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J Appl Physiol 98: 2316-2319, 2005. First published February 3, 2005; doi:10.1152/japplphysiol.00355.2004
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Relationship between intracellular PO2 recovery kinetics and fatigability in isolated single frog myocytes

Casey A. Kindig, Brandon Walsh, Richard A. Howlett, Creed M. Stary, and Michael C. Hogan

University of California-San Diego, Department of Medicine, Physiology Division, La Jolla, California

Submitted 1 April 2004 ; accepted in final form 25 January 2005

ABSTRACT

In single frog skeletal myocytes, a linear relationship exists between "fatigability" and oxidative capacity. The purpose of this investigation was to study the relationship between the intracellular PO2 (PIO2) offset kinetics and fatigability in single Xenopus laevis myocytes to test the hypothesis that PIO2 offset kinetics would be related linearly with myocyte fatigability and, by inference, oxidative capacity. Individual myocytes (n = 30) isolated from lumbrical muscle were subjected to a 2-min bout of isometric peak tetanic contractions at either 0.25- or 0.33-Hz frequency while PIO2 was measured continuously via phosphorescence quenching techniques. The mean response time (MRT; time to 63% of the overall response) for PIO2 recovery from contracting values to resting baseline was calculated. After the initial square-wave constant-frequency contraction trial, each cell performed an incremental contraction protocol [i.e., frequency increase every 2 min from 0.167, 0.25, 0.33, 0.5, 1.0, and 2.0 Hz until peak tension fell below 50% of initial values (TTF)]. TTF values ranged from 3.39 to 10.04 min for the myocytes. The PIO2 recovery MRT ranged from 26 to 146 s. A significant (P < 0.05), negative relationship (MRT = –12.68TTF + 168.3, r2 = 0.605) between TTF and PIO2 recovery MRT existed. These data demonstrate a significant correlation between fatigability and oxidative phosphorylation recovery kinetics consistent with the notion that oxidative capacity determines, in part, the speed with which skeletal muscle can recover energetically to alterations in metabolic demand.

muscle fiber; fatigue; metabolic control; oxidative capacity

AN INVERSE, PROPORTIONAL RELATIONSHIP has been proposed to exist between the speed of phosphocreatine (PCr) resynthesis and muscle mitochondrial capacity (18, 22). Previous investigations in both animals (23) and humans (e.g., Refs. 14, 20, 21) have confirmed this postulation. Specifically, Paganini and colleagues (23) demonstrated a linear relationship between PCr recovery times after electrically stimulated contractions and citrate synthase activity in rat skeletal muscle.

O2 uptake (O2) onset kinetics are considered dependent, at least in part, on maximal aerobic capacity. Indeed, aerobic exercise training that induces significant increases in muscle oxidative capacity also speeds O2 kinetics (4, 8, 10). However, this can occur before any discernible increase in maximal O2 and muscle oxidative capacity (24). To date, no rigorous investigation studying the relationship between maximal aerobic capacity and O2 off-kinetics exists. However, given that PCr and O2 kinetics appear to be reasonably well matched (1, 19, 26), it is possible that a strong relationship exists between maximal aerobic capacity and O2 off-kinetics.

The purpose of the present investigation was to study the relationship between single myocyte fatigability, which we reported previously to be linearly related to mitochondrial volume density (29) and oxidative phosphorylation recovery [assessed via intracellular PO2 (PIO2) kinetics; Ref. 11] after a bout of repeated tetanic contractions. We tested the hypothesis that the time course for PIO2 recovery after a 2-min bout of moderate-intensity contractions would be inversely proportional to the time to fatigue (TTF) during an incremental contraction protocol.

METHODS

Female adult Xenopus 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 = 30) were isolated and prepared as described previously (11). Briefly, frogs were doubly pithed and the lumbrical muscles (II-IV) were removed from the hind feet. Single myocytes were dissected with tendons intact in a chamber of physiological Ringer solution at a pH = 7.0. Cells were injected via micropipette pressure injection (PV830 pneumatic picopump, World Precision Instruments, Sarasota, FL) with a solution consisting of 0.5 mM Pd-meso-tetra (4-carboxyphenyl) porphine bound to bovine serum albumin and the Ca2+ indicator dye fura 2.

Experimental protocol.   Platinum clips were attached to the tendons of each myocyte to facilitate fiber positioning within the Ringer solution-filled chamber. One tendon was fixed, whereas the contralateral was attached to an adjustable force transducer (model 400A, Aurora Scientific, Aurora, Ontario, Canada), allowing the muscle to be set at optimum muscle length. 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 solution equilibrated with 5% CO2 and 4–5% O2 in N2 balance. Constant perfusion was maintained throughout the protocol to maintain the extracellular PO2 and to reduce the occurrence of an appreciable unstirred layer surrounding the cell. Tetanic contractions were elicited using direct (8–10 V) stimulation of the muscle (model S48, Grass Instruments, Warwick, RI). The stimulation protocol consisted of ~250 ms trains of 70-Hz impulses of 1-ms duration.

