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Am J Physiol Regul Integr Comp Physiol 282: R1122-R1132, 2002. First published December 21, 2001; doi:10.1152/ajpregu.00671.2001
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Vol. 282, Issue 4, R1122-R1132, April 2002

Temperature dependency of force loss and Ca2+ homeostasis in mouse EDL muscle after eccentric contractions

Gordon L. Warren1,2, Christopher P. Ingalls2, and R. B. Armstrong2

1 Department of Physical Therapy, Georgia State University, Atlanta, Georgia 30303; and 2 Department of Health & Kinesiology, Texas A&M University, College Station, Texas 77843


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The goals of this study were first to determine the effect of temperature on the force loss that results from eccentric contractions in mouse extensor digitorum longus (EDL) muscles and then to evaluate a potential role for altered Ca2+ homeostasis explaining the greater isometric force loss observed at the higher temperatures. Isolated muscles performed five eccentric or five isometric contractions at either 15, 20, 25, 30, 33.5, or 37°C. Isometric force loss, caffeine-induced force, lactate dehydrogenase (LDH) release, muscle accumulation of 45Ca2+ from the bathing medium, sarcoplasmic reticulum (SR) Ca2+ uptake, and resting muscle fiber free cytosolic Ca2+ concentration ([Ca2+]i) were measured. The isometric force loss after eccentric contractions increased progressively as temperature rose; at 15°C, there was no significant loss of force, but at 37°C, there was a 30-39% loss of force. After eccentric contractions, caffeine-induced force was not affected by temperature nor was it different from that of control muscles at any temperature. Loss of cell membrane integrity and subsequent influx of extracellular Ca2+ as indicated by LDH release and muscle 45Ca2+ accumulation, respectively, were minimal over the 15-25°C range, but both increased as an exponential function of temperature between 30 and 37°C. SR Ca2+ uptake showed no impairment as temperature increased, and the eccentric contraction-induced rise in resting fiber [Ca2+]i was unaffected by temperature over the 15-25°C range. In conclusion, the isometric force loss after eccentric contractions is temperature dependent, but the temperature dependency does not appear to be readily explainable by alterations in Ca2+ homeostasis.

lengthening; injury; damage


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

UNACCUSTOMED eccentric muscle contractions, in which muscles lengthen while active, result in a loss of strength. The strength loss can be dramatic (typically >40-50%) and long-lasting (often >4 wk) (32). The mechanisms underlying this strength loss are not completely resolved, although damage to force-bearing elements, excitation-contraction (E-C) coupling failure, and a loss of contractile proteins have been shown to play a role in some experimental models (32). However, the causes of the damage, the E-C coupling failure, and the protein loss are poorly understood.

The general aim of this study was to manipulate muscle temperature during the eccentric contractions in hope of further elucidating the mechanisms underlying the reduction in strength. For example, numerous studies have shown hypothermia to provide protection against skeletal muscle injury induced by the Ca2+ paradox, ischemia-reperfusion, and metabolic overload (4, 8, 22, 27, 34). Even small reductions in muscle temperature (from 37°C to 32-35°C) provide protection against these injuries as assessed histologically, biochemically, and/or functionally (8, 22, 34). The protective effect at lower muscle temperatures may be due to better maintenance of plasmalemmal and/or t-tubular integrity, and as a consequence, improved intracellular Ca2+ homeostasis (27).

The effect of temperature on eccentric contraction-induced injury, the most common type of muscle injury, is unknown. We are aware of only one preliminary report comparing the eccentric contraction-induced force loss at two temperatures (35). Therefore, the first objective of this study was to determine the effect of temperature over the range of 15 to 37°C on the force loss that results from eccentric contractions performed in an isolated mouse extensor digitorum longus (EDL) muscle preparation.

