The present study examined the effect of elevated temperature on muscle energy turnover during dynamic exercise. Nine male subjects performed 10 min of dynamic knee-extensor exercise at an intensity of 43 W (SD 10) and a frequency of 60 contractions per minute. Exercise was performed under normal (C) and elevated muscle temperature (HT) through passive heating. Thigh oxygen uptake (V̇o2) was determined from measurements of thigh blood flow and femoral arterial-venous differences for oxygen content. Anaerobic energy turnover was estimated from measurements of lactate release as well as muscle lactate accumulation and phosphocreatine utilization based on analysis of muscle biopsies obtained before and after each exercise. At the start of exercise, muscle temperature was 34.5°C (SD 1.7) in C compared with 37.2°C (SD 0.5) during HT (P < 0.05). Thigh V̇o2 after 3 min was 0.52 l/min (SD 0.11) in C and 0.63 l/min (SD 0.13) in HT, and at the end of exercise it was 0.60 l/min (SD 0.14) and 0.61 l/min (SD 0.10) in C and HT, respectively (not significant). Total lactate release was the same between the two temperature conditions, as was muscle lactate accumulation and PCr utilization. Total ATP production (aerobic + anaerobic) was the same between each temperature condition [505.0 mmol/kg (SD 107.2) vs. 527.1 mmol/kg (SD 117.6); C and HT, respectively]. In conclusion, within the range of temperatures studied, passively increasing muscle temperature before exercise has no effect on muscle energy turnover during dynamic exercise.
- ATP turnover
- energy production
- mechanical efficiency
a number of factors have been suggested to influence energy turnover during exercise. These include contraction frequency (15) and intensity of exercise (23). Another potential candidate is an increase in the temperature of the working muscles. The temperature-related mechanisms responsible for the change in energy turnover during exercise could include changes in cross-bridge cycling activity. Indeed, it has been demonstrated that ATP turnover, indicative of a greater rate of cross-bridge cycling, was higher during intense isometric contractions (12) and dynamic exercise (13) when muscle was heated. This is supported by the observation of an elevated myofibrillar ATPase activity in single human muscle fibers at increasing temperatures (30). Other mechanisms could include temperature-related alterations in mitochondrial efficiency through changes in the phosphorous-to-oxygen ratio (9, 32).
Some human studies have examined the influence of temperature per se on aspects of energy turnover during whole body dynamic exercise, with conflicting results. For example, no changes in pulmonary oxygen uptake (V̇o2) were observed during submaximal cycling exercise as a result of prior passive elevations in muscle temperature (10, 21). In these studies, the subjects were allowed to self-select a pedal cadence of ∼75–90 rpm. On the other hand, Ferguson et al. (16) demonstrated that passive elevation of muscle temperature, also before submaximal cycling exercise, increased pulmonary V̇o2 when the exercise was performed at a pedal rate of 60 rpm but that this decreased V̇o2 when performed at 120 rpm. However, it is unclear whether the high temperature affects V̇o2 of the contracting muscles. Febbraio et al. (13) found that glycogenolysis and high-energy phosphate degradation during short-duration intense (115% of maximal V̇o2) cycling exercise was greater when muscle temperature was elevated. Thus it may be that high temperature increases both the aerobic and anaerobic muscle energy production during exercise.
To gain further insight about the effect of temperature on human skeletal muscle energy turnover and efficiency, exercise with an isolated muscle group, in which both the mechanical power output and total energy turnover can be accurately quantified, may be used. The single-legged dynamic knee-extensor exercise model can be utilized, because the muscle mass can be readily determined and the exercise is confined largely to the quadriceps muscle group (2, 26, 27). The exercise model enables an accurate estimation of the total energy turnover from measurements of thigh blood flow; arterial-venous difference for O2 content and metabolites, such as lactate; as well as changes in metabolites in the active muscle during exercise (3, 7). Furthermore, the temperature of the exercising muscle can be manipulated by wrapping the thigh in a blanket through which hot water can be perfused (see Refs. 17, 23).
