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Am J Physiol Regul Integr Comp Physiol 290: R1707-R1713, 2006. First published January 19, 2006; doi:10.1152/ajpregu.00687.2005
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ENVIRONMENTAL, EXERCISE, AND RESPIRATORY PHYSIOLOGY

Measurement of activation energy and oxidative phosphorylation onset kinetics in isolated muscle fibers in the absence of cross-bridge cycling

B. Walsh, R. A. Howlett, C. M. Stary, C. A. Kindig, and M. C. Hogan

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

Submitted 23 September 2005 ; accepted in final form 16 January 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study utilized N-benzyl-p-toluene sulfonamide (BTS), a potent inhibitor of cross-bridge cycling, to measure 1) the relative metabolic costs of cross-bridge cycling and activation energy during contraction, and 2) oxygen uptake kinetics in the presence and absence of myosin ATPase activity, in isolated Xenopus laevis muscle fibers. Isometric tension development and either cytosolic Ca2+ concentration ([Ca2+]c) or intracellular PO2 (PFormula) were measured during contractions at 20°C in control conditions (Con) and after exposure to 12.5 µM BTS. BTS attenuated tension development to 5 ± 0.4% of Con but did not affect either resting or peak [Ca2+]c during repeated isometric contractions. To determine the relative metabolic cost of cross-bridge cycling, we measured the fall in PFormula ({Delta}PFormula; a proxy for VO2) during contractions in Con and BTS groups. BTS attenuated {Delta}PFormula by 55 ± 6%, reflecting the relative ATP cost of cross-bridge cycling. Thus, extrapolating {Delta}PFormula to a value that would occur at 0% tension suggests that actomyosin ATP requirement is ~58% of overall ATP consumption during isometric contractions in mixed fiber types. BTS also slowed the fall in PFormula (time to 63% of overall {Delta}PFormula) from 75 ± 9 s (Con) to 101 ± 9 s (BTS) (P < 0.05), suggesting an important role of the products of ATP hydrolysis in determining the VO2 onset kinetics. These results demonstrate in isolated skeletal muscle fibers that 1) activation energy accounts for a substantial proportion (~42%) of total ATP cost during isometric contractions, and 2) despite unchanged [Ca2+]c transients, a reduced rate of ATP consumption results in slower VO2 onset kinetics.

oxygen consumption; N-benzyl-p-toluene sulfonamide; sarcoplasmic reticulum; sarco(endo)plasmic reticulum calcium-adenosine triphosphatase; adenosine 5'-triphosphate

DURING EXERCISE, skeletal muscle ATP consumption rates can increase several hundredfold over resting rates of ATP consumption. The majority of ATP consumed in skeletal muscle during contraction is believed to either directly satisfy the increased rates of cross-bridge cycling (via myosin ATPase) or sarcoplasmic reticulum (SR) Ca2+ handling [via sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)] (12, 24), with a relatively small ATP cost for membrane ion transport. Because of the close functional coupling of SR Ca2+ handling and cross-bridge cycling, estimates of the energetic requirements of either myosin ATPase or SERCA must be conducted under conditions in which one or the other has been inactivated. Several investigators have measured actomyosin ATP utilization in the absence of SERCA activity (34, 42, 44). However, these studies are limited to nonintact systems (e.g., skinned muscle fibers and isolated vesicles) that allow external regulation of cross-bridge cycling in the absence of SR function. Although such measurements have provided important information, the potential for altered cellular function that exists when the cell is disrupted makes it ultimately desirable to study muscle energetics in an intact cell.

Although cross-bridge cycling is intimately linked to Ca2+ handling, SR Ca2+ release and reuptake can occur independently of cross-bridge cycling. Therefore, it is possible to measure activation energy (i.e., the energy required to activate, but not directly drive, cross-bridge cycling) in an intact cell under conditions in which cross-bridge cycling has been inhibited. Traditionally, the technique most commonly used to measure activation energy is that of overstretching the muscle to prevent actin-myosin interaction and subsequently estimating energy consumption during stimulation via measurement of heat production, 31P-NMR, or biochemical assays of cellular metabolites. Such measurements have demonstrated that the activation energy can account for ~20–50% of the total energy requirement depending on factors such as fiber type (4, 12), type of stimulation (12, 19), fatigue state (4, 5), and temperature (10, 33).

