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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 1, 2004 as doi:10.1096/fj.03-0762fje. |
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Department of Physiology, Division of Pathobiology, School of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands; and
* Department of Molecular Cell Physiology, BioCentrum, Vrije Universiteit, Amsterdam, the Netherlands
2Correspondence: Department of Physiology, Division of Pathobiology, School of Veterinary Medicine, Androclus gebouw, Yalelaan 1, 3508 TD, Utrecht, the Netherlands. E-mail: j.jeneson{at}vet.uu.nl
SPECIFIC AIMS
It is generally thought that intracellular pH (pHi) of skeletal muscle falls at least 0.5 units during intense activity, but all evidence on natural [i.e., voluntary, two-legged (2L)] locomotor exercise in humans has come from invasive biopsy studies. To provide the missing in vivo data, we collected 31P NMR spectroscopic data on the energetics and homeostatic pHi control of human quadriceps muscle during and after incremental bicycling exercise to exhaustion.
PRINCIPAL FINDINGS
1. Intracellular pH of human quadriceps muscle falls linearly and no more than 0.2 units during incremental bicycling exercise to exhaustion
We measured 31P nuclear magnetic resonance amplitudes and frequencies at 1.5 Tesla of phosphocreatine (PCr), inorganic phosphate (Pi), and ATP in the medial head of the right quadriceps muscle of six normally active human subjects during incremental supine bicycling exercise to exhaustion. The average pHi of quadriceps muscle cells at steady-states of ATP metabolism was determined from the chemical shift difference between PCr and Pi resonances in the corresponding 31P NMR spectrum using a standard titration curve. We found that acidification of the quadriceps muscle progressed linearly (r=0.82) during ramp bicycling exercise to exhaustion and was surprisingly moderate: pHi dropped only 0.24 ± 0.03 units from 7.08 ± 0.01 at rest to 6.84 ± 0.02 at maximal power output (Fig. 1
, upper panel). Any heterogeneity of pHi among fibers within the sampled quadriceps mass was only minor, evidenced from the similar line widths of the PCr and Pi resonances in 31P NMR spectra of exercising muscle.
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2. Cellular energy charge of human quadriceps muscle falls linearly during incremental bicycling exercise to exhaustion
To assess the energetics of quadriceps muscle performing natural locomotor exercise, we computed the [PCr]/[PCr]+[Pi]) concentration ratio [cellular energy charge (CEC)] and the cytosolic free energy of ATP hydrolysis (
Gp) at steady-states of ATP metabolism during ramp exercise. Near-equilibrium of the creatine kinase reaction was assumed for this condition, and an ATP concentration of 8.2 mM and standard free energy of ATP hydrolysis at pH 7.0 (Gpo’) of –32.8 kJ/mol were used. Quadriceps CEC dropped linearly (r=0.90) from 0.92 ± 0.01 in resting state to 0.16 ± 0.03 at maximal power output (Fig. 1
, lower panel). The Gp in quadriceps muscle fell linearly (r=0.95) from –64.8 ± 0.4 kJ/mol at rest to –51.2 ± 0.7 kJ/mol at maximal sustained bicycling work rate, yielding a thermodynamic operational range of ATP free energy conversion supporting contractile work of 13.6 ± 1.1 kJ/mol.
3. Human quadriceps pHi drops 0.2 units within 8 s after termination of exercise
To understand the discrepancy between our observations on quadriceps end-exercise pHi during exhaustive dynamic bicycling exercise and current understanding of this variable based on classic biopsy data, we measured metabolic recovery after termination of exercise with a time resolution of 8 s. Within 40 s, quadriceps pHi had dropped an additional 0.24 units toward the textbook value of 6.60; the majority of this pHi drop occurred within the first 8 s of recovery. Analysis of the kinetics of recovery of resting state CEC (=0.92) by reversal of the Lohman reaction showed that the accumulated cytosolic proton buffer Pi (end-exercise concentration: 33.5±2.4 mM) was consumed at an initial rate of 1.5 ± 0.3 mM/s after termination of exercise causing net release of protons into the cytosol at an initial rate of 107 nmol/l/s. These findings reconciled our observation of only moderate quadriceps acidification during exhaustive bicycling exercise with classic biopsy estimates of quadriceps end-exercise pHi.