On finding a progressively greater isometric force loss as temperature increased from 15 to 37°C, we attempted to elucidate the mechanism(s) underlying the effect. We (16) and others (1, 2, 21) have reported muscle fiber free cytosolic calcium levels ([Ca2+]i) to be elevated after eccentric contractions. It is generally accepted that a loss of Ca2+ homeostasis contributes to eccentric contraction-induced injury (e.g., Ref. 7), although it is not clear how the Ca2+ effect might be mediated; possibilities include activation of Ca2+-sensitive proteases and/or phospholipases (7), and depression of sarcoplasmic reticulum (SR) Ca2+ release and, subsequently, force production (19). We postulated that the temperature-dependent force loss after eccentric contractions may reflect a temperature-dependent effect on Ca2+ homeostasis. Thus the second objective of this study was to investigate the potential cause(s) of the temperature-dependent isometric force loss, focusing on the possibility that intracellular Ca2+ homeostasis may be better maintained after eccentric contractions done at lower temperatures.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Female ICR mice (n = 104) purchased from Harlan Laboratories were used. The mice were 8-12 wk old and weighed 30.5 ± 2.6 (SD) g. They were housed at 20-23°C with a 12:12-h light-dark cycle. Before the EDL muscles were excised, the mice were anesthetized with pentobarbital sodium (90 mg/kg ip); supplemental doses (15-20 mg/kg ip) were given when indicated by return of pedal or palpebral reflexes. After excision of the muscles, the mice were euthanized with an overdose of pentobarbital sodium (200 mg/kg ip). The experimental protocols and the animal care procedures were approved by the institutional animal care and use committee and complied with guidelines set by the American Physiological Society.

Experimental Procedures

Experiment 1: effect of muscle temperature on eccentric contraction-induced isometric force loss and caffeine force. The isolated mouse EDL muscle preparation and eccentric contraction-induced injury protocol used were similar to those described previously (e.g., Ref. 31). After the EDL muscle was excised, it was mounted in a 6.5-ml bath assembly maintained at one of six temperatures (i.e., 15, 20, 25, 30, 33.5, or 37°C) within ± 0.1°C. The temperatures selected for use were based on the following logic. First, superficial and deep muscle temperatures of rats at rest are normally 36-37°C (10). Similar values have been observed for human superficial muscle, but temperatures in the 30-35°C range are not uncommon, and temperatures of 25-30°C can occur in cold and wet environments (24, 26). The two lower muscle temperatures investigated (i.e., 15 and 20°C) are outside the normal physiological range in homeotherms but are used in studies of contractile function in mammalian muscle (11, 25). Although muscle temperature during exercise can exceed 40°C, experiments were not performed at temperatures above 37°C because of concerns about viability of the in vitro muscle preparation.

After mounting the muscle in the bath assembly, the muscle was set at its anatomic Lo (i.e., a length halfway between the muscle's minimum and maximum in vivo lengths). This was done by adjusting muscle length to produce a resting force of 4.4 mN, a force previously determined to correspond to the anatomic Lo in mouse EDL muscle (31). We opt for setting muscle length to the anatomic Lo as opposed to the physiological Lo (i.e., the length for which tetanic force is maximized) because 1) the mouse EDL anatomic Lo is more reliably set using our procedure, 2) the process of determining physiological Lo for the fast EDL muscle at warmer temperatures induces fatigue because of the repeated tetani, and 3) it is ensured that the muscle is not stretched outside its in vivo range of motion (i.e., 87-113% of anatomic Lo) during the eccentric contractions that employed a length change from 90 to 110% of the anatomic Lo. This last point is an important one because physiological Lo often does not lie within a muscle's in vivo range of motion (6). There were no differences in anatomic Lo among the six muscle temperatures (P = 0.26); Los at the six temperatures were 15°C, 14.98 ± 0.08 mm; 20°C, 15.02 ± 0.07 mm; 25°C, 14.99 ± 0.06 mm; 30°C, 14.84 ± 0.08 mm; 33.5°C, 14.78 ± 0.09 mm; and 37°C, 14.96 ± 0.09 mm.