The above-described techniques rely on the assumption that overstretching the cell does not alter normal Ca2+ handling. Although several studies have supported this contention (9, 24), there are data to suggest that Ca2+ handling may be altered during stretch (see Ref. 24). Therefore, when estimating the relative energetic requirements of cross-bridge cycling and activation energy, it may be desirable to use alternative techniques to dissociate Ca2+ handling from cross-bridge cycling (especially in isolated muscle fibers; see DISCUSSION).

Using a chemical inhibitor of cross-bridge cycling (2,3-butanedione 2-monoximine, BDM) in single frog skeletal muscle fibers, Buschman et al. (12) found activation heat to be 34–48% of total stabile heat. However, the chemical blocker used by Buschman et al. (BDM) does not completely inhibit cross-bridge cycling unless used in concentrations high enough to interfere with Ca2+ handling (>5 mM) (18, 43). Therefore, investigations designed to dissociate normal Ca2+ handling from cross-bridge cycling (e.g., Refs. 12, 30) require the use of lower concentrations of BDM that do not completely inhibit cross-bridge cycling. Recently, it was demonstrated that another inhibitor of cross-bridge cycling, N-benzyl-p-toluene sulfonamide (BTS), can strongly inhibit cross-bridge cycling (~95% inhibition) in intact cells (15, 16, 48) without affecting Ca2+ handling (15, 16). Therefore, the use of BTS allows for the study of intact cellular ATPase rates with and without cross-bridge cycling.

A benefit of the single-fiber model used by Buschman et al. (12) is that it allows for the precise control of the extracellular environment and the simultaneous measurement of tension development and either intracellular PO2 (PFormula) or cytosolic Ca2+ concentration ([Ca2+]c). Measurements of [Ca2+]c transients can be used to verify that Ca2+ handling is not affected in the experimental condition, whereas PFormula can be used as a proxy for O2 consumption (VO2; see METHODS). Because oxidative phosphorylation accounts for virtually all ATP production during submaximal steady-state contractions, measurement of PFormula allows a novel method of estimating ATP consumption to compare with previously used techniques (i.e., heat measurements, 31P-NMR, and biochemical assays of frozen tissue).

In addition, PFormula measurements allow study of onset kinetics of oxidative phosphorylation at the single-fiber level, in the absence of issues of O2 delivery. Presently, the mechanism of control of oxidative phosphorylation is under debate, with both feedback (i.e., control by the products of ATP consumption) and parallel activation (control of contraction and oxidative phosphorylation by a common controller, e.g., Ca2+) mechanisms currently proposed. Because BTS should alter the normal response of phosphorylation potential (ATP/ADP + Pi), but not [Ca2+]c transients, to a given rate of stimulation, measurements of PFormula kinetics in the absence and presence of BTS will allow investigation of the importance of these proposed mechanisms of control (i.e., control in response to changes in ADP, Pi, or phosphorylation potential vs. Ca2+).

In the present study, isolated single skeletal muscle cells were subjected to a moderate intensity contraction protocol in the absence or presence of BTS to investigate the relative metabolic requirement of cross-bridge cycling vs. activation energy. Isometric tension development and PFormula (a surrogate for VO2), were monitored continuously during contractions to determine the relative contribution of actomyosin ATPase to total ATP consumption during contractions and the effect of dissociating potential controllers of mitochondrial function on the onset kinetics of oxidative phosphorylation.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Female adult Xenopus laevis were used in this investigation. All procedures were approved by the University of California, San Diego, Animal Care and Use Committee and conform to National Institutes of Health standards.