4. Homeostatic control of human quadriceps pHi is robust during two-legged but not one-legged cycling exercise
One more discrepancy remained between our results and previous noninvasive observations on human quadriceps performing cycling exercise. Specifically, Richardson and co-workers observed textbook acidification of 0.5 pH units in human quadriceps muscle during incremental one-legged (1L) knee extension exercise to exhaustion. To address this issue, we performed comparative co-response analysis of scaled pHi and CEC changes in quadriceps muscle in response to work rate (WR) increments for 1L and 2L cycling. The analysis used the 1L cycling data of Richardson et al. and involved computation of the response coefficients RWRH+ and RWRCEC, and their ratio, the co-response coefficient 
WRH+,CEC, as a function of scaled WR for each exercise regimen. We found that the (WR, WRH+,CEC) relation for bicycling exercise was linear over the full span of work rates between resting and maximal (Fig. 2
). For 1L cycling exercise, however, this relation was curvilinear (Fig. 2)
. Specifically, progressive draining of CEC to support WR increments in the domain above 50% of WRmax was accompanied by exponentially progressive acidification of the quadriceps (Fig. 2)
. These findings showed that, unlike 1L cycling, homeostatic control of quadriceps pHi during 2L cycling exercise is robust and does not break down at high work rates.
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CONCLUSIONS AND SIGNIFICANCE
The present 31P MRS data represent the first comprehensive in vivo study of intramuscular energetics and pH in the quadriceps during natural (i.e., voluntary, bipedal) locomotor exercise in humans. We found that integrative homeostatic control of quadriceps pHi during bicycling exercise is robust. Moreover, our analyses of these and previously reported 31P MRS data on quadriceps energetics and pHi during 1L cycling by Richardson and co-workers show that this emergent property of the integrative set of physicochemical and physiological control mechanisms in acid base balance in humans is unique to natural locomotor exercise and may be important to sustain this vital activity.
The physicochemical and physiological control mechanisms in acid base balance tending to minimize the fall of tissue pH and remove CO2 during exercise affect the following three parameters in tissue [H+] regulation: 1) the net charge difference between strong ions (e.g., lactate), termed the strong ion difference (SID), 2) the total concentration of weak electrolyte ([Atot]), and 3) total CO2 content ([CO2 tot]) (Fig. 3
). As such, cardiovascular control of blood flow and respiratory control of alveolar ventilation are effective systemic means to regulate intramuscular pH affecting SID and [CO2 tot] via washout of lactic acid and CO2, and gas exchange, respectively (Fig. 3)
. The primary cellular means to regulate pHi during contractile work is production of H+ buffer in the form of the ATP hydrolysis product Pi affecting [Atot] (Fig. 3)
. The latter explains our finding of ancillary acidification of the quadriceps to textbook pH values of 6.6 after termination of exercise when the Lohmann reaction is reversed toward Pi consumption and PCr resynthesis. The fast kinetics of this process in maximally activated quadriceps muscle (initial rate 1.5 mM/s) reconciled our novel finding of moderate acidification of the quadriceps muscle during exhaustive dynamic exercise with classic biopsy results of quadriceps end-exercise pHi of 6.6. Alternatively, by scaling for [Pi] effects on [Atot] (comprised in the term CEC), the outcome of the co-response analysis of scaled quadriceps pHi and CEC changes in response to WR increments for 2L vs. 1L cycling must be explained in terms of SID and [CO2 tot] control in quadriceps muscle in each exercise regimen. It has been well documented that quadriceps perfusion reaches similar peak values in 1L and 2L cycling, if not higher in 1L cycling. We therefore conclude that the identified superior robustness of homeostatic control of working quadriceps pHi during 2L cycling compared with 1L cycling must result from a higher capacity to remove CO2 from the working quadriceps ultimately by alveolar ventilation (Fig. 3)
. However, this remains to be objectified.
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The benefits of the apparent optimal matching cardiovascular and respiratory response to sustained quadriceps performance during bipedal exercise are considerable. First, the inhibitory H+ sensitivity of Ca2+ activation of force-generating cross-bridge cycling between myofibrillar thick and thin filaments is significant and linear in the acidic pH range. Second, the absolute thermodynamic operational range of ATP free energy conversion supporting muscular work, typically 10 kJ/mol for excitable cells, is expanded by another 4 kJ/mol to theoretical limits set by the equilibrium potential of the ATP-driven Ca2+ pumps of the sarcoplasmic reticulum. More important, control of changes in the cytosolic ATP free energy potential during WR increments, reflected in the response coefficient RWRCEC (see also Fig. 1
, lower panel), is robust during 2L but not 1L cycling. Indeed, Richardson and co-workers using 1H MRS in addition to 31P MRS observed rapid myoglobin desaturation at low-to-moderate 1L cycling work rates concomitant with a steep drop in CEC to levels observed here only at bicycling work rates >80% of maximal. Moreover, they proposed that their observations might apply to other forms of dynamic exercise in humans. Although this far-reaching hypothesis has been embraced by some, the present observations on the in vivo energetics of bicycling exercise render such an outcome unlikely.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0762fje; 
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