The purpose of the present investigation was to test the hypothesis that an elevation of muscle temperature would increase skeletal muscle energy turnover during dynamic exercise. Single-legged knee-extensor exercise was performed at a frequency of 60 contractions per minute (cpm) under normal muscle temperature and after passive heating to increase muscle temperature before exercise.
Nine healthy male subjects volunteered to participate in the investigation. All the subjects were physically active, but none was specifically trained. Mean (SD) age, height, and body mass were 24 yr (SD 2), 178.8 cm (SD 5.7), and 73.2 kg (SD 6.8), respectively. The subjects were fully informed of the risks and discomfort associated with the experiment before providing their consent to volunteer. The Copenhagen Ethics Committee approved the study.
Before the experiments, the subjects were familiarized with the knee-extensor exercise and performed a preliminary trial to ascertain their peak knee-extensor work rate. On the experimental day, the subjects arrived at the laboratory at 8:00 AM after consuming a standard breakfast (consisting of fruit juice and cereal) and rested in the supine position. During this time, under local anesthesia, a catheter to collect arterial blood samples was placed antegrade into the femoral artery of the nonexercising (left) leg with the tip positioned ∼2 cm proximal to the inguinal ligament. A second catheter for collection of venous blood samples was placed antegrade in the femoral vein of the experimental leg (right) with the tip positioned 2 cm distal to the inguinal ligament proximal to the saphenous vein. A thermistor (Edslab, T.D. Probe, 94-030-2.5F, Baxter, Allerod, Denmark) for measurement of blood temperature was advanced ∼8 cm beyond the tip of the venous catheter for measurement of venous blood temperature for the calculation of thigh blood flow. The catheter was perforated with four side holes to facilitate infusate dispersion.
For measurements of muscle temperature, the technique described by González-Alonso et al. (17) was used. Briefly, two to three flexible thermistors were inserted 3–4 cm into the muscles of the quadriceps. The thermistors were inserted through a flexible Venflon cannula (18 gauge) and advanced ∼0.5 cm beyond the end of the cannula into the muscle. At least one thermistor was positioned in the vastus lateralis, one in the rectus femoris, and one in the vastus medialis. The probes were inserted at different angles (30, 45, and 60°) with respect to the longitudinal direction of muscle fibers. This ensured varying depths of muscle temperature measurement (2–4 cm) and minimized the movement of the thermistors and discomfort to the subjects. Skin thermistors (Ellab, Copenhagen, Denmark) were attached to the skin next to the insertion points of muscle thermistors, and all were secured with micropore tape.
Single-leg dynamic knee-extension exercise (2) was performed at 60 cpm in the supine position (3). After preparation, the subject rested and muscle temperature was either maintained at a normal temperature (C; i.e., 34–35°C) or elevated by use of a water-perfused cuff that surrounded the thigh (HT). Muscle temperature was elevated in the experimental condition by perfusing water at ∼45°C through the cuff for ∼60 min, which increased muscle temperature to ∼37°C. During exercise, the water temperature of the cuff was maintained at 37°C. Before exercise, blood flow was measured, blood samples were obtained, and a muscle biopsy was taken. After this, subjects performed a 10-min exercise bout at an external power output of 43 W (SD 10), corresponding to ∼85% of peak knee-extensor work rate. Fifteen seconds before the onset of exercise, the leg was passively moved to accelerate the ergometer flywheel and ensure a constant power output from the onset of exercise. Blood was drawn from the femoral artery and vein during the passive exercise 10 s before the onset of exercise and 15 s after exercise had commenced. Further samples were taken at 30 s and 1, 2, 3, 5, 7, and 9.5 min of exercise. To account for the transit time of blood from the artery, through the muscle capillary bed and to the collection point at the vein (6), in the first 15 s of exercise the arterial sample was taken ∼10 s before the venous sample, 6 s before for the next 60 s of exercise, and 5 s before for the remainder of the exercise. Blood flow was measured at 2.5, 5, 7, and 9.5 min. After 10 min, the leg was rapidly stopped (<2 s), and a muscle biopsy taken within 5 s. Because of the experimental procedure, the concurrent measurement of blood flow and sampling for blood metabolites was not possible during the first 2 min of exercise; therefore, 60 min after the first bout of exercise, a 2-min bout at the same intensity was performed during which blood flow was measured. Care was taken to ensure muscle temperature was the same as before the 10-min exercise bout. Again, the leg was moved passively for 15 s before exercise. During this exercise period, blood flow was measured during the transition from passive leg movement to active exercise as well as at 15, 30, 60, and 90 s of exercise. The experimental protocol was repeated for the two muscle temperature conditions on the same day in a randomized order, separated by at least 60 min.