Single skeletal muscle fiber preparation. Single muscle cells (n = 19) were isolated and prepared as described previously (23). Briefly, frogs were doubly pithed, and the lumbrical muscles (II–IV) were removed from the hind feet. Single muscle fibers were dissected with tendons intact in a chamber of physiological Ringer solution consisting of (in mM) 112 NaCl, 1.87 KCl, 0.82 CaCl2, 2.38 NaHCO3, 0.07 NaH2PO4, and 0.1 EGTA, pH 7.0. Cells were injected via micropipette pressure injection (PV830 pneumatic picopump; World Precision Instruments, Sarasota, FL) with either a solution consisting of 0.5 mM Pd-meso-tetra(4-carboxyphenyl)porphine bound to bovine serum albumin (for phosphorescence quenching measurements of PFormula) or 10 mM fura-2 (for fluorescence microscopy measurements of intracellular Ca2+; Molecular Probes, Eugene, OR) (20, 23). After microinjection, cells were given a minimum of 30 min for recovery.

Experimental protocol. Platinum clips were attached to the tendons of each fiber to facilitate fiber positioning within the Ringer solution-filled chamber. One tendon was fixed, whereas the contralateral tendon was attached to an adjustable force transducer (model 400A; Aurora Scientific, Aurora, ON, Canada), allowing the muscle to be set at optimum muscle length (i.e., length at which maximal tetanic force was 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 solution equilibrated with 5% CO2 and ~5% O2 in N2 balance at ~20°C. Constant perfusion was maintained throughout the protocol to maintain the extracellular PO2 at ~50 mmHg and to reduce the occurrence of an unstirred layer surrounding the cell. Tetanic contractions were elicited using direct (8–10 V) stimulation of the muscle from end to end (model S48; Grass Instruments, Warwick, RI). The stimulation protocol consisted of ~250-ms trains of 70-Hz impulses of 1-ms duration. Fibers were subjected to trials of 100–200 s at a stimulation frequency of 0.167–0.5 Hz with a 15-min recovery period between trials.

In pilot studies, it was determined that tension would not return to control values after exposure to BTS and subsequent washout. Therefore, order randomization was not possible. We have previously shown that tension development and baseline [Ca2+]c are unchanged (P > 0.05) and peak [Ca2+]c is reduced by <10% (P < 0.05) of the initial control trial during a second bout of contractions under control conditions after 15 min of recovery (a similar time between measurements was used in the present study) (29). Thus the current experiments were performed with a control trial (Con) followed by a similar trial in which 12.5 µM BTS (demonstrated in preliminary experiments to be the lowest concentration that would cause maximal inhibition of cross-bridge cycling) was added to Ringer solution 10 min before stimulation to inhibit cross-bridge cycling while measuring either [Ca2+]c (n = 10) or PFormula (n = 9).

Cytosolic [Ca2+] measurement. [Ca2+]c was measured using an epifluorescent microscope system that consisted of a Nikon inverted microscope with a x40 fluor objective and a DeltaScan illumination and detection system (Photon Technology International, South Brunswick, NJ) as described previously (41). Fibers injected with fura-2 were illuminated sequentially (20 Hz) with two excitation wavelengths of 340 and 380 nm, and the resulting fluorescence emission was measured at 510 nm. The ratio of 340- to 380-nm fluorescence was used to obtain the Ca2+-dependent signal (20).

Assessment of PFormula. Isolated fibers were observed with a Nikon x40 fluor objective (0.70 numerical aperture). The phosphorescence quenching of the porphyrin compound within the fiber was measured using 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, we averaged the phosphorescent decay curves from a series of 20 flashes (15 Hz) and fit a monoexponential function 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

Formula 1(1)

thus

Formula 2(2)

where {tau}o and {tau} are the phosphorescence lifetimes at anoxia and a given PO2, respectively, and kq, the quenching constant (in mmHg/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 kq and {tau}o were set at 690 mmHg/s and 100 µs for Pd-meso-tetra(4-carboxyphenyl)porphine bound to albumin in solution for this preparation as established previously (23). Phosphorescent decay curves for measurement of PFormula 2 were recorded every 4 s from each cell throughout the experimental period.

Data analysis. The fall in PFormula 2 ({Delta}PFormula 2) was determined as the difference between the initial PFormula 2 and the steady-state PFormula 2 as measured in mmHg. This difference is proportional to contraction intensity (and thus Formula 2O2), as demonstrated by Howlett and Hogan (26), and is appropriate to use for comparisons of ATP consumption. Therefore, the relative difference in {Delta}PFormula 2 between control and BTS groups was used to calculate the relative contribution of actomyosin ATPase activity to total ATP consumption.