Initially, myocytes were subjected to a trial of ~2 min at either 0.25- or 0.33-Hz stimulation frequency, during which time PIO2 was measured continuously and throughout the PIO2 recovery to baseline levels. After the initial "square-wave" contraction trial, each cell performed an incremental contraction protocol [i.e., frequency increase every 2 min from 0.167, 0.25, 0.33, 0.5, 1.0, and 2.0 Hz until peak tension fell below 50% of initial values (TTF)] under ambient conditions. After the incremental trial, TTF was then determined offline as the time to the first contraction with a peak isometric tension that was below 50% of the initial (nonfatigued) isometric tension.

Assessment of PIO2.   PIO2 was measured via phosphorescence quenching techniques as described previously (11). Briefly, each myocyte was observed with a Nikon x40 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). Phosphorescent decay curves were recorded every 4 s from each cell throughout the experimental period.

Data and statistical analysis.   After experimental procedures, the mean response time (MRT) was calculated as the time to 63% of the recovery in PIO2 from contracting values to resting baseline. Time to fatigue was denoted as the time at which peak tension fell to 50% of initial peak values. Data are presented as means ± SE. MRT and TTF data were regressed linearly by standard least-squares procedures. Statistical significance was accepted at P < 0.05.

RESULTS

Peak tension development over the duration of the incremental frequency contraction protocol is shown in Fig. 1 for two representative myocytes with widely different TTF values. In the incremental frequency protocol, TTF values for all cells (n = 30) ranged from 3.39 to 10.04 min (mean = 7.17 ± 0.35 min). PIO2 recovery responses from the nadir PIO2 after the brief (2 min) constant-frequency (0.25 or 0.33 Hz) contraction bout are shown in Fig. 2 for the same two representative myocytes. The PIO2 recovery MRT averaged 77.4 ± 5.7 s (range = 26–146 s). During the constant-frequency (2 min) protocol, over the course of the contraction bout, peak tension fell to 80 ± 2.0% of initial peak values. No significant relationship existed between the constant-frequency end-to-peak tension ratio and TTF (r2 = 0.285). The mean difference between end-contracting PIO2 and PIO2 recovery to extracellular values (i.e., {Delta}PIO2) was 26.6 ± 2.2 Torr. There existed no significant relationship between {Delta}PIO2 and TTF (r2 = –0.152). Figure 3 demonstrates that a significant (P < 0.05), negative relationship (MRT = –12.68TTF + 168.3, r2 = 0.605) between TTF and PIO2 recovery MRT existed.



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  		Fig. 1. Representative peak isometric tension data for 2 single isolated frog myocytes during the incremental frequency protocol (increasing contraction frequency every 2 min) demonstrating the wide differences in fatigability. Developed tension for a fatigue-resistant fiber (A) and for a fast-fatiguing fiber (B). In the incremental frequency protocol, time-to-fatigue (TTF) values for all cells (n = 30) ranged from 3.39 to 10.04 min (mean = 7.17 ± 0.35 min).

 


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  		Fig. 2. Intracellular PO2 (PIO2) recovery data for the same representative fast-fatiguing ({circ}) and fatigue resistant ({bullet}) single myocytes as presented in Fig. 1. The recovery kinetics represent the change in PIO2 profile from the end of a bout of tetanic contractions (time = 0) until recovery to resting values. The PIO2 recovery mean response time (MRT) averaged 77.4 ± 5.7 s (range = 26–146 s) whereas the difference between end-contracting PIO2 and PIO2 recovery to extracellular values (i.e., {Delta}PIO2) was 26.6 ± 2.2 Torr.

 


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  		Fig. 3. A significant (P < 0.05), negative relationship existed (n = 30) for the PIO2 recovery time (MRT) after a 2-min constant-frequency contraction bout and TTF (assessed as time for peak tension to fall to 50% of initial levels) in a subsequent incremental-frequency contraction bout.

 
DISCUSSION

It is generally accepted that PCr recovery kinetics provide an index of muscle oxidative capacity (e.g., Refs. 14, 18, 20–23). However, less is known about the relationship between O2 off-kinetics and muscle oxidative capacity. To our knowledge, this is the first investigation to study the relationship between individual myocyte fatigability (considered a proxy for muscle fiber oxidative capacity as discussed below) and PIO2 recovery kinetics. Our findings demonstrate that TTF and PIO2 recovery MRT in isolated single myocytes were significantly correlated in that myocytes that fatigue rapidly and have low oxidative capacity require a longer time period for PIO2 recovery. These data indicate that, much like PCr resynthesis kinetics, O2 off-kinetics are at least partly dependent on muscle oxidative capacity.

