a peak value of 13.0 ± 1.59 ml O₂ · min¹ · 100 ml leg volume¹. The ³¹P spectra indicated that phosphocreatine decreased to 32% of its resting value, whereas intracellular pH decreased linearly with power output, reaching 6.86. Muscle ATP concentration, however, remained constant through- out the exercise protocol. The ¹H NMR deoxymyoglobin signal, reflecting the cellular PO₂, decreased in proportion to increments in power output and O₂. At the highest exercise intensity and peak O₂, myoglobin was ~50% desaturated. These findings, taken together, suggest that the O₂ gradient from hemoglobin to the mitochondria can modulate the O₂ flux to meet the increased O₂ in exercising muscle, but declining cellular PO₂ during enhanced mitochondrial respiration suggests that O₂ availability is not limiting O₂ during exercise.

	LETTER

Intracellular PO₂ and bioenergetic measurements in skeletal muscle: the role of exercise paradigm

To the Editor: Intracellular PO₂ measurements in exercising skeletal muscle have far reaching implications, from the determination of maximal oxygen consumption (5) to the production of lactate (7). Thus we recognize and are in full support of the recently reported and commendable efforts of Molé et al. (4) to further investigate the relationship between exercise intensity and intracellular partial pressure of O₂ (PO₂). However, as I too am immersed in this area of research, I feel that it is important to raise several issues that arise from reading this manuscript.

The manuscript makes it clear that the findings of Molé et al. (4) contrast with our previous findings (5, 7) and that this discrepancy "may" originate from differences in the muscle group studied, the subject population, and/or the nuclear magnetic resonance (NMR) acquisition/processing methodology. However, significant evidence of these differences are poorly highlighted and, in some cases, inappropriately used as an indication of similarity between previous investigations. Specifically, the statement that "At the highest work output of 15 W, the cellular pH is 6.87 ± 0.16, which is consistent with the value of 6.554 ± 0.325 observed by Richardson et al. (6)" is misleading. In fact, the pH reported in the original paper at maximal exercise was 6.571 ± 0.012. Although we recognize our large variations in standard error (SE) between work rates, the latter pH and SE are the correct comparison as both studies assume these to be maximum work rates. As we know, pH = log[H+], thus a numerically small change in pH is indicative of a large change in [H+]. In this particular case, at maximal exercise, Molé et al. (4) reported a pH indicative of a [H+] of 135 neq/l vs. our value of 269 neq/l at maximal exercise. I would hardly consider these two intracellular environments "consistent." This is undoubtedly a result of the muscle groups studied and not NMR methodology as we too have found similar pH values in the gastrocnemius during maximal effort of ~6.9 (3), whereas we have again recorded pH values of 6.47 ± 0.16 in untrained subjects during maximal knee-extensor exercise. With the size of surface coil (5 inch diameter) used by Molé et al. (4), it is most likely that a significant contribution to the signal was attained from the soleus as well as the gastrocnemius (both myoglobin and phosphorus). With this regard, it has previously been demonstrated that line width for the P_i, which reflects relative homogeneity of the pH values within the tissue being observed, can alter significantly during progressive plantar flexion exercise, indicating functional heterogeneity (2). This heterogeneity has previously been reported as an important limitation in the interpretation of MR data in the calf muscle (8). This potential overlap into a muscle rich in slow-twitch fibers (soleus) is additionally supported by the observation that at maximum work levels, the percentages of phosphagen shifts reported by Molé et al. (4) were the same as previously observed at only 40-50% of aerobic maximum (1, 6).

Molé et al. (4) report a P_i-to-PCr ratio of 2.4 and state that our study using knee-extensor exercise (5) reported very high P_i-to-PCr ratios approaching 10 and that the "unacceptably large SE precludes any comparative analysis." This is incorrect on two counts. We did not report or calculate P_i-to-PCr, but rather the PCr-to-P_i ratio, which fell to an extremely low level at maximal exercise, and thus the SE was large in comparison to the data themselves. We provided both the PCr (3.0 ± 0.3) and P_i (29.5 ± 1.6) millimolar values, and thus the calculation and comparative analysis of these results was not precluded. In fact, the contrasting P_i-to-PCr ratios between the two exercise modalities (again confirmed by our recent studies in untrained subjects during knee-extensor exercise, ratio = 6.3) are in fact added evidence that the region of skeletal muscle under study in these two exercise paradigms is vastly different. In contrast to calf exercise, during knee-extensor exercise, the complete region of interest appears taxed to extreme levels at maximal effort.