The kinetics of the {Delta}PFormula 2 were described as mean response time (MRT). MRT was calculated as the time to 63% of both the overall {Delta}PFormula 2 with contractions (on) and PFormula 2 recovery following contractions (off). Both peak tension and [Ca2+]c data were normalized to the initial control point of the first trial.

Statistical analysis. Data are presented as means ± SE. Differences between trials in regard to the PFormula 2 and MRT were tested using a paired t-test. The ratio of {Delta}PFormula 2 in Con vs. BTS groups was calculated for individual fibers, and the mean of this ratio was taken to be the relative contribution of cross-bridge cycling to total energy consumption. Changes in peak tension and [Ca2+]c were tested using a repeated-measures one-way ANOVA. When significant F values were present, the Bonferroni post hoc test was employed for determination of between-group differences. Statistical significance was accepted at P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Peak tension and cytosolic Ca2+. Exposure to BTS resulted in an almost complete attenuation in peak tension that was present in the first contraction (5 ± 0.4% of Con; P < 0.05) and lasted throughout the entire contraction bout (Fig. 1). Baseline and peak [Ca2+]c remained stable throughout the contraction period in Con and were not significantly affected by the addition of BTS (Fig. 2). Therefore, BTS was effective in attenuating tension development without affecting Ca2+ transients. Magnified views of representative tension development and [Ca2+]c during the first two contractions of a bout are presented in Fig. 3.


Figure 1
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  		Fig. 1. A: representative tracing of tension development (arbitrary units, AU) in a single Xenopus laevis fiber before (Con; left) and after (right) exposure to 12.5 µM N-benzyl-p-toluene sulfonamide (BTS). B: tension development in single X. laevis fibers (n = 9; normalized to initial contraction in Con conditions) in the absence (bullet) or presence ({circ}) of 12.5 µM BTS. Values are means ± SE.

 

Figure 2
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  		Fig. 2. A: representative tracing of cytosolic Ca2+ concentration ([Ca2+]c) in a single X. laevis fiber during contractions, assessed as the ratio of 340- to 380-nm fluorescence (340/380 ratio) in the absence (Con) and presence of 12.5 µM BTS. B: peak and baseline [Ca2+]c in single X. laevis fibers (n = 10), assessed as the 340/380 fluorescence ratio in the absence (Con) and presence of 12.5 µM BTS. Values are means ± SE and are normalized to the initial peak [Ca2+]c in Con.

 

Figure 3
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  		Fig. 3. Representative magnified views of tension development (A) and [Ca2+]c (B) during the initial 2 contractions during Con and BTS conditions in single X. laevis fibers.

 
Metabolic cost of cross-bridge cycling (assessed via {Delta}PFormula 2). Starting PFormula 2 for all fibers was 50.7 ± 2.9 mmHg and did not differ between Con (52.6 ± 4.8 mmHg) and BTS groups (48.9 ± 3.5 mmHg). During steady-state contractions, PFormula 2 fell to 15.3 ± 4.7 mmHg in Con and 35.1 ± 2.6 mmHg in the BTS group (P < 0.01, control vs. BTS). BTS attenuated {Delta}PFormula 2 by 55 ± 6% compared with Con (Fig. 4B; P < 0.01). Therefore, the metabolic rate of the cell when cross-bridge cycling was inhibited by 95% (BTS condition) can be assumed to be ~55% lower than the total metabolic rate in Con. Because {Delta}PFormula 2 has been demonstrated to be linearly related to contraction intensity (26), extrapolation of these results to 0% force demonstrates that ATP consumption directly attributable to cross-bridge cycling is ~58% of total ATP consumption in Con (i.e., {Delta}PFormula 2 is decreased ~58% compared to Con when force is extrapolated to 0% Con).


Figure 4
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  		Fig. 4. A: representative data for intracellular PO2 (PFormula 2) during contractions in Con and BTS conditions. B: change in intracellular PO2 ({Delta}PFormula 2) and mean response time (MRT; time to 63% of the total change in PFormula 2) in single X. laevis fibers (n = 9) during contractions in the absence (Con) and presence of 12.5 µM BTS. Values are means + SE. *P < 0.01, significant difference compared with Con.