The relationship between O2 and PIO2 for single myocytes lacking myoglobin, such as in Xenopus muscle, is described by Fick's law of diffusion as:

where DO2 is the muscle O2 diffusion constant and PO2mito and PeO2 represent mitochondrial and extracellular PO2, respectively. Assuming little or no gradient between cytosolic and mitochondrial PO2, the difference between PeO2 and PIO2 is proportional to the net increase in O2 (12).

Recently, our laboratory (29) demonstrated a very strong linear relationship (r = 0.93; P < 0.0001) between TTF (utilizing the same variable frequency protocol as the present study) and mitochondrial volume density in Xenopus lumbrical single myocytes (the same type of myocytes as utilized in the present investigation). This association, which confirms similar results reported previously for single muscle cells (30), demonstrates that fatigue profiles from a single cell can be used to predict oxidative capacity (29). Thus, for the purposes of the present investigation, fatigability profiles in response to the incremental frequency protocol can be considered analogous to the intrinsic oxidative capacity of the cell.

In the present investigation, PIO2 recovery kinetics were shown to be correlated (r2 = 0.605) with TTF and, thus, by inference from earlier work (29), oxidative capacity, although the strength of this relationship is not known. There are a number of putative mechanisms for the association between recovery kinetics and oxidative capacity. First, it would be expected that the myocytes with the highest oxidative capacity would evoke less PCr depletion for a given amount of work (5); thus there would be less oxidative cost associated with PCr repletion. Second, whereas the O2 cost per unit work should be similar between myocytes (although some slight variations in this may exist because of fiber type differences; Ref. 6), the work of Mahler (18) and Meyer (22) would suggest that the muscle cells with the greatest mitochondrial density, and thereby the greatest oxidative capacity, can recover most rapidly after an elevated metabolic demand, in concert with first-order respiratory control.

Factors associated with O2 availability may confound peak aerobic capacity and O2 kinetics data obtained in whole muscle and whole body preparations. First, previous investigations have reported that exercise training significantly speeds O2 kinetics (4, 8, 10). However, a tight relationship exists between mitochondrial volume density and fiber capillarization (25) such that it is difficult to dissociate the effects of increased oxidative capacity from the augmented capacity for capillary-to-myocyte O2 flux (i.e., muscle DO2) and possibly other factors (24, 28). Second, peak O2 (often considered analogous to muscle oxidative capacity) may be differentially contingent on O2 availability based on fitness level. Specifically, it has been demonstrated that breathing hyperoxic gas increases peak leg O2 from normoxic levels in trained subjects (16) but may not in untrained individuals (3). Third, not only may peak muscle O2 values be dependent on O2 availability, both PCr (e.g., Refs. 9, 13) and O2 (e.g., Ref. 7) off-kinetics can be modulated by O2 concentration. One of the particularly powerful aspects of the present investigation utilizing single myocytes was that fatigue protocols were run under supraphysiological O2 levels and the constant-frequency protocols under highly controlled PO2 conditions demonstrated previously not to affect PIO2 kinetics (15) such that the potentially confounding effects of O2 availability were avoided.

Previous seminal work has demonstrated intrinsic differences in mitochondrial function and respiratory control between muscles of differing fiber type (e.g., Refs. 2, 6, 17, 27). However, ambiguities in fiber typing (i.e., metabolic, myosin heavy chain, etc.) along with differences in recruitment, fatigability, and efficiency confound metabolic control inferences derived from investigations studying whole body and whole muscle. Thus it was interesting to note that the linear relationship between TTF and PIO2 off-kinetics (Fig. 3) is not based on any fiber-type classifications per se.

The present investigation was undertaken to study the relationship between TTF and PIO2 off-kinetics, independent of fiber type, in single myocytes isolated from frog muscle under a highly controlled and homogeneous O2 environment. Our findings demonstrate a significant negative relationship between TTF in response to an incremental frequency protocol (a known proxy for mitochondrial volume density) and PIO2 recovery kinetics after a 2-min bout of moderate-intensity contractions. The findings suggest that O2 recovery kinetics are dependent in part on the intrinsic oxidative capacity of the skeletal muscle performing that work.

GRANTS

This study was supported, in part, by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-40155 and AR-48461. C. A. Kindig and R. A. Howlett are Parker B. Francis pulmonary fellows.

FOOTNOTES


Address for reprint requests and other correspondence: R. A. Howlett, Univ. of California-San Diego, Dept. of Medicine, Physiology Division, 9500 Gilman Dr., MC0623a, La Jolla, CA 92093-0623 (E-mail: rhowlett{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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