Finally, although there are some exercise testing protocols on treadmills that hold a constant grade and increase the running speed (usually reserved for elite distance runners possessing fast leg speeds), it is not typical to increase work rate by altering the rate of contraction while keeping the load constant, as performed in the study by Molé et al. (4). The concerns here are both the limited capacity to perform a given motion at increasingly rapid speeds and the reduction in the relaxation period of the duty cycle, perhaps limiting perfusion during the exercise. Either may result in an end-of-the-exercise test that does not coincide with physiological maximal effort.

In summary, I suggest that the discrepancy between the report by Molé et al. (4) and our previous observations (6, 7) clearly originate from fundamental differences in the intracellular perturbations achieved within the muscles studied and the type of exercise employed. Again, I wish to emphasize that the present comments are voiced because of my interest in the data presented by Dr. Molé and colleagues and the vastly different implications for oxygen transport and respiratory control in exercising skeletal muscle, dependent on the issue of whether intracellular PO₂ falls (4) or remains constant across the higher levels of muscular work (6, 7).

	REFERENCES

1.   Allen, P, Matheson G, Zhu G, Gheorgiu D, Dunlop R, Falconer T, Stanley C, and Hochachka P. Simultaneous ³¹P MRS of soleus and gastrocnemius in Sherpes during graded calf muscle exercise. Am J Physiol Regulatory Integrative Comp Physiol 273: R999-R1007, 1997[Abstract/Free Full Text].

2.   Barstow, T, Buchthal S, Zanconato S, and Cooper D. Changes in potential controllers of human skeletal muscle respiration during incremental calf exercise. J Appl Physiol 77: 2169-2176, 1994[Abstract/Free Full Text].

3.   Hogan, MC, Richardson RS, and Haseler L. Human muscle performance and PCr hydrolysis with varied inspired oxygen fractions: a ³¹P-MRS study. J Appl Physiol 86: 1367-1374, 1999[Abstract/Free Full Text].

4.   Molé, P, Chung Y, Tran T, Sailasuta N, Hurd R, and Jue T. Myoglobin desaturation with exercise intensity in human gastrocnemius muscle. Am J Physiol Regulatory Integrative Comp Physiol 277: R173-R180, 1999[Abstract/Free Full Text].

5.   Richardson, RS, Leigh JS, Wagner PD, and Noyszewski E. Cellular PO₂ as a determinant of maximal mitochondrial O₂ consumption in trained skeletal muscle. J Appl Physiol 87: 325-331, 1999[Abstract/Free Full Text].

6.   Richardson, RS, Noyszeski EA, Kendrick KF, Leigh JS, and Wagner PD. Myoglobin O₂ desaturation during exercise: evidence of limited O₂ transport. J Clin Invest 96: 1916-1926, 1995.

7.   Richardson, RS, Noyszewski EA, Leigh JS, and Wagner PD. Lactate efflux from exercising human skeletal muscle: role of intracellular PO₂. J Appl Physiol 85: 627-634, 1998[Abstract/Free Full Text].

8.   Vanderbourne, K, McCully K, Kakihira H, Prammer M, Bolinger L, Detre J, De Meirlier K, Walter G, Chance B, and Leigh J. Metabolic heterogeneity in human calf muscle during maximal exercise. Proc Natl Acad Sci USA 88: 5714-5718, 1991[Abstract/Free Full Text].

Russ S. Richardson, Department of Medicine University of California, San Diego La Jolla, CA 92093 E-mail: rrichardson{at}ucsd.edu

To the Editor: Dr. Richardson contends that the comparative analysis in Molé et al. (6) is misleading and speculates that the discrepancy in the myoglobin (Mb) O₂ desaturation response arises from differences in muscle work output.

At issue are the Richardson data in Tables 1 and 2, indicating quadricep exercise intensity at rest, 50%, 64%, 77%, 95%, and 100% of O_{2 max} (7). The observed pH values are 7.073 ± 0.017 (at rest), 6.867 ± 0.036, 6.745 ± 0.414, 6.617 ± 0.187, 6.554 ± 0.325, and 6.571 ± 0.012. Molé et al. (6) report a pH of 6.87 ± 0.16 at O_{2 max}, consistent with the values in exercise levels two to four and with Richardson's observation "in the gastrocnemius during maximal effort."

The corresponding PCr/P_i values are 11.5 ± 4.1 (at rest), 0.6 ± 1.3, 0.2 ± 0.7, 0.1 ± 0.4, 0.1 ± 0.2, and 0.1 ± 0.02. Both the pH and PCr/P_i data have very large standard errors and certainly preclude any rigorous comparative analysis. However, one cannot simply choose the values at O_{2 max} and disregard the values at 95% O_{2 max}.