 
Kinetics of Formula 2O2 at onset and cessation of contractions. The kinetics of the fall in PFormula 2, assessed as the time to 63% of {Delta}PFormula 2 (MRT), at the onset of contractions were significantly faster in Con (75 ± 9 s) compared with the BTS group (101 ± 9 s; P < 0.01) (Fig. 4B), whereas the speed of recovery of PFormula 2 after cessation of contractions was not significantly different in Con (100 ± 12 s) compared with the BTS group (118 ± 15 s). Given the larger {Delta}PFormula 2 and the more rapid kinetics of the fall in PFormula 2, the initial absolute rate of change in PFormula 2 ({Delta}PFormula 2/MRT; in mmHg/s) was significantly faster in the Con group compared with the BTS group (0.36 ± 0.08 vs. 0.09 ± 0.01 mmHg/s; P < 0.01).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The primary findings of the study were that 1) 12.5 µM BTS was a highly effective inhibitor of cross-bridge cycling in an intact cell, resulting in 95% attenuation of tension production with no effect on Ca2+ handling; 2) the metabolic cost of cross-bridge cycling in mixed fiber types of X. laevis estimated from {Delta}PFormula 2 was ~58%, whereas activation energy accounted for ~42%, of total ATP cost during moderate intensity contractions; and 3) the speed of the onset of oxidative phosphorylation (both the kinetics of the response and the absolute rate of increase in O2 consumption) was slowed when metabolic demand was lowered, despite unchanged [Ca2+]c transients.

Use of BTS to measure activation energy. Many of the previous studies designed to measure activation energy rely on overstretching of the cell to prevent cross-bridge cycling and are therefore dependent on the assumption that overstretching the cell does not alter activation energy. Although several investigations suggest that stretching does not alter Ca2+ uptake/release (9, 24), there are data to suggest that Ca2+ handling may be inhibited upon stretch (see Ref. 24). Indeed, it may be particularly important to avoid overstretch when studying isolated fibers, because sarcomeres close to the tendons are not fully stretched, resulting in unequal heat production by cross-bridge cycling along the fiber (see Ref. 12). It also has been demonstrated that in single muscle fibers, Ca2+ release is affected in a stretched state (6). Therefore, other methods for dissociating cross-bridge cycling from Ca2+ handling may be desirable, particularly in the single-fiber model.

Although BDM has previously been used to dissociate Ca2+ handling from cross-bridge cycling, BDM only incompletely inhibits cross-bridge cycling (requiring large extrapolations to zero tension development) when used in concentrations that do not interfere with Ca2+ handling (12, 18, 30, 43). Recently, BTS was shown to inhibit Ca2+-stimulated cross-bridge gliding motility (up to 97% inhibition) in type II mammalian muscle primarily through a decrease in the rate of Pi and ADP release from myosin and a decrease in the affinity of S1 subunit of myosin for actin (15, 38). In addition, Young et al. (48) have shown that BTS almost completely inhibits force generation in skinned muscle fibers with virtually no effect on Ca2+ handling, and similar results have been shown in intact muscle (15, 16). These results were duplicated in the present study (i.e., 12.5 µM BTS caused a 95% inhibition of tension development with no change in [Ca2+]c transients; see Figs. 1 and 2). Therefore, BTS allows for the near complete dissociation of SR function from cross-bridge cycling in intact cells and offers a mechanism by which to investigate cellular function in the absence of actomyosin ATPase activity.

Oxidative cost of activation energy/cross-bridge cycling. During submaximal steady-state work, ATP demand is met almost exclusively though oxidative phosphorylation once the mitochondria have been sufficiently activated. Therefore, measurements of O2 consumption directly reflect muscle energetic (ATP) demand. Previously, it was demonstrated in single cells that, as with Formula 2O2 (17), the {Delta}PFormula 2 is linearly related with contraction intensity (26). Thus measurement of the {Delta}PFormula 2 during contractions can be used as an indication of metabolic rate and, therefore, ATP consumption.