Seven minutes into the experiment, an isometric twitch (0.2-ms pulse duration) was done, and a second twitch followed 30 s later. Thirty seconds after the second twitch, an isometric tetanus (200-ms train at the optimal tetanic stimulation frequency) was performed. A second tetanus was done 2 min later. These four contractions are referred to as the preprotocol contractions. Optimal tetanic stimulation frequencies were determined in pilot work for each of the six temperatures (i.e., 62, 114, 175, 244, 278, and 313 Hz for 15, 20, 25, 30, 33.5, and 37°C, respectively). The stimulator (Grass Instruments models S8800 and SIU-5) and muscle length were controlled by computer (Pentium 120 MHz) using a Keithley-MetraByte DAS1802 ST/DA interface board, Keithley-MetraByte VTX (version 1.02) with Microsoft Visual Basic software, and a Cambridge Technology 300B servomotor. Force and length outputs from the servomotor were sampled at 5 kHz. Analog outputs from the computer to the stimulator and servomotor were also done at 5 kHz.

Beginning 3 min after the second isometric tetanus in the preprotocol measurements, one of two protocols was initiated, an isometric protocol (Iso) with five isometric contractions or an eccentric protocol (Ecc) with five eccentric contractions. For an eccentric contraction, the muscle was passively shortened from Lo to 0.9 Lo over 3 s, stimulated tetanically for 133 ms as the muscle was lengthened to 1.1 Lo at 1.5 Lo/s, and then returned passively to Lo over 3 s. For an isometric contraction, the muscle was stimulated tetanically for 133 ms while remaining at Lo. There were 3 min between contractions, and thus protocol duration was 15 min.

Three minutes after the fifth contraction in the Ecc or Iso protocol, an isometric twitch was done, followed 30 s later by an isometric tetanus. The percentage change in maximal isometric tetanic force (Po) from pre- to postprotocol was used to indicate the magnitude of the protocol-induced loss of muscle strength. Po was not measured at later times because we know Po does not recover in the hour after an in vitro eccentric contraction protocol done by a mouse EDL muscle (31). Immediately after the postprotocol isometric tetanus, the water perfusing the temperature-controlled water jacket on the bath assembly was switched to that coming from a 37°C recirculating bath. Muscle temperature reached 37°C in <= 5 min in this bath assembly. (Muscle temperature was measured using a 0.23-mm-diameter thermocouple wire implanted in the muscle. Muscles in which the thermocouple wire was implanted were not included in the data analysis.)

Eight minutes after switching to the 37°C recirculating bath, the muscle was exposed to Krebs-Ringer solution containing 50 mM caffeine. Caffeine is known to bypass the normal E-C coupling pathway by acting directly on the SR Ca2+ release channel to promote an increase in [Ca2+]i (13). If the caffeine-induced force in injured muscles was relatively low, then one may conclude that the SR had a diminished ability to release Ca2+ and/or that there had been a disruption or alteration of force-generating and/or -transmitting structures within the muscle (32).

After maximal caffeine-induced force was attained (i.e., ~5 min after initiating the caffeine exposure), the muscle was removed from the bath assembly, trimmed, blotted dry, and weighed. With the muscle weight and length data, the isometric, eccentric, and caffeine force data were normalized to physiological cross-sectional area as described previously (31). Eighty-four muscles were used in this experiment. For the Ecc protocol, eight muscles were studied at each of the six temperatures. For the Iso protocol, six muscles were studied at each temperature.

Experiment 2: importance of muscle temperature during contractions vs. that in between contractions. After finding greater eccentric contraction-induced isometric force deficits at the higher temperatures in experiment 1, we tested whether it was the muscle temperature during the contractions or the temperature in between contractions that was more important. If the temperature during the contractions was more important, it would suggest that the strength of some structure or component of the muscle (e.g., sarcolemma, Z-line) is temperature sensitive and that the failure of that structure during eccentric contractions is the mechanism of force loss. On the other hand, if the temperature between contractions is more important, it would suggest that a secondary event (or sequence of events) is temperature sensitive and causes the force loss. An enzymatic degradative pathway (e.g., phospholipase A2, Ca2+-activated neutral protease, reactive oxygen species production) would be a likely candidate.

In this experiment, a smaller version of the bath assembly was used (the inner chamber was a 4.1-mm-diameter by 30-mm-long cylinder with a volume of 300 µl). Rapid changes in muscle temperature (15 to 37°C and vice versa in 30 s) were possible by switching the water jacket's supply between two recirculating baths set at different temperatures. Switching between the two baths was done by computer control of an electromechanical relay that drove two solenoid valves regulating the inflow and outflow lines. In preliminary experiments, muscle temperatures were measured during the temperature changes using a 0.23-mm-diameter thermocouple wire implanted in the muscles.