The contention that the PCr decline reported in Molé et al. (6) indicates only submaximal work, about "40-50% of aerobic maximum" is unfounded. The decline of PCr by itself is not a direct index of respiratory control. Rather, the P_i/PCr ratio, which reflects the ADP level, is the appropriate parameter (3). The reported P_i/PCr value of 2.4 is consistent with maximal work. NMR studies have reported a P_i/PCr between 1.3 and 4.1 during maximum work in gastrocnemius muscle (1, 10).

Similarly, the speculation of a perfusion limitation imposed by a frequency-dependent exercise protocol does not square with the experimental evidence. A linear relationship exists between O₂ and work output (5). Toe-lifting exercise at constant frequency (30 rpm) or frequency varying (30-60 rpm) at constant load (30% maximal voluntary contraction) yields exactly the same O_{2 max}. No blood flow impediment is apparent.

NMR localization study of the deoxy Mb signal in gastrocnemius muscle also does not support any significant soleus contribution (9). In fact, aerobic myocardial tissue shows no MbO₂ desaturation with enhanced respiration (4). Any significant soleus contribution to the NMR signal would actually diminish the extent of Mb desaturation with increasing O₂.

Finally, the results reported in Molé et al. (6) match closely the near-infrared spectroscopy (NIRS) observations. Mb/Hb desaturates with O₂ and does not plateau after 50% O_{2 max} (2). If the NIRS signal has a significant contribution from Mb, then the NIRS findings indicate also that Mb desaturates linearly with increasing O₂ (8).

Why the two studies show a different response in Mb desaturation during exercise is intriguing (6, 7). The discussion, stimulated by the Richardson study, opens a very important dialogue in respiratory physiology and will lead to a deeper understanding of the fundamental mechanisms regulating O₂ consumption and transport in muscle.

	REFERENCES

1.   Barstow, TJ, Buchthal SD, Zanconato S, and Cooper DM. Changes in potential controllers of human skeletal muscle respiration during incremental calf exercise. J Appl Physiol 77: 2169-2176, 1994.

2.   Belardinelli, R, Barstow TJ, Porszasz J, and Wasserman K. Changes in skeletal muscle oxygenation during incremental exercise measured with near infrared spectroscopy. Eur J Appl Physiol 70: 487-492, 1995[Web of Science].

3.   Chance, B, Leigh JS, Kent J, McCully K, Nioka S, Clark BJ, Maris TM, and Graham T. Multiple controls of oxidative metabolism in living tissues as studied by phosphorous magnetic resonance. Proc Natl Acad Sci USA 83: 9458-9462, 1986[Abstract/Free Full Text].

4.   Kreutzer, U, Mekhamer Y, Tran TK, and Jue T. Role of oxygen in limiting respiration in the in situ myocardium. J Mol Cell Cardiol 30: 2651-2655, 1998[Web of Science][Medline].

5.   Laughlin, MH, and Armstrong RB. Muscle blood flow during locomotory exercise. Exerc Sport Sci Rev 13: 95-136, 1985[Medline].

6.   Molé, P, Chung Y, Tran TK, Sailasuta N, Hurd R, and Jue T. Myoglobin desaturation with exercise intensity in human gastrocnemius muscle. Am J Physiol Regulatory Integrative Comp Physiol 277: R173-R180, 1999.

7.   Richardson, RS, Noyszewski EA, Kendrick KF, Leigh JS, and Wagner PD. Myoglobin O₂ desaturation during exercise. J Clin Invest 96: 1916-1926, 1995.

8.   Tran, KT, Sailasuta N, Kreutzer U, Hurd R, Chung Y, Molé P, Kuno S, and Jue T. Comparative analysis of NMR and NIRS measurements of intracellular PO₂ in human skeletal muscle. Am J Physiol Regulatory Integrative Comp Physiol 276: R1682-R1690, 1999[Abstract/Free Full Text].

9.   Tran, TK, Sailasuta N, Hurd R, and Jue T. Spatial distribution of deoxymyoglobin in human muscle: an index of local tissue oxygenation. NMR Biomed 12: 26-30, 1999[Web of Science][Medline].

10.   Vandenborne, K, McCully K, Kakihira H, Prammer M, Bolinger L, Detre JA, De Meirlier K, Walter G, Chance B, and Leigh JS. Metabolic heterogeneity in human calf muscle during maximal exercise. Proc Natl Acad Sci USA 88: 5714-5718, 1991.

Thomas Jue, Department of Biological Chemistry University of California Davis, CA 95616-8635