The control of glycolysis in contracting muscle is not fully understood. The aim of the present study was to examine whether activation of glycolysis is mediated by factors related to the energy state or by a direct effect of Ca2+ on the regulating enzymes. Extensor digitorum longus muscles from rat were isolated, treated with cyanide to inhibit aerobic ATP production and stimulated (0.2 s trains every 4 s) until force was reduced to 70% of initial force (control muscle, referred to as Con). Muscles treated with BTS (N-benzyl-p-toluene sulfonamide), an inhibitor of cross-bridge cycling without affecting Ca2+ transients, were stimulated for an equal time period as Con. Energy utilization by the contractile apparatus (estimated from the observed relation between ATP utilization and force–time integral) was 60% of total. In BTS, the force–time integral and ATP utilization were only 38 and 58% of those in Con respectively. Glycolytic rate in BTS was only 51% of that in Con but the relative contribution of ATP derived from PCr (phosphocreatine) and glycolysis and the relation between muscle contents of PCr and Lac (lactate) were not different. Prolonged cyanide incubation of quiescent muscle (low Ca2+) did not change the relation between PCr and Lac. The reduced glycolytic rate in BTS despite maintained Ca2+ transients, and the unchanged PCr/Lac relation in the absence of Ca2+ transients, demonstrates that Ca2+ is not the main trigger of glycogenolysis. Instead the preserved relative contribution of energy delivered from PCr and glycolysis during both conditions suggests that the glycolytic rate is controlled by factors related to energy state.

INTRODUCTION

Skeletal-muscle energy turnover can increase more than 100-fold during high-intensity exercise. Thus the muscle faces intricate problems related to fuel homoeostasis and metabolic regulation. The precision in adjusting the rate of ATP generating processes to the energy requirements is remarkable and is achieved by both feedforward and feedback control [1]. Carbohydrate utilization is of major importance for ATP regeneration during exercise and therefore a precise control of the rate of glycolysis is required. The rate of glycogen utilization is in the order of 3–4 mmol glycosyl units·kg−1·min−1 at 100% V̇O2max, and can increase to 30–50 mmol glycosyl units kg−1 min−1 during maximal dynamic or static contractions [2]. Despite its key importance in metabolism and despite intensive research, the control of glycolysis during exercise is not fully understood.

The conventional view of glycogenolytic/glycolytic control in muscle is that GP (glycogen phosphorylase) and PFK (phosphofructokinase) are the key rate-limiting steps. GP is activated by increases in the concentration of allosteric modulators [AMP and IMP (inosine mono-phosphate)] and substrate Pi and by phosphorylation of the less active form (GPb) into the more active form (GPa). Phosphorylation of GPb is catalysed by phosphorylase kinase, which in turn is activated by the cAMP cascade and/or Ca2+. PFK activity is regulated by the concentrations of adenine nucleotides, Pi, citrate and fructose bisphosphates. Furthermore, Ca2+ and calmodulin have been shown to increase the activity of PFK and to act synergistically with increased levels of AMP and ADP [3]. However, the allosteric effect of Ca2+ and calmodulin were measured in vitro with diluted enzyme and the role of Ca2+ in activating PFK needs to be established at physiological enzyme concentrations or alternatively confirmed in whole muscle.

Glycolytic flux, which is intimately related to glycogenolytic flux, increases severalfold during muscle contraction in close relation to the frequency of muscle activation but is immediately shut down when the contraction terminates even during anaerobic conditions [46]. This suggests that the control of glycolysis is exerted by some factor(s) intimately related to the contraction process. The control of glycolysis in contracting muscle has been studied extensively and two main theories have emerged. One theory is that the rate of glycolysis is under feedback control by the energy demand through increased concentrations of the products of ATP hydrolysis (ADP, AMP and Pi). Another possibility is that glycolysis is under feedforward control by cytosolic Ca2+, which changes rapidly in concert with contractile activity [4,7,8]. The relative importance of these two factors (Ca2+ or energy status) in the regulation of glycolysis has been disputed. Conley et al. [7,8] used 31P MRS (magnetic resonance spectroscopy) to measure the kinetics of PCr (phosphocreatine), Pi, ATP, sugar phosphates and pH in contracting human muscle. Glycolytic rate, calculated from measured parameters, was similar during ischaemic and non-ischaemic conditions and thus was independent of metabolic feedback by Pi, ADP and AMP. The hypothesis that glycolysis is controlled by products of ATP hydrolysis was therefore questioned [7]. The dependence of glycolytic rate on muscle stimulation frequency [8] and the independence of glycolytic rate on the feedback control mechanism are consistent with the view that glycolysis is controlled solely by Ca2+ and not by the cellular energy status. However, there are limitations and pitfalls with estimating the glycolytic rate with the MRS method. It has been pointed out that the MRS method can result in incorrect calculation of glycolytic rate due to overestimation of changes in pHm (muscle pH) in a mixed fibre muscle and an unjustified assumption that Lac (lactate) accumulation has a lag phase during the initial period of contraction [9]. Therefore the role of Ca2+ in the control of the glycolytic rate needs to be reinvestigated with alternative experimental techniques.

Several textbooks convey the message that increases in Ca2+ is the major factor in the control of glycogenolysis during exercise [1013], whereas others conclude that metabolic feedback by energy status is an important part of the control [14]. Control of glycolysis in contracting muscle is a fundamental issue of great importance for our understanding of muscle energetics. It is therefore important to clarify this point by further experimental work.

A possible experimental approach to investigate this issue would be to dissociate the normal Ca2+ transients in a contracting muscle from energy turnover. A potential tool to maintain normal Ca2+ transients during excitation while decreasing ATP turnover is the cross-bridge cycle blocker, BTS (N-benzyl-p-toluene sulfonamide). More specifically, BTS suppresses force production by inhibiting the release of Pi together with a decrease in the apparent affinity for S1*ADP*Pi and S1*ADP (S1 is subfragment 1 of myosin) for actin, and BTS appears to act specifically on the myosin heavy-chain type II isoform [15,16]. Furthermore, BTS does not alter the Ca2+ transients of electrically stimulated single fibres from rabbit [15] or mouse [17,18], it does not impair normal muscle excitability [19], and it does not affect the SR (sarcoplasmic reticulum) Ca2+ pump ATP utilization [20]. The ability to inhibit cross-bridge cycling with BTS, thereby decreasing ATP turnover in the muscle cell, without altering any of the upstream processes in the E–C (excitation–contraction) coupling such as SR Ca2+ transients, makes BTS an excellent tool for the study of the role of Ca2+ and/or metabolites in the activation of skeletal-muscle glycogenolysis.

In previous studies, it was reported that there is a strong correlation between muscle Lac accumulation and PCr levels during both static and dynamic contractions [21]. The PCr/Lac ratio links glycolytic flux and the creatine kinase reaction and is therefore an ideal tool to distinguish abnormalities in energy metabolism. Contracting muscle incubated with BTS has a low energy turnover, whereas Ca2+ transients are maintained similar to those in muscles not treated with BTS. If the hypothesis that Ca2+ is an important trigger of glycolysis is correct, one would expect that the relative ATP provision by glycolysis would increase in BTS-treated muscles. Another approach is to inhibit aerobic ATP formation in quiescent muscle by cyanide and thus to increase the rate of anaerobic ATP formation (PCr utilization and glycolysis) without increases in Ca2+. If the hypothesis that Ca2+ is an important trigger of glycolysis is correct, one would, due to the absence of Ca2+ transients, expect that the relation would change towards a lower Lac and a higher PCr utilization. Conversely, if glycolysis is controlled by energy status one would expect that the relation between PCr and Lac would be similar to that in stimulated control muscles both in muscles treated with BTS and during prolonged cyanide treatment.

It is generally accepted that actomyosin ATPase accounts for most of the ATP consumed during muscle contractions at maximal or near-maximal force, i.e. 50–80% of total energy consumption [18,2230]. BTS selectively inhibits the cross-bridge cycling, while muscle ion pumping remains unchanged, thereby enabling precise estimation of the relative energy utilization by the contractile apparatus. Using BTS, the cross-bridge ATP requirement during isometric contractions has been reported to be 20–30% of overall ATP consumption [31], which advocates for further estimates of the ATP utilization by the relative cross-bridge.

The aim of the present study was, therefore, to test the hypothesis that the glycolytic and glycogenolytic rates are controlled by cellular energy status and not by a direct effect of Ca2+ on the regulating enzymes. We have specifically investigated the effect of modified energy turnover, while maintaining unchanged Ca2+ transients by using the cross-bridge cycling blocker BTS and the effect of blunted Ca2+ transients by incubating muscles with cyanide for a prolonged period. A second purpose of the study was to determine the relative ATP turnover by the cross-bridge cycling during intermittent static contractions. Our findings demonstrate that the glycogenolytic rate is controlled by factors related to energy state, but not directly by increases in Ca2+ and that cross-bridge cycling accounts for 60% of skeletal-muscle total energy turnover during intermittent static contractions.

EXPERIMENTAL

Animals, preparation and incubation of muscles

All handling and use of animals complied with Danish animal welfare regulations. Experiments were performed using 12-week-old Wistar rats, weighing approx. 250 g, which were kept in a thermostatically maintained environment at 21 °C with a 12 h dark/12 h dark cycle and fed ad libitum. The animals were killed by cervical dislocation, followed by decapitation. Intact EDL (extensor digitorum longus) muscles were prepared and incubated in standard KR buffer (Krebs–Ringer bicarbonate buffer) containing the following (in mM): 122.1 NaCl, 25.1 NaHCO3, 2.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.3 CaCl2 and 5.0 D-glucose (pH 7.4). All incubations were performed at 25 °C under continuous gassing with a mixture of 95% O2 and 5% CO2.

Force measurements

Muscles were mounted for isometric contractions in thermostatically maintained chambers containing standard KR buffer and adjusted to optimal length for force production. After a 30 min rest, muscle contractility was tested once, using a short 0.5 s train of 60 Hz, 0.2 ms, 12 V supramaximal pulses (initial F). Muscles were then incubated in a standard KR buffer for 90 min or with 50 μM BTS for a matching time. As BTS was dissolved in DMSO, an equivalent amount of DMSO (0.05%) was added to control muscles. For the final 5 min of the 90 min incubation, standard KR buffer was replaced with glucose-free KR buffer containing 2 mM NaCN, which prevented glucose transport into the muscle cell and mitochondrial oxygen utilization respectively. Muscles then underwent fatiguing stimulation using 0.2 s trains of 60 Hz, 0.2 ms, 12 V supramaximal pulses every 4 s until force was reduced to 70% of initial force response [Con (control muscles)]. BTS-treated muscles were stimulated for an equal time period as Con muscle. Resting values were obtained from the corresponding contralateral muscles (Con rest and BTS rest), which were treated as above, i.e. incubated with or without BTS for 90 min and 2 mM NaCN for the last 5 min plus the corresponding time of the stimulation protocol (88 s). Furthermore, a series of muscles underwent fatiguing stimulation until force was reduced to 85% of initial force response (Con½, n=4). In order to elucidate the effect of an increased energy turnover on muscle metabolite levels, during resting [Ca2+] (Ca2+ concentration), an additional series of resting muscles (n=12) was incubated with 2 mM cyanide for 10 min, at a higher temperature (30 °C).

Force was measured using force displacement transducers and recorded with a chart recorder and digitally on a computer. Results are expressed as a percentage of initial F.

Immediately after fatiguing stimulation, the chamber was lowered, leaving the muscle suspended in air, before the muscles were rapidly detached and frozen in liquid N2 while stirring and stored at –80 °C until assessment of metabolites. This entire procedure was completed in less than 5 s. A sample of the buffer (1 ml from a total of 7 ml) was taken for determination of Lac that has been released from the muscle into the solution during the fatiguing stimulation.

Metabolite measurements

Muscles were frozen and later freeze-dried, dissected free of non-muscle tissue, powdered and extracted with HClO4 as previously described [32]. Neutralized extracts were analysed for Lac, ATP, ADP, AMP, PCr, Cr (creatine), glucose, G-1-P (glucose 1-phosphate), G-6-P (glucose 6-phosphate), F-6-P (fructose 6-phosphate) and Pyr (pyruvate), as previously described [32]. In order to adjust for variability in solid non-muscle constituents and weighing variability, all values are normalized by dividing by the total amount of creatine (PCr+Cr) and multiplied by the mean of total creatine for the whole material [145.1 mmol·kg−1 dm (dry muscle)]. In addition the buffer solution was analysed for Lac in the same way as the muscle extract in order to estimate the total muscle Lac production. Muscle glycogen was analysed from a separate portion of the freeze-dried muscle by the method of Lowry and Passonneau [33]. The metabolic changes after stimulation of Con and BTS muscles were calculated from the difference from the non-stimulated contralateral muscles in each animal.

Calculations

GI (glycolytic intermediates) were calculated as the sum of glucose, G-6-P, G-1-P and F-6-P. Lac was determined from the sum of muscle Lac and Lac released to the buffer. ATP utilization was estimated from substrate phosphorylation as: –ΔPCr–(2ΔATP–ΔADP)+1.5ΔLac+1.5ΔPyr, where Δ refers to the difference between metabolite concentration in the stimulated muscle and the contralateral resting muscle. Glycolytic flux was calculated from (ΔLac+ΔPyr)/2, and glycogenolytic flux was calculated from the sum of glycolytic flux and accumulated GI. pHm was determined using the equation: pHm=7.06–0.00532 (Lac+pyruvate) [34]. Muscle-free ADP and free AMP are the metabolically active forms, and have been estimated from the creatine kinase and adenylate kinase equilibrium [35].

The metabolic rates are given per total time of stimulation, 88 s (Con) and 90 s (BTS). The force–time integral, calculated as the area below the force tracings, was used as an estimate of the isometric ‘work’.

Chemicals

All chemicals were of analytical grade and, unless otherwise stated, were obtained from Sigma–Aldrich, with BTS obtained from Toronto Research Chemicals.

Statistics

Statistical comparisons were made using either a Student's t test or one-way ANOVA, where appropriate. Significant differences between means were located using the Bonferroni post hoc test. Statistical significance was accepted at P<0.05. All values are given as means±S.E.M.

RESULTS

Force production and force–time integrals during fatiguing isometric stimulation

Summarized data of force production during fatiguing stimulation are shown in Figure 1. Muscles were stimulated intermittently at 60 Hz until force was reduced to 70% (Con) of initial force response, which corresponded to 88.4±8.6 s (Figure 1). Initial force averaged 0.81±0.05 N for Con, which corresponds to 83% of maximum tetanic force (100 Hz). The initial force, in stimulated BTS muscle, was 17±2.1% of that in Con and did not significantly change during the 90 s stimulation (Figure 1). The isometric ‘work’ (force–time integral) in BTS muscles was only 38% of that in Con muscles (1.25±0.00 N s and 3.30±0.24 N s respectively). Thus BTS muscles had a much lower force production and isometric ‘work’ than Con. There was no difference in force–time integrals between Con½ muscles, stimulated until the force was reduced to 85% of the initial force response (41.6±3.4 s), and BTS-treated muscles.

Effect of BTS on force production during fatiguing stimulation

Figure 1
Effect of BTS on force production during fatiguing stimulation

Muscles were stimulated using supramaximal 60 Hz, 0.2 s pulses every 4 s, in the absence (controls, ●) or presence (▲) of BTS. BTS muscles were incubated in 50 μM BTS for 90 min prior to fatiguing stimulation. Control muscles were stimulated until force was reduced to 70% of initial force. BTS muscles were stimulated for the time corresponding to the average time of stimulation for control muscles. Data points correspond to means±S.E.M.; n=5–8 and 8 for control and BTS muscles respectively and are normalized to the initial force produced 90 min prior to fatiguing stimulation. *Statistically significant difference from initial force.

Figure 1
Effect of BTS on force production during fatiguing stimulation

Muscles were stimulated using supramaximal 60 Hz, 0.2 s pulses every 4 s, in the absence (controls, ●) or presence (▲) of BTS. BTS muscles were incubated in 50 μM BTS for 90 min prior to fatiguing stimulation. Control muscles were stimulated until force was reduced to 70% of initial force. BTS muscles were stimulated for the time corresponding to the average time of stimulation for control muscles. Data points correspond to means±S.E.M.; n=5–8 and 8 for control and BTS muscles respectively and are normalized to the initial force produced 90 min prior to fatiguing stimulation. *Statistically significant difference from initial force.

Muscle metabolites

Muscle metabolites measured in stimulated muscles and in the respective contralateral non-stimulated muscles incubated for the same time and under the same conditions are presented in Tables 1 and 2. There was no difference between conditions in the metabolite contents of non-stimulated muscles. Thus, in agreement with previous reports, BTS did not alter resting metabolite levels [36]. Muscle contents of ATP, PCr and Lac are in the same order as those previously reported for rat muscle [36] as well as for muscles immediately excised and frozen from a similar sized rat [37]. This demonstrates that pre-incubation of muscles in oxygenated Krebs–Ringer solution for 90 min with addition of cyanide during the final 5 min of incubation had a minor influence on muscle metabolites. However, when non-stimulated muscles were incubated with cyanide at 30 °C for 10 min (n=12), muscle Lac increased by 7-fold [35.4±1.7 mmol·kg−1 dw (dry weight)] and PCr decreased to 39% of initial level (40.0±2.8 mmol·kg−1 dw). In Con½ muscles ATP levels did not change during the stimulation in either of the protocols, whereas ADP was significantly increased and the ATP/ADP ratio was significantly decreased after the stimulation in all protocols. However, a large fraction of ADP and AMP is considered to be bound to proteins or otherwise sequestered in the cell; thus the muscle concentration of the metabolic active forms was estimated. Both [ADPfree] and [AMPfree] were significantly increased after contractions, with a 2- and 4-fold higher concentration of [ADPfree] and [AMPfree] respectively in Con compared with BTS muscles (Table 1). PCr decreased by 73% in Con muscles, with a significantly less severe decrease in BTS (48%) and Con½ (51%) muscles. The decrease in PCr was paralleled by a similar increase in Cr. The relative ATP provision from PCr and glycolysis to the total ATP utilization was not significantly different between protocols (Table 3). Both Con and BTS muscles showed an increase in GI (GI=glucose+G-6-P+G-1-P+F-6-P) after stimulation, with the most pronounced being the 5–6-fold increases in G-6-P and F-6-P. Further, there was a significant higher concentration of both total GI and G-6-P after stimulation in Con than in BTS. Lac and pyruvate levels increased significantly during both conditions, with the increase being approx. 1.5-fold higher in Con than in BTS muscles. In line with the increase in Lac, there was a larger decrease in muscle glycogen levels in Con muscles (22%) than in BTS muscles (12%), and no difference in resting levels. Using the values for Lac and pyruvate, pHm was estimated after stimulation (Table 2). The pHm decreased from the resting value after both treatments, but reached a lower level in Con muscles than in BTS-treated muscles.

Table 1
Content of ATP, ADP, AMP, PCr and Cr in stimulated and non-stimulated muscles

The values shown are means±S.E.M. and are given as mmol·kg−1 dw, except for ADPfree and AMPfree, which are given as μmol·kg−1 dw; n=8 for all conditions. ADPfree and ADPfree are the calculated concentrations of free ADP and AMP respectively.

Con non-stimulatedCon stimulatedBTS non-stimulatedBTS stimulated
ATP 29.6±0.2 29.4±0.4 28.6±0.4 28.4±0.4 
ADP 3.3±0.1 4.0±0.1* 3.3±0.1 4.0±0.1* 
AMP 0.1±0.02 0.1±0.01 0.1±0.01 0.2±0.03 
TAN 33.0±0.2 33.6±0.4 32.0±0.4 32.5±0.4 
ADPfree 79.8±6.2 554.4±24.2* 85.5±2.8 272.5±12.1* 
AMPfree 0.2±0.04 10.1±0.9* 0.3±0.02 2.5±0.2* 
PCr 103.3±2.5 27.4±1.0* 99.9±1.3 52.2±1.1* 
Cr 41.8±2.5 117.7±1.0* 45.2±1.3 92.9±1.1* 
PCr+Cr 143.7±3.1 141.5±2.2 146.7±4.2 142.7±3.0 
Con non-stimulatedCon stimulatedBTS non-stimulatedBTS stimulated
ATP 29.6±0.2 29.4±0.4 28.6±0.4 28.4±0.4 
ADP 3.3±0.1 4.0±0.1* 3.3±0.1 4.0±0.1* 
AMP 0.1±0.02 0.1±0.01 0.1±0.01 0.2±0.03 
TAN 33.0±0.2 33.6±0.4 32.0±0.4 32.5±0.4 
ADPfree 79.8±6.2 554.4±24.2* 85.5±2.8 272.5±12.1* 
AMPfree 0.2±0.04 10.1±0.9* 0.3±0.02 2.5±0.2* 
PCr 103.3±2.5 27.4±1.0* 99.9±1.3 52.2±1.1* 
Cr 41.8±2.5 117.7±1.0* 45.2±1.3 92.9±1.1* 
PCr+Cr 143.7±3.1 141.5±2.2 146.7±4.2 142.7±3.0 
*

Significantly different from the corresponding resting value.

Significantly different from Con.

Table 2
Muscle content of glycogen and glycolytic intermediates at rest and after stimulation

Values are means±S.E.M. and metabolites are expressed as mmol·kg−1 dw; n=8 for all conditions. Glycogenolytic flux was calculated from ΔGI+0.5(ΔLac+ΔPyr), where GI is the sum of glucose, G-6-P, G-1-P and F-6-P and ΔLac is the sum of Lac accumulated in the muscle and Lac released to the buffer.

Con non-stimulatedCon stimulatedBTS non-stimulatedBTS stimulated
Glycogen 120.8±3.6 94.5±3.5* 111.2±4.3 97.6±4.6* 
GI 1.8±0.1 4.2±0.6* 2.4±0.2 3.6±0.4* 
Glucose 1.2±0.1 1.3±0.2 1.6±0.1 1.5±0.2 
G-6-P 0.5±0.04 2.6±0.4* 0.7±0.1 1.8±0.1* 
G-1-P 0.01±0.01 0.01±0.01 0.03±0.01 0.03±0.01 
F-6-P 0.04±0.02 0.24±0.05* 0.08±0.02 0.23±0.04* 
Pyr 0.3±0.02 1.1±0.05* 0.3±0.02 0.8±0.07* 
Lac, muscle 5.3±0.5 47.8±1.1* 4.5±0.3 26.3±2.4* 
Lac, released 0.7±0.2 4.6±0.4* 1.9±0.04 4.7±0.5* 
Muscle pH 7.0±0.01 6.8±0.01* 7.0±0.01 6.9±0.01* 
Δ Glycogen − 26.3±0.9 − 13.6±1.4* 
Calculated glycogenolytic flux − 26.0±0.9 − 13.7±1.4* 
Con non-stimulatedCon stimulatedBTS non-stimulatedBTS stimulated
Glycogen 120.8±3.6 94.5±3.5* 111.2±4.3 97.6±4.6* 
GI 1.8±0.1 4.2±0.6* 2.4±0.2 3.6±0.4* 
Glucose 1.2±0.1 1.3±0.2 1.6±0.1 1.5±0.2 
G-6-P 0.5±0.04 2.6±0.4* 0.7±0.1 1.8±0.1* 
G-1-P 0.01±0.01 0.01±0.01 0.03±0.01 0.03±0.01 
F-6-P 0.04±0.02 0.24±0.05* 0.08±0.02 0.23±0.04* 
Pyr 0.3±0.02 1.1±0.05* 0.3±0.02 0.8±0.07* 
Lac, muscle 5.3±0.5 47.8±1.1* 4.5±0.3 26.3±2.4* 
Lac, released 0.7±0.2 4.6±0.4* 1.9±0.04 4.7±0.5* 
Muscle pH 7.0±0.01 6.8±0.01* 7.0±0.01 6.9±0.01* 
Δ Glycogen − 26.3±0.9 − 13.6±1.4* 
Calculated glycogenolytic flux − 26.0±0.9 − 13.7±1.4* 
*

Significantly different from resting value.

Significantly different from Con.

Table 3
ATP-turnover rates during stimulation

The values shown are means±S.E.M. ATP-turnover data are given in mmol ATP, calculated from the following formula: −ΔPCr–(2×ΔATP–ΔADP)+1.5×(ΔLac+ΔPyr), where Δ is the difference between stimulated and corresponding non-stimulated contralateral muscle; n=8 for both Con and BTS muscles.

ConBTS
ATP turnover (mmol ATP·kg−1 dw) 147.7±3.0 86.3±2.5 
mmol ATP·kg−1 dw s−1 32.6±2.4 18.4±0.5 
mmol ATP·kg−1 dw N·s−1 46.5±3.4 73.6±2.7 
ATP derived from PCr (% of total) 51.4±0.8 55.8±2.6 
ATP derived from glycolysis (% of total) 47.9±0.7 43.2±2.4 
ConBTS
ATP turnover (mmol ATP·kg−1 dw) 147.7±3.0 86.3±2.5 
mmol ATP·kg−1 dw s−1 32.6±2.4 18.4±0.5 
mmol ATP·kg−1 dw N·s−1 46.5±3.4 73.6±2.7 
ATP derived from PCr (% of total) 51.4±0.8 55.8±2.6 
ATP derived from glycolysis (% of total) 47.9±0.7 43.2±2.4 

Significantly different from Con.

ATP turnover

Using the metabolite values it was possible to calculate the ATP utilization, ATP turnover and the rate of ATP utilization during contraction (Table 3). ATP utilization in BTS was 58% of that in Con and the turnover of ATP was significantly higher (77%) in Con than in BTS muscles despite similar duration of stimulation. In line with this, the ATP turnover per stimulation time was significantly lower in BTS-treated muscles compared with Con muscles (Table 3). When the ATP utilization was related to the amount of isometric ‘work’, BTS muscles had a significantly higher ATP utilization than Con muscles, due to the inhibition of cross-bridge cycling, while maintaining normal SR and sarcolemma/t-tubule ion-pumping (Table 3).

Glycolytic flux and glycolytic rates

There was a significantly lower glycolytic flux in BTS (12.5± 1.1 mmol glycosyl units·kg−1 dw) muscles, averaging 54% of that in Con muscles (23.6±0.5 mmol glycosyl units·kg−1 dw). The effect of inhibiting cross-bridge cycling, while maintaining normal Ca2+ transients, on the glycolytic rate is presented in Figure 2(A). In BTS-treated muscle, the glycolytic rate was approximately half of that in Con muscles.

Glycolytic and glycogenolytic rates

Figure 2
Glycolytic and glycogenolytic rates

Stimulation of glycolytic (A) and glycogenolytic (B) rates in Con and BTS muscles with supramaximal 60 Hz, 0.2 s pulses every 4 s. BTS muscles were incubated with the cross-bridge cycle inhibitor BTS for 90 min prior to stimulation; n=8 for Con and BTS muscles. Glycolytic flux is calculated by (ΔLac+Δpyr)/2, and glycogenolytic flux is estimated from direct measures of the difference in muscle glycogen content at rest and after stimulation. Rates are given per stimulation period. †Significantly different from Con.

Figure 2
Glycolytic and glycogenolytic rates

Stimulation of glycolytic (A) and glycogenolytic (B) rates in Con and BTS muscles with supramaximal 60 Hz, 0.2 s pulses every 4 s. BTS muscles were incubated with the cross-bridge cycle inhibitor BTS for 90 min prior to stimulation; n=8 for Con and BTS muscles. Glycolytic flux is calculated by (ΔLac+Δpyr)/2, and glycogenolytic flux is estimated from direct measures of the difference in muscle glycogen content at rest and after stimulation. Rates are given per stimulation period. †Significantly different from Con.

Glycogenolytic flux and glycogenolytic rates

Glycogenolytic flux was estimated both from direct measures of the decrease in muscle glycogen content after stimulation (observed glycogenolysis), as well as from calculations based on the increase in Lac, pyruvate and GI (see the Calculations subsection). There was good agreement between observed and calculated glycogenolytic flux (Table 2). Glycogenolytic flux was almost 2-fold higher in Con than in BTS-treated muscles. Despite differences in glycogenolytic flux, there were no differences between groups, in relation to the relative contribution of Lac, pyruvate and GI in the calculated glycogenolytic flux, accounting on average for 88, 1 and 11% of the glycosyl units respectively. The glycogenolytic rate was on average 2-fold lower in BTS-treated muscles compared with Con (Figure 2B).

PCr versus Lac levels

In a previous study, it was reported that there was a strong logarithmic correlation between human muscle Lac accumulation and PCr levels [21]. We wanted to test if this correlation would also fit with BTS-treated muscles, having an approx. 2-fold lower energy turnover while having the same Ca2+ transients as Con muscles. The curvilinear relationship between PCr and Lac derived from Con rest, Con stimulated and Con½ muscles can be described by the two-phase exponential decay equation: PCr=59.6e−0.035Lac+54.8e−0.035Lac (Figure 3). Even in our data, using rat muscle, there is a strong relation between PCr and Lac (r2=0.95), with the BTS-treated muscles fitting well with the correlation. In line with this, the calculated contribution of PCr to the total energy turnover was not significantly different between conditions (Table 3). This demonstrates that treatment with BTS does not affect the relation between PCr and Lac. If the hypothesis that Ca2+ is an important trigger of glycolysis is correct, one would expect that the relative ATP provision by glycolysis would increase in BTS-treated muscles. PCr and Lac levels fitted well with the correlation when the aerobic ATP formation was inhibited by cyanide in quiescent muscle, i.e. increased rate of anaerobic ATP formation (PCr utilization and glycolysis) without increasing intracellular Ca2+. This observation supports the idea that glycolysis is controlled by energy status and argues against the hypothesis that Ca2+ is an important trigger of glycolysis.

Comparison of muscle PCr content with that of Lac

Figure 3
Comparison of muscle PCr content with that of Lac

Values at rest (■, Con; ●, BTS; ▲, 30 °C incubation for 10 min) and stimulated (□, Con; ○, BTS; Δ, half work). The relationship between PCr and Lac is curvilinear and can be described by the two-phase exponential decay equation: PCr=59.6e−0.035Lac+54.8e−0.035Lac (derived from Con rest, Con stimulated and half work data); r2=0.95.

Figure 3
Comparison of muscle PCr content with that of Lac

Values at rest (■, Con; ●, BTS; ▲, 30 °C incubation for 10 min) and stimulated (□, Con; ○, BTS; Δ, half work). The relationship between PCr and Lac is curvilinear and can be described by the two-phase exponential decay equation: PCr=59.6e−0.035Lac+54.8e−0.035Lac (derived from Con rest, Con stimulated and half work data); r2=0.95.

Relative ATP utilization by cross-bridge cycling

A second purpose of the study was to determine the relative ATP utilization by cross-bridge cycling during intermittent static contractions. The relationship between isometric ‘work’ and ATP utilization in Con and BTS-treated muscles is shown in Figure 4. Extrapolating the line defining the relationship to zero isometric ‘work’ gives an estimate of the ATP utilization by processes other than cross-bridge cycling. Energy utilization by Con muscles averaged 146 mmol ATP·kg−1 dw (Table 3) and the intercept, i.e. non-contractile energy utilization, was 59 mmol ATP·kg−1 dw, with a 95% confidence interval of 43–74. Thus, under our conditions of intermittent static contractions, non-contractile energy utilization on average accounted for 40% (29–51%) and the cross-bridge cycling corresponds to 60% (49–71%) of the total energy utilization.

Relation between force–time integral and ATP utilization

Figure 4
Relation between force–time integral and ATP utilization

The linear relationship between ATP utilization and the force-time integral (isometric ‘work’) with 95% confidence interval. The relation is best fitted by the following equation: ATP utilization=58.7+25.0×force–time integral (R2=0.86). The intercept denotes non-contractile energy utilization and was 59 (43–74) mmol ATP, and total energy utilization was on average 146 mmol ATP·kg−1 dm. Thus, under our conditions of intermittent static contractions for 90 s, non-contractile energy utilization on average accounted for 40% (29–51%) and the cross-bridge cycling corresponds to 60% (49–71%) of the total energy utilization.

Figure 4
Relation between force–time integral and ATP utilization

The linear relationship between ATP utilization and the force-time integral (isometric ‘work’) with 95% confidence interval. The relation is best fitted by the following equation: ATP utilization=58.7+25.0×force–time integral (R2=0.86). The intercept denotes non-contractile energy utilization and was 59 (43–74) mmol ATP, and total energy utilization was on average 146 mmol ATP·kg−1 dm. Thus, under our conditions of intermittent static contractions for 90 s, non-contractile energy utilization on average accounted for 40% (29–51%) and the cross-bridge cycling corresponds to 60% (49–71%) of the total energy utilization.

DISCUSSION

The major observation of the present study is that the rates of glycogenolysis and glycolysis in contracting muscle are reduced when energy turnover is reduced, despite maintenance of the same normal Ca2+ transients as during contractions. The relative glycolytic rate during prolonged cyanide treatment was also independent of Ca2+ transients. This demonstrates that glycolytic flux is controlled by factors related to energy state, but not by Ca2+ directly. A second observation is that, using the cross-bridge cycle inhibitor BTS, estimates of the ATP turnover demonstrate that the cross-bridge cycling accounts for approx. 60% of the total energy turnover by the skeletal muscle during intermittent static contractions.

Regulation of glycolytic and glycogenolytic rates

Our findings are consistent with previous studies showing a close relation between the rate of glycolysis and energy turnover [6]. By using BTS we were able to construct an experimental model where changes in energy turnover were disconnected from parallel changes in Ca2+ transients, i.e. reduced the energy turnover but maintained Ca2+ transients. Several lines of observation from the present study demonstrate that the glycolytic rate is controlled by factors other than a direct effect of Ca2+ on the flux controlling enzymes. First, the glycolytic rate in the BTS-treated muscles was only half of that in the Con muscles (Table 3). Since muscles treated with BTS maintain normal Ca2+ transients but have reduced ATP turnover, this strongly indicates that the glycolytic rate is controlled by energy state and not by a direct effect of Ca2+. Secondly, the relationship between PCr and Lac was independent of BTS (Figure 3) and the relative contribution of PCr and glycolysis to the energy turnover was not different between conditions (Table 3). If Ca2+ is the key factor in the control of glycolytic rate, one would expect that Lac concentration in BTS muscles would be higher than that observed, resulting in a higher relative glycolytic ATP production versus Con and a rightward shift of the relation shown in Figure 3. Thirdly, using prolonged cyanide treatment of quiescent muscle, intracellular Ca2+ levels remain low, while energy supply is covered by anaerobic ATP formation. With this experimental approach the relation between PCr and Lac was not changed. If Ca2+ is an important trigger of glycolysis, one would, due to the absence of Ca2+ transients in resting muscle, expect that the relation would change towards a lower Lac and a higher PCr utilization. On the basis of our results, we conclude that the glycolytic rate is controlled by factors related to energy state but not by Ca2+ directly.

Furthermore, the theoretical base for Ca2+ being the trigger of increased glycolysis is weak. Although the activation of phosphorylase b kinase by Ca2+ and the contraction-induced transformation of GPb into the more active GPa are well established, there is no clearcut relation between the fraction of GP in the form of GPa and glycogenolytic rate. First, GPa levels are already high in resting muscle (approx. 10% of total GP) despite a low rate of glycogenolysis [38]. Secondly, GPa increases transiently at the onset of contraction, but is reverted back to the basal level during sustained contraction despite high rates of glycogenolysis [38]. Thirdly, when GP is almost totally converted into GPa by adrenaline administration, the glycolytic rate only increases to a minor extent, and is maintained below that during intensive exercise [39]. It can be concluded that if Ca2+ is the main trigger of glycogenolysis and glycolysis, this must occur through a mechanism different from conversion of GPb into GPa. In line with this contention, Crowther et al. [40] showed that hexose phosphate levels remained high when exercise and glycolytic flux ceased and they concluded that glycolysis was controlled independently of glycogenolysis. As an alternative to activation of GP by Ca2+ they suggested that glycolytic rate was directly influenced by Ca2+ through Ca2+-initiated phosphorylation of glycolytic enzymes and/or via Ca2+-mediated binding of these enzymes to the cytoskeleton. The findings in the present study give no support for such a mechanism but, on the contrary, demonstrate that glycolytic flux is not controlled by Ca2+ directly. However, the possibility that Ca2+ has a permissive role and that there is a ‘dual-control’ model [41] to achieve high rates of glycolysis cannot be excluded.

An important assumption for the above conclusion is that skeletal-muscle intracellular Ca2+ transients ([Ca2+]i), are the same during the various conditions, i.e. not affected by BTS and/or contractions. BTS has been identified as a highly specific inhibitor of myosin II ATPase that does not interfere with [Ca2+]i, which has consistently been shown by direct measures of [Ca2+]i in rabbit psoas muscle [15], mouse toe muscle [17,31], and as in the present study, rat fast-twitch muscle [42]. Further there is no effect of BTS on muscle excitability [19,36] or SR Ca2+ pump ATP utilization [20]. Although tetanic [Ca2+]i is relatively stable in isolated fibres, stimulated for several minutes using a similar protocol as in present study (350 ms every 2.5–4 s; [43]), it is conceivable that [Ca2+]i is decreased at fatigue during the 90 s stimulation in Con. Importantly, this would strengthen our proposition that the glycolytic rate is controlled by other factors than by a direct effect of Ca2+ on the flux controlling enzymes. Thus BTS muscles not developing fatigue, i.e. no decrease in [Ca2+]i, have a lower glycolytic rate compared with Con muscles with a conceivably decreased [Ca2+]i, while still having a substantially higher glycolytic rate.

The present study demonstrates that the rates of glycolysis and glycogenolysis are more closely related to energy state than to intracellular Ca2+ transients. An elevated rate of ATP turnover leads to increased muscle concentration of ADP, and due to the presence of the adenylate kinase reaction, to even larger increases in AMP. Calculated ADPfree increased approx. 7 times after stimulation in Con, which was 2-fold higher than that in stimulated BTS (Table 1). AMP is a more potent activator of GP and PFK than ADP [2] and increases the affinity of GP for Pi and of PFK for F-6-P. Calculated AMPfree increased approx. 50 times after stimulation in Con, which was approx. 4 times higher than that in BTS (Table 1). The concentration of AMP after stimulation was 3.4 and 0.8 μM in Con and BTS respectively and is within the range where GP is activated [6]. An increase in AMP is therefore an attractive candidate for linking ATP turnover to increased rates of glycolysis.

The glycolytic flux is immediately shut off when the contraction terminates. This is the case even during ischaemic conditions when the metabolic state is clamped with elevated levels of ADPfree and AMPfree [4,21,40,41,44]. Studies using MRS, which has a high temporal resolution, have confirmed the finding of an immediate arrest of glycolytic rate during ischaemic conditions [5]. The low rate of glycolysis in ischaemic quiescent muscle, despite elevated ADP and AMP levels, has been taken as evidence that glycolysis is not solely controlled by elevated metabolites [4,40,41]. However, the concentrations of ADPfree and AMPfree are calculated under the presumption that the creatine kinase and adenylate kinase reactions are at equilibrium without cellular concentration gradients. This is likely to be an oversimplified model and it was suggested that rapid local changes occur in the concentration of ADP and AMP in a contracting muscle at the ATP-utilizing sites and that these are the triggers of glycogenolysis and glycolysis [45]. The hypothesis of transient changes in phosphate metabolites is supported by theoretical modelling [46] and measurement in beating heart with high-resolution MRS [47]. Supporting evidence also comes from the observation that deamination of AMP to inosine monophosphate and ammonia is a process that occurs in a fatigued muscle during contraction but not during the subsequent ischaemic recovery [37]. AMP deaminase is stimulated by increases in AMP and the close link between AMP deamination and contraction therefore gives indirect evidence of transient increases in AMP in contracting muscle.

Cross-bridge ATP utilization

The present data show that the muscle cross-bridge cycling accounts for 60% (49–71%) of skeletal-muscle total energy turnover during intermittent static contractions under our conditions. This is in line with the general acceptance that actomyosin ATPase accounts for 50–80% of the ATP consumed during muscle contractions of maximal or near maximal force [18,2230]. In the present study we have used BTS, which strongly inhibits cross-bridge cycling without affecting Ca2+ handling [15,17,18]. Using BTS, Walsh et al. [18] determined the relative metabolic cost of cross-bridge cycling by estimating the fall in intracellular pO2 in contracting isolated fibres from Xenopus laevis muscle. During intermittent isometric contractions (250 ms every 2–6 s for 100–200 s) at 20 °C, the relative ATP cost used in cross-bridge cycling was 58% of overall ATP consumption [18]. Recently, Barclay et al. [30] estimated cross-bridge cycling accounts for slightly less than two-thirds of overall ATP consumption, with no difference in estimates using the reduced filament overlap or BTS. These estimates are very much in line with the present data of 60% using a more direct approach to determine metabolic cost in similar conditions in mammalian muscle. All the above studies, including the present study, have estimated the relative energy turnover by cross-bridge cycling, by comparing the energy utilization with and without actomyosin ATP turnover (i.e. stretch or BTS). This assumes that cross-bridge cycling efficiency is unaltered during the contractile period. However, muscle fibres increase efficiency with contraction time, most pronounced in fast-twitch muscle [48], which is thought to be a result of decreased actomyosin turnover. Thus the estimated relative ATP consumption by cross-bridge cycling is an average for the contractile period and, as a consequence, underestimates ATP consumption for shorter stimulation periods and overestimates for longer stimulation periods.

Recently, Zhang et al. [31] reported that cross-bridge cycling accounts for only 18–25% of the consumed ATP during repeated tetani for 100 s. The difference in the reported relative myosin ATPase energy utilization may in part be explained by the methods used to estimate the total ATP turnover. Both the present study and Zhang et al. [31] have used cyanide to inhibit oxidative phosphorylation, enabling the estimation of ATP utilization by using conventional biochemical methods. There are, however, some limitations and pitfalls with the technique. First, inhibiting the mitochondrial oxidative phosphorylation at 30 °C may, in contrast with that at 25 °C (used in the present study), lead to perturbation of the resting muscle metabolite levels, i.e. increased Lac and reduced PCr. This may lead to overestimation of total ATP utilization in both Con and BTS and thus reduced relative ATP utilization by the cross-bridge cycling. However, with this in mind, the differences between studies in temperature and relative force during contraction, i.e. 25 °C and 83% Fmax (the present study) versus 30 °C and 35% Fmax, may, as discussed by Zhang et al. [31], be an important part of the explanation for the observed differences in relative cross-bridge ATP utilization.

In conclusion, the present study demonstrates that reducing the energy turnover during contractions in fast-twitch muscles, while keeping normal Ca2+ transients, results in proportional decreases in the glycogenolytic and glycolytic rates. This clearly reveals that the rates of glycogenolysis and glycolysis are controlled by factors related to energy turnover but not to Ca2+ directly. Furthermore, estimates of the ATP turnover with and without inhibiting the cross-bridge cycle reveal that the cross-bridge cycling accounts for 60% of the total energy turnover by the skeletal muscle during intermittent static contractions.

We thank Professor Torben Clausen and Associate Professor Ole Baekgaard Nielsen for the use of their laboratory for the muscle stimulation experiments and Marianne Stürup Johansen and Chris Christensen for skilled technical assistance.

Abbreviations

     
  • Δ

    the difference between metabolite concentrations in the stimulated muscle and the contralateral resting muscle

  •  
  • BTS

    N-benzyl-p-toluene sulfonamide

  •  
  • [Ca2+]

    Ca2+ concentration

  •  
  • Con

    control muscle

  •  
  • Cr

    creatine

  •  
  • dm

    dry muscle

  •  
  • dw

    dry weight

  •  
  • F-6-P

    fructose 6-phosphate

  •  
  • G-1-P

    glucose 1-phosphate

  •  
  • G-6-P

    glucose 6-phosphate

  •  
  • GI

    glycolytic intermediates

  •  
  • GP

    glycogen phosphorylase

  •  
  • KR buffer

    Krebs–Ringer bicarbonate buffer

  •  
  • Lac

    lactate

  •  
  • MRS

    magnetic resonance spectroscopy

  •  
  • PCr

    phosphocreatine

  •  
  • PFK

    phosphofructokinase

  •  
  • pHm

    muscle pH

  •  
  • Pyr

    pyruvate

  •  
  • SR

    sarcoplasmic reticulum

FUNDING

This study was supported by grants from the Ministry of Culture Committee on Sports Research [grant number TKIF 2005-021].

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Author notes

1

Present address: School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, WA, Australia.