After excising the EDL muscle, it was mounted in the bath at 15°C. Six minutes later, the water perfusing the bath assembly's water jacket was switched to that from a 37°C bath. One minute later (i.e., after the muscle temperature had reached 37°C), the preprotocol contraction sequence was initiated. Immediately after the second tetanus, the water perfusing the bath assembly's water jacket was switched to that from the 15°C recirculating bath.

The EDL muscle then performed the Ecc protocol under one of four conditions. In the "constant 15°C" condition, the muscle stayed at 15°C for the duration of the Ecc protocol. In the "constant 37°C" condition, the water perfusing the bath assembly's water jacket was switched to 37°C starting 1 min before the first eccentric contraction. Muscle temperature then remained at 37°C for the remainder of the experiment. In the "15°C contractions:37°C incubation" condition, the muscle performed the eccentric contractions at a muscle temperature of 15°C, but in between contractions the temperature was switched to 37°C. The time-averaged muscle temperature over the Ecc protocol for this condition was 33.7°C. In the "37°C contractions:15°C incubation" condition, the muscle performed the eccentric contractions at a muscle temperature of 37°C, but in between contractions the temperature was switched to 15°C. The time-averaged muscle temperature over the Ecc protocol for this condition was 18.3°C. After an eccentric contraction at 37°C and switch to 15°C, it took 7 s to reduce muscle temperature to <25°C. As described below, temperatures of <= 25°C were found to confer protection against lactate dehydrogenase (LDH) release and muscle Ca2+ accumulation. It then took another 23 s to get down to <16°C. Figure 1 shows the rapid changes in muscle temperature that occurred in the "37°C contractions:15°C incubation" condition.


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  		Fig. 1.   Muscle temperature over a 4-min period in the "37°C contractions:15°C incubation" condition (i.e., when the eccentric contractions were done at 37°C but the muscle was incubated at 15°C in between contractions). Muscle temperature was measured using a 0.23-mm-diameter thermocouple wire implanted in the muscle. To raise muscle temperature from 15 to 37°C in 30 s, the warmer recirculating water bath was set to 38.3°C. For the "15°C contractions:37°C incubation" condition, the temperature-time profile was the inverse of that for the "37°C contractions:15°C incubation" condition.

The Ecc protocol used was identical to that described for experiment 1 with two exceptions. First, there were 4 min between contractions instead of 3 min. The longer time between contractions was done to exacerbate any "incubation" effect. Second, when the eccentric contractions were done at 37°C, the muscles were stimulated at either 313 Hz (the frequency eliciting maximal force production at 37°C) or 170 Hz (the frequency eliciting a peak eccentric force matching that produced by maximal eccentric contractions done at 15°C).

Three minutes after the fifth eccentric contraction, the water perfusing the bath assembly's water jacket was switched to 37°C (if the muscle was not already being maintained at 37°C). One minute later an isometric twitch was done, followed 30 s later by an isometric tetanus. The muscle was then removed from the bath and weighed.

Thirty-six muscles were used in this experiment. For the "constant 15°C" and "15°C contractions:37°C incubation" conditions, six muscles were studied in each condition. For the "constant 37°C" and "37°C contractions:15°C incubation" conditions, 12 muscles were studied in each condition; in each of these two conditions, 6 muscles were stimulated at 313 Hz and 6 muscles at 170 Hz during the eccentric contractions.

Experiment 3: effect of muscle temperature on eccentric contraction-induced loss of cell membrane integrity and influx of extracellular Ca2+. The purpose of this experiment was to determine how muscle temperature affects the Ecc protocol-induced loss of cell membrane integrity, as reflected by LDH release into the bathing medium, and influx of extracellular Ca2+ (as indicated by accumulation of 45Ca2+ from the bathing medium). The experiment was conducted as in experiment 1 with the following exceptions. The 300-µl muscle chamber was used. Following the preprotocol contractile measurements, a 10-µl sample of the bath medium was taken to determine the baseline LDH activity. Also at this time, 45CaCl2 at a specific activity of 450 Ci/mol was added to the bath medium so that the final concentration was 4 µCi/ml. The Ecc protocol was then performed at one of the six temperatures. Three minutes after the fifth eccentric contraction, another sample of the bath medium was taken to assay LDH activity.