In the present study, it was demonstrated that 12.5 µM BTS was highly effective in inhibiting cross-bridge cycling while having no effect on SR function, as demonstrated by the decrease in force to 5% of Con and unchanged Ca2+ handling (Figs. 1B and 2B). Extrapolating the 55% attenuation in {Delta}PFormula 2 that occurred in BTS (5% Con force) to 0% Con force suggests that during moderate-intensity exercise, cross-bridge cycling accounts for ~58% of total metabolic cost. Assuming the majority of activation energy during exercise is due to SR function, these data suggest that SR Ca2+ handling is responsible for ~42% of ATP consumption during exercise. This estimation is based on the assumption that the efficiency of Ca2+ handling is not altered in the experimental condition. However, it is possible that a greater rate of overall ATP hydrolysis in Con conditions resulted in higher cellular free ADP concentration ([ADP]free) compared with the BTS condition. Given that high [ADP] has been shown to increase Ca2+ leak from the SR (32), a decrease in [ADP]free at the SR in the BTS condition may attenuate Ca2+ leak and lead to a an underestimation of the relative contribution of SR Ca2+ handling to overall energy consumption.

Buschman et al. (12) measured heat production during metabolic inhibition of contractile cycling with BDM and found activation energy in fused contractions of intact X. laevis isolated myocytes to be between 34 and 48% (depending on fiber type 1 and 3, respectively), which is similar to the results of the present study (activation energy in mixed muscle from X. laevis was 42% of total ATP cost of contraction). The results of the present study are also nearly identical to the estimates of Baker et al. (1), who demonstrated using 31P-NMR that activation energy accounts for 43% of total ATP consumption in frogs. Although the estimate of the relative contribution of activation energy in the present study is somewhat higher than that found in frogs by other investigators (25–30%) (5, 25, 39), it should be noted that those experiments were performed between 0 and 6°C. In contrast, the present study, as well as the studies of Buschman et al. (12) and Baker et al. (1), was performed at room temperature. Therefore, given the well-known effect of temperature on activation energy (10, 33), it is likely that these differences can be explained by differing experimental temperatures.

When extrapolating the present finding to human muscle during exercise, a few considerations should be made. First, the present study was conducted using an amphibian model. However, studies in both rat (47) and mouse (4) have led to similar estimations of activation energy (i.e., ~40%), suggesting that there may not be major species differences. Second, during exercise, in vivo muscle fibers shorten during contraction; however, isometric contractions were performed in the present study. Thus, it should be considered that the energetic requirement during maximal shortening can be severalfold higher than during isometric contractions (i.e., Fenn effect) (22, 40). Although this effect has been shown to be considerably lower or absent at low stimulation frequencies (11), estimates of activation heat obtained during isometric contractions may overestimate the energetic requirement of activation in vivo during shortening.

Kinetics of oxygen consumption in the absence of actomyosin ATPase function. It is well known that work intensity dictates steady-state Formula 2O2 and, in turn, the absolute rate of change in Formula 2O2 at the onset of increased rates of work. Numerous whole muscle experiments (in addition to single-fiber experiments) have demonstrated that despite the dependence of the absolute rate of change in Formula 2O2 on metabolic demand, the relative work of the muscle does not affect the kinetics of the rise (i.e., MRT) in Formula 2O2 (e.g., Ref. 13). In other words, the initial rate of activation of oxidative phosphorylation is increased/decreased in proportion to changes in workload, but the time to achieve steady-state mitochondrial respiration mandated by the work intensity is unchanged.

It has been proposed that the regulation of oxidative phosphorylation is based on feedback from alterations in cellular metabolites associated with an increased ATPase activity (i.e., [ADP], ADP/ATP, phosphorylation potential, Pi, phosphocreatine/creatine). According to this model, increased ATPase activity resulting from an increased rate of work will alter the concentrations of these metabolites (ADP, Pi, etc.) and activate oxidative phosphorylation (3, 7, 8). The simplest of these models (first-order control by ADP) was proposed by Chance and Williams (14) in 1955, and it repeatedly has been demonstrated that ADP is a potent stimulator of mitochondrial respiration.