The mass of the quadriceps femoris muscle group was estimated using measurements of thigh length, multiple circumferences of the thigh, and skinfold thickness (19), and the mass was corrected based on a comparison between anthropometric measurements and MRI scan determinations (ratio 1:0.78; Ref. 24). The mean quadriceps muscle mass of the experimental leg was 2.36 kg (SD 0.18).
Thigh blood flow.
Femoral venous blood flow (i.e., thigh blood flow) was measured by the constant-infusion thermodilution technique (1) and as previously modified (17). Briefly, venous and infusate temperatures were measured continuously before and during ice-cold saline infusion into the femoral vein (10–15 s) at a rate of 120 ml/min. This achieved a drop in venous blood temperature of ∼0.6–2°C. Resting blood flow measurements were made with an infusion rate of ∼30 ml/min for 30–45 s. Venous temperature was measured with the thermistor positioned through the venous catheter. Infusate temperature (0–4°C) was measured at the site of entry to the catheter. An occlusion cuff placed below the knee was inflated (250 mmHg) 30 s before the exercise and remained inflated throughout exercise to avoid contribution of blood from the lower leg.
All arterial and venous blood samples were immediately analyzed for Po2, O2 saturation, and hemoglobin (model ABL510, Radiometer, Copenhagen, Denmark) from which O2 content was calculated. For the determination of blood lactate and glucose (YSI 2300, Yellow Spring Instruments, Yellow Springs, OH), 200 μl of whole blood were hemolyzed within 10 s of sampling by adding to 200 μl of buffer (Yellow Spring Instruments; 0.5% Triton X-100).
Muscle samples were taken from the medial part of the vastus lateralis under local anesthesia (1% lidocaine) using the needle biopsy technique (8) with suction. Biopsy samples were immediately frozen in liquid nitrogen and stored at −80°C for subsequent analysis. The frozen samples were weighed before and after freeze-drying to determine water content. The freeze-dried samples were dissected free of blood and connective tissue and prepared for metabolite analysis. Metabolites were extracted in a solution of 0.6 M perchloric acid and 1 mM EDTA, neutralized to pH 7.0 with 2 M KHCO3, and stored at −80°C. Phosphocreatine (PCr) and lactate were analyzed fluorometrically (25). Glycogen was extracted in 1 M HCl, hydrolyzed at 100°C for 3 h, and assayed fluorometrically (25).
Arterial, venous blood, and muscle temperatures as well as saline infusate temperatures were recorded at 400-Hz analog-to-digital sampling rate (Powerlab 16s data-acquisition system, Chart v4.13 software, ADInstruments, Sydney, Australia) onto the hard drive of a computer. Muscle thermistors were connected to the Powerlab via a custom-made interface (17).
Thigh V̇o2 and lactate release were calculated by multiplying blood flow with arterial-venous O2 difference and venous-arterial lactate difference, respectively. A continuous blood flow curve was constructed for each subject by linear interpolation of the measured blood flow data points to obtain time-matched values of blood flow with the blood variables. To obtain measurements of V̇o2 and release of lactate at the capillary level, corrections were made for the blood transit time from the capillaries to the collection points in the femoral artery and vein (6). This correction has significant importance during the initial phase of exercise, where blood flow, oxygen extraction, and blood temperatures increase progressively. Total thigh V̇o2 and thigh lactate release were calculated as the area under the uptake-release curve.
Thigh V̇o2 was converted to aerobic ATP production using a mole volume of 22.4 l/mol O2 and a phosphorous-to-oxygen ratio of 2.5 mmol ATP mmol/molecular oxygen (O) (18). Thigh oxygen uptake was converted into kilojoules by multiplying with 20.7 kJ/l O2, assuming a respiratory quotient value of 0.9 during exercise (20). Muscle anaerobic ATP production was calculated as change in (Δ) muscle creatine phosphate + 3/2·Δmuscle lactate + 3/2·lactate release + others. Others, which accounted for <1% of the anaerobic ATP turnover during C and HT, represents ATP production related to accumulation of pyruvate assumed to be of accumulated muscle lactate (29), lactate uptake by inactive tissues of the exercising leg (5), and accumulation of glycolytic intermediates (29). Anaerobic energy turnover was determined from the net change in reactant levels and average values of energy produced in each of the reactions determined in vitro. ΔH values of 55 and 67 kJ/mol of ATP produced were used for the creatine kinase reaction and glycolysis leading to lactate formation, respectively (11, 31). Muscle ATP production (mmol ATP) and the mean rate of energy turnover (J/s) were determined as the sum of aerobic and anaerobic ATP production and energy turnover, respectively. Mechanical power output was determined as the sum of external work and internal work. A value of 17 W was used for the internal work that is related to the gravitational and inertial forces acting on the lower leg (14). Mechanical efficiency was calculated as the ratio between total mechanical power output (J/s) and total energy turnover (J/s). Mechanical efficiency was also calculated as the ratio between the total ATP production (mmol ATP) and the total work performed (J).
Data were analyzed by either paired t-tests or two-way (muscle temperature condition and time) ANOVA with repeated measures, where appropriate. When a significant effect was observed, differences were located with post hoc paired t-tests with an appropriate Bonferonni correction. Significance was accepted at P < 0.05, and data are presented as means (SD).
Temperature of the quadriceps muscle (Fig. 1) immediately before exercise was higher (P < 0.05) in HT than in C [37.2°C (SD 0.5) vs. 34.5°C (SD 1.7), respectively]. In both C and HT, muscle temperature increased (P < 0.05) progressively during exercise, reaching 38.3°C (SD 0.7) in HT and 37.4°C (SD 1.2) in C, with the temperature on average being 1.5°C (SD 0.3) higher (P < 0.05) throughout exercise in HT than in C [37.9°C (SD 0.7) vs. 36.4°C (SD 1.7)].
Immediately before exercise in both conditions, arterial-venous oxygen difference was ∼35 ml/l and increased to a similar extent (P < 0.05) in C and HT to 88.0 ml/l (SD 20.7) and 89.4 ml/l (SD 23.7) after 15 s, respectively (Fig. 2A). There were further increases (P < 0.05) after 40 s to 131.7 ml/l (SD 17.5) and 125.3 ml/l (SD 17.5) in C and HT, respectively. After 3 min, arterial-venous oxygen difference was 144.2 ml/l (SD 17.4) in C and 137.4 ml/l (SD 11.8) in HT. Thereafter, arterial-venous oxygen difference remained constant and was not different between C and HT.
At the end of 15 s of passive exercise, thigh blood flow was the same in C and HT (Fig. 2B). After 15 s of voluntary exercise, blood flow had increased (P < 0.05) to similar levels in C and HT [2.5 l/min (SD 1.0) and 2.4 l/min (SD 0.6), respectively]. After 2 min and 20 s, blood flow had increased further (P < 0.05) to 3.7 l/min (SD 1.1) and 4.4 l/min (SD 0.8) in C and HT, respectively, and remained unchanged for the duration of the exercise with no difference between C and HT.
Before exercise, thigh V̇o2 was the same in C and HT. After 15 s, V̇o2 had increased (P < 0.05) by the same extent to 0.20 l/min (SD 0.08) and 0.22 l/min (SD 0.09) in C and HT, and it increased further to 0.38 l/min (SD 0.09) and 0.40 l/min (SD 0.11) after 40 s (Fig. 3). After 3 min, thigh V̇o2 had increased (P < 0.05) to 0.52 l/min (SD 0.11) in C and 0.63 l/min (SD 0.13) in HT. By the end of exercise, thigh V̇o2 was 0.60 l/min (SD 0.14) and 0.61 l/min (SD 0.10) in C and HT, respectively. There were no differences in thigh V̇o2 between C and HT, and total thigh V̇o2 was also the same [5.0 liters (SD 0.9) vs. 5.2 liters (SD 1.2)].
Thigh Lactate Release
Immediately before exercise, no venous-arterial lactate difference was observed under either condition. After 3 min, venous-arterial lactate difference had increased (P < 0.05) to equivalent levels in C and HT [1.8 mmol/l (SD 0.8) and 1.5 mmol/l (SD 0.8), respectively]. Thereafter, venous-arterial lactate difference was unchanged at ∼1.2 mmol/l for the rest of exercise in both C and HT.
Before exercise, there was no net exchange of lactate under either condition. Lactate release increased (P < 0.05) during exercise and was 6.2 mmol/min (SD 2.6) in C and 7.0 mmol/min (SD 4.2) in HT after 3 min (Fig. 4). At the end of exercise, lactate release was 4.6 mmol/min (SD 2.5) and 4.3 mmol/min (SD 1.9) in C and HT, respectively, and there were no differences between C and HT. The total release of lactate during exercise was also the same [50.1 mmol (SD 19.7) vs. 47.6 mmol (SD 25.4)].
Muscle PCr decreased (P > 0.05) by similar amounts in C and HT [33.2 mmol/kg dry wt (SD 15.2) and 38.4 mmol/kg dry wt (SD 17.6), respectively]. Muscle lactate increased (P < 0.05) by 23.7 mmol/kg dry wt (SD 22.5) and 32.4 mmol/kg dry wt (SD 25.2) in C and HT, respectively (Table 1). Muscle glycogen declined (P < 0.05) by 86 mmol/kg dry wt (SD 59) and 100 mmol/kg dry wt (SD 107) in C and HT, respectively (Table 1).
ATP Production, Energy Turnover, and Efficiency
Total net lactate production (lactate release and lactate accumulation) was the same in C and HT [25.5 mmol/kg (SD 10.6) and 26.3 mmol/kg (SD 14.0), respectively]. Also, anaerobic [46.3 mmol/kg (SD 17.6) vs. 48.0 mmol/kg (SD 24.4)] and aerobic [458.7 mmol/kg (SD 110.3) vs. 479.1 mmol/kg (SD 112.7)] ATP production were similar in C and HT. Thus total ATP production was the same in C [505.0 mmol/kg (SD 107.2)] and HT [527.1 mmol/kg (SD 117.6)]. Similarly, there was no difference in the rate of energy turnover (aerobic + anaerobic) between the temperature conditions [198.9 J/s (SD 28.3) in C and 209.0 J/s (SD 39.8) in HT]. The average mechanical efficiency was 30.6% (SD 7.2) in C and 29.5% (SD 6.9) in HT (not significant). Mechanical efficiency, when expressed as work per ATP production, was 28.6 J mmol/ATP (SD 6.6) and 27.5 J mmol/ATP (SD 6.4) in C and HT, respectively (not significant).
The main finding of the present study was that passive elevation of muscle temperature before dynamic knee-extensor exercise did not influence muscle energy turnover during exercise. Therefore, mechanical efficiency was not influenced by muscle temperature in the range of 34 to 38°C.
This study has demonstrated that neither aerobic nor anaerobic energy turnover during exercise changed as a result of prior elevation of muscle temperature. The present findings suggest that the observation of a 5% higher pulmonary V̇o2 when muscle temperature was elevated during cycle exercise at the same contraction frequency (60 rpm; Ref. 16) was caused by an elevated energy turnover in nonmuscle tissues. However, it cannot be excluded that the higher body temperatures (39°C) obtained in the latter study, achieved through immersion of the exercising limbs in hot water, influences energy production of the contracting muscles. With respect to the anaerobic contribution to energy turnover, our data are in contrast to the work by Febbraio et al. (13), where they observed a higher degradation of glycogen, higher accumulation of lactate, and high-energy phosphate degradation during intense cycle exercise when muscle temperature was elevated. It is unclear what caused the difference between the two studies. The relative intensity used in their study (13) was ∼115% of maximal V̇o2 for 2 min, compared with ∼85% of peak knee-extensor work rate for 10 min in the present study. The finding of an about fourfold higher lactate accumulation in the study by Febbraio et al. suggests that the anaerobic contribution was significantly larger than in the present study. However, whether this has affected the effect of temperature on the rate of glycogenolysis and glycolysis and whether the elevated anaerobic energy contribution in their study was compensated by a reduced aerobic energy production are uncertain, because the V̇o2 was not measured (13). It might be suggested that the different findings in the present study, compared with those using cycle exercise, are related to the relatively higher perfusion that occurs during knee-extensor exercise compared with two-legged cycling (28), which may cause a higher heat release to the blood and reduce the temperature differences between trials. Nonetheless, a significant difference in muscle temperature (∼1°C) was observed at the end of exercise in the present study.
In the present study and that of Ferguson et al. (15), muscle efficiency was estimated to be 28–31 J/mmol ATP at moderate work rates of 37–43 W, which are similar to what has been previously observed (23). These values are higher than those reported for intense (work rates of ∼65 W) knee-extensor exercise (25–28 J/mmol ATP; see Refs. 4, 7, 23), indicating that the intensity of exercise influences the energy cost of muscular work. Muscle efficiency has also been shown to change with time during constant-load knee-extensor exercise (7, 22, 23). For example, muscle efficiency declined from 51 to 33% from the first 15 s to the last 15 s of a 3-min intense exercise bout (22). Therefore, considering that all the knee-extensor exercise studies reported muscle temperatures within the investigated temperature range (∼34.5–38°C; see Refs. 7, 15, 22, 23), the present study has demonstrated that factors other than muscle temperature must have been responsible for the observed intensity- and duration-dependent variations in muscle efficiency.
The difference in several variables such as rate of energy turnover, PCr utilization, and lactate accumulation between C and HT was 5, 15, and 36%, respectively. It should be considered whether the lack of statistical significance could have been caused by a type II error. In most cases, five subjects had a higher value in HT, two had a lower value, and two had no difference. Although the use of retrospective power calculations is controversial, we have performed such a calculation based on the significance level obtained, the actual observed difference, and the original sample size. The estimated sample size in most cases was found to be >70, indicating that a sample size of this magnitude would be required to obtain a minimal detectable difference. Therefore, based on this analysis, we are confident that we are not making a type II error.
In conclusion, we have demonstrated that an elevation in muscle temperature had no effect on muscle V̇o2 and energy turnover throughout dynamic exercise, suggesting that, in the conditions used, muscle temperature does not influence energy turnover.
The study was supported by Danish National Research Foundation Grant 504-14. In addition, support was obtained from The Sports Research Council (Idrættens Forskningsråd) and Team Danmark.
The authors thank Merete Vannby, Ingelise Kring, and Winnie Taagerup for excellent technical assistance.
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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