The muscle was then removed from the bath assembly and rinsed in unlabeled ice-cold Krebs-Ringer solution three times for 1 min each. A rinse of this duration probably does not remove all extracellular 45Ca2+, but we wanted to minimize leaching of 45Ca2+ from fibers with damaged plasmalemma and/or t-tubules. The control muscles should have accounted for any partial removal of 45Ca2+ from the extracellular space as well as any normal exchange through Ca2+ channels.

LDH activity in the bath medium was assayed as described previously (30); all assays were run at 37°C. Muscle 45Ca2+ content was assayed by placing the muscle in a 6.5-ml polyethylene scintillation vial and adding 0.75 ml Solvable (Packard Instruments). To solubilize the muscle, the vial was heated to 60°C for 3 h with occasional swirling. After cooling, 5 ml of liquid scintillation cocktail (Packard Instruments Ultima Gold) was added. After a >= 1-h light and temperature adaptation, the sample was counted in a liquid scintillation counter (Beckman model 3801).

A total of 44 muscles was used for the LDH release and 45Ca2+ accumulation measurements. The Ecc protocol was performed on six muscles at each of the six temperatures. In addition, there were eight control muscles that did no contractions after the preprotocol measurements; four were incubated at 15°C and four at 37°C. Additional controls, i.e., muscles performing the Iso protocol at the two or more temperatures, were not used because the Iso protocol muscles in experiment 1 showed minimal changes in isometric force at any temperature.

Because we observed a marked increase in 45Ca2+ accumulation as temperature rose above 30°C, we tested whether an increased activation of the Ca2+-sensitive protease calpain could explain the greater isometric force losses at the higher temperatures. Muscles (n = 6) were exposed to calpain inhibitors (i.e., 50 µM leupeptin, 100 µM calpeptin, or 120 µM E-64d) for 20 min before and during Ecc protocols done at 37°C.

Experiment 4: effect of temperature on the rate of SR Ca2+ uptake. The results of experiment 3 showed that as temperature rose above 30°C, there was a marked increase in the influx of extracellular Ca2+ after the Ecc protocol. We wondered if the SR had the capacity to sequester Ca2+ at a rate sufficient to offset this increased influx. There are data indicating that the rate of SR Ca2+ uptake decreases as temperature increases above 37°C (12, 28). Warmington and co-workers (28) reported 26-28% decreases on warming of rat soleus and EDL muscles to 40°C for just 1 min. Geimonen and co-workers (12) showed a linear decrease in the rate of rabbit muscle SR Ca2+ uptake as temperature increased above 37°C. SR Ca2+ uptake was reduced by 90% after an increase of only 8°C (i.e., at 45°C). Unfortunately, it is not possible from these two studies to determine at what temperature the decrease in SR Ca2+ uptake begins. We hypothesized that the SR Ca2+ uptake rate in the mouse EDL muscle may begin to level off or decrease in the 30-37°C range. Thus the purpose of this experiment was to determine if the rates of SR Ca2+ uptake in the 30-37°C range fall below values predicted from a Q10 extrapolation of Ca2+ uptake rates at lower temperatures.

The rate of SR Ca2+ uptake was measured in homogenates of uninjured muscles at the six temperatures using a Ca2+-selective minielectrode as described previously (16). Uninjured muscles were used in this experiment because SR Ca2+ uptake in the injured muscle is not appreciably different from control muscles until 3-5 days postinjury (16). Muscles were homogenized on ice in 10 volumes of isolation buffer (20 mM HEPES, 250 mM sucrose, 0.2% NaN3, and 0.2 mM phenylmethylsulfonyl fluoride at pH 7.5). Aliquots (40 µl) of the homogenates were pipetted into 2-ml microcentrifuge tubes, frozen in liquid N2, and stored at -80°C. Six EDL muscles pooled together and nine tibialis anterior (TA) muscles were used for this experiment. Pooling of the six EDL muscles was necessary because a single EDL muscle homogenate yields enough volume to run duplicate assays at only one temperature. On the other hand, a TA muscle homogenate yields enough volume to run duplicate assays at each of the six temperatures. The SR Ca2+ uptake data obtained from the TA muscles should be generalizable to the EDL muscles because of their similarity in function (i.e., both are anterior crural muscles) and fiber-type composition (i.e., both are >= 97% fast twitch).