Another proposed mechanism of mitochondrial activation is a system of parallel activation. In this model, mitochondrial respiration is not activated in direct response to an increased rate of ATP hydrolysis. Instead, mitochondrial respiration is activated in concert with increased rates of ATP consumption. Thus, the controller of mitochondrial respiration will also control muscle contraction. Because Ca2+ is not only critical in initiating cross-bridge cycling but also has been shown to activate many mitochondrial enzymes, it has been suggested to be a potential metabolite responsible for control of oxidative phosphorylation via parallel activation (2, 21).

As discussed above, the metabolic rate of muscle does not affect the onset kinetics of oxidative phosphorylation. However, altering the rate of work will result in concomitant changes in both [Ca2+]c and phosphorylation potential, making it difficult to deduce the relative importance of these putative controllers of mitochondrial respiration. In the current experimental conditions, Ca2+ handling was unchanged from control conditions, resulting in a dissociation of the response of [Ca2+]c from adenine nucleotide concentration to a given rate of contraction. Previously, we used this paradigm to demonstrate that a modest reduction in metabolic demand (40% reduction in tension development via BDM) lowered steady-state Formula 2O2 and slowed the initial rate of change in oxygen consumption despite an unaltered [Ca2+]c during contraction. Interestingly, Formula 2O2 kinetics (MRT) were unchanged (time to achieve 63% of the reduced steady-state Formula 2O2 was unaltered because of the slower the rate of change in Formula 2O2), supporting the importance of adenine nucleotides on the control of respiration (i.e., feedback control of respiration) (30). If Ca2+ were a primary controller for respiration, it would be expected that the unchanged [Ca2+]c response to contractions with BDM would have mandated an unaltered absolute rate of change in respiration and resulted in more rapid onset kinetics to achieve the lower steady-state Formula 2O2.

In the present study, the initial rate of change in oxygen consumption was slowed after exposure to BTS. Assuming that BTS does not directly alter mitochondrial function, these data support the contention that Ca2+ (which was unchanged in the presence of BTS) does not play a critical role in setting Formula 2O2 onset kinetics in this single-fiber model. Interestingly, exposure to BTS resulted in a proportionally greater reduction in the initial rate of change in Formula 2O2 than steady-state Formula 2O2, resulting in slowed Formula 2O2 onset kinetics (71% of Con, P < 0.01; Fig. 4B). Although the reasons for this difference remain uncertain, it does imply that respiratory control is not solely dependent on first-order control by adenine nucleotides, which dictates that for any given change in metabolic rate, onset kinetics remain invariable. Alternatively, it should be considered that the greater degree of cross-bridge cycling inhibition in the present study compared with that reported by Kindig et al. (30) (95% attenuation in tension development vs. 40% via BDM) resulted in a much more drastic shift in the region of the cell in which ADP + Pi is generated (from primarily actomyosin ATPases to almost completely SERCA). Given the evidence that compartmentation and direct channeling of metabolites occurs throughout the cell (27, 28, 31, 35–37, 45, 46), it is possible that mitochondria, and thus Formula 2O2 onset kinetics, respond more rapidly to the products of actomyosin ATPase compared with SERCA.

In conclusion, the exposure of isolated single skeletal muscle fibers to BTS allowed direct estimation of the relative contribution of actomyosin and nonactomyosin ATPase activity to overall ATP consumption in a contracting intact cell. Our results demonstrate that cross-bridge cycling accounted for ~58% of total ATP consumption during moderate-intensity contractions, suggesting that Ca2+ handling accounts for ~42% of the ATP cost during contractions. In addition, the results of the present study demonstrate that almost completely abolishing ATP consumption via actomyosin ATPase resulted in slower onset kinetics of oxidative phosphorylation, suggesting that in this single-fiber model, Ca2+ is not the primary controller of oxidative phosphorylation onset kinetics.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported, in part, by National Institutes of Health Grants AR-40155 (to M. C. Hogan), AR-053219 (to B. Walsh), AR-48461 (to C. A. Kindig), and HL-17731 (to M. C. Hogan). C. A. Kindig was a Parker B. Francis pulmonary fellow.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. Walsh, Division of Physiology, Dept. of Medicine, MC0623A, Univ. of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0623 (e-mail: bjwalsh{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.


   REFERENCES
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 DISCUSSION
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