For calibration of the Ca2+-selective minielectrode, seven CaEGTA standards with 1 mM free Mg2+ (Molecular Probes C-3722) were used. The standards ranged from 5 to 9.8 mM CaEGTA with a total EGTA concentration of 10 mM. After determining the pH values of the standards at each temperature, the EGTA Kd for Ca2+ was calculated for each temperature using MaxChelator software (version 2.0) (3, 23). The [Ca2+] of a given standard was calculated as the product of the EGTA Kd for Ca2+ and the [CaEGTA]:[EGTA] ratio. For all calibrations, the correlation of log [Ca2+] to electrode potential exceeded 0.996.

The SR Ca2+ uptake assay began by adding 1 ml of assay medium (100 mM KCl, 7.5 mM tetrasodium pyrophosphate, 20 mM HEPES, 1 mM MgCl2, 10 mM NaN3, and 3.5 µM free Ca2+) to a frozen aliquot of muscle homogenate. Five minutes after initiation of the assay, 10 µl of 100 mM Na2ATP was added. The assay proceeded for 12 min or until the assay medium [Ca2+] had decreased to <= 1.0 µM. Figure 2 depicts representative tracings for SR Ca2+ uptake obtained at each of the six temperatures.


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  		Fig. 2.   Representative tracings of assay medium Ca2+ concentration ([Ca2+]) as a function of time at each of the 6 temperatures during the sarcoplasmic reticulum (SR) Ca2+ uptake measurement. ATP (1 mM) was added at 30 s into the data acquisition.

For each set of "assay medium [Ca2+] vs. time" data, an analysis program searched for the >= 20-s long segment of data yielding the highest rate of decrease in [Ca2+]. The search was not initiated until >= 15 s after the injection of ATP. A criterion for selection of the data segment was that the correlation between [Ca2+] and time had to be >= 0.99. Thus, for noisier data (i.e., at 15°C), only longer-duration segments could meet the criterion. Response properties of the Ca2+-selective minielectrode do not affect the calculated SR Ca2+ uptake rates. For example, at 25°C, a step reduction in assay medium [Ca2+] of one order of magnitude elicits an electrode response that is 90% complete within 80 ms.

Experiment 5: effect of muscle temperature on eccentric contraction-induced changes in muscle fiber resting [Ca2+]i. From the results of experiments 3 and 4, there was evidence of a temperature-dependent loss of cell membrane integrity and subsequent influx of extracellular Ca2+ after eccentric contractions, but no evidence that the fibers' Ca2+ buffering capacity was not adequate to maintain intracellular Ca2+ homeostasis. The purpose of this experiment was to determine how temperature affects the resting muscle fiber [Ca2+]i after eccentric contractions. An increase in muscle fiber resting [Ca2+]i at higher temperatures would be interpreted as a diminished ability to maintain intracellular Ca2+ homeostasis at those temperatures.

Measurement of [Ca2+]i was based on the use of ratiometric confocal laser scanning microscopy (CLSM) and visible-wavelength Ca2+-sensitive dyes as described previously (15, 16). EDL muscles were incubated for 20 min at room temperature with 10 µM fluo 3-acetoxymethyl ester (AM) and 10 µM fura red-AM (Molecular Probes) in an oxygenated, amino acid-free Krebs-Ringer solution containing 3 mg/ml of Pluronic F127. The muscle was then mounted horizontally in a superfused Plexiglas chamber on the microscope stage. One end of the muscle was attached via suture to the arm of a Cambridge Technology 300B servomotor. The other end of the muscle was attached to a chamber wall. The muscle then did the Ecc protocol as described for experiment 1. Experiments were attempted at only three temperatures (i.e., 15, 25, and 37°C) because of the difficulty associated with these measurements.

CLSM images were acquired immediately before the first eccentric contraction and 2 min after each eccentric contraction. The muscle was imaged using a NORAN Odyssey XL CLSM with a 50-mW argon-krypton laser attached to a Zeiss Axioskop microscope. The dyes were excited using the 488-nm laser line, and the fluorescence was collected into the two channels using 515- to 545-nm (i.e., primarily the fluorescence of fluo 3) and 635- to 685-nm (i.e., primarily the fluorescence of fura red) band-pass filters. A Zeiss Achroplan 10× water-immersion objective (NA 0.30) was used to acquire the optical sections. Optical sections (i.e., 765 × 589 µm) were acquired from a standardized location on the muscle. Depending on the muscle, 6-14 muscle fibers were resolved in a given optical section.

For estimation of muscle fiber [Ca2+]i, background fluorescence was first subtracted from images from the eight channels. Voxel intensities in the fluo 3 channel were then divided by the corresponding voxel intensities in the fura red channel to obtain an eight-bit ratio image of [Ca2+]i. To estimate [Ca2+]i, an in vitro calibration was applied to the eight-bit gray-scale voxel intensity. After determination of intracellular fluo 3 and fura red concentrations, fluo 3 and fura red salts were added at those concentrations to CaEGTA standards (i.e., 1-10 mM) and imaged to determine the relationship between the fluorescence ratio and [Ca2+]; EGTA Kd values for Ca2+ were temperature and pH corrected as in experiment 4. Fiber fluorescence ratio values were then converted to [Ca2+]i using the Hill equation. This in vitro calibration method does not take into account changes in dye properties when bound to intracellular proteins.

There are two limitations associated with this experiment. First, the muscles that did the eccentric contractions at 37°C apparently lost cytosolic fluo 3 and fura red throughout the protocol. This conclusion is based on the observations that fluorescence in both the fluo 3 and fura red channels tended to decrease over the Ecc protocol at 37°C, and these muscles did not show the typical large increase in the fluo 3:fura red fluorescence ratio on exposure to 10 µM beta -escin at the end of the experiment. Thus the data for the four muscles done at 37°C were excluded from the analyses. Second, peak eccentric force in the dye-loaded muscles was less (i.e., by 21-24%) than that observed in unloaded muscles at the 15 and 25°C temperatures. Thus the amount of injury sustained by the dye-loaded muscles was probably less than that sustained in other studies (29). In all, [Ca2+]i was determined on 41 fibers from four muscles at 15°C and 28 fibers from three muscles at 25°C.

Statistical Analyses

For experiment 1, the effects of protocol (Ecc vs. Iso) and temperature on the dependent variables were analyzed using a two-way ANOVA. For experiments 2 and 3, the effect of temperature was analyzed using a one-way ANOVA. The effect of temperature on SR Ca2+ uptake in experiment 4 was analyzed using a one-way ANOVA with repeated measures. For experiment 5, the effects of temperature and time into the Ecc protocol on [Ca2+]i were analyzed using a two-way ANOVA. When assumptions of normality or equal variance were violated, Kruskal-Wallis one-way ANOVAs were employed. When significant main effects or interactions were found, differences in group means were tested using Student-Newman-Keuls post hoc tests. All statistical testing was done using Jandel SigmaStat software (version 1.02/2.0). An alpha -level of 0.05 was used for all analyses. Values presented in RESULTS are means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mean weight of the 177 EDL muscles studied was 10.03 ± 0.07 mg. During the preprotocol measurements, the mean Po of the 60 EDL muscles studied at 37°C was 426.2 ± 4.3 mN. The mean weight of the nine TA muscles used in experiment 4 was 63.2 ± 2.4 mg.

Experiment 1: Effect of Muscle Temperature on Eccentric Contraction-Induced Isometric Force Loss and Caffeine Force

The changes in Po after the Ecc and Iso protocols are shown for the six temperatures in Fig. 3. For muscles performing the Ecc protocol at 15°C, the percent reduction in Po was not significantly different from zero or from that observed for muscles performing the Iso protocol. However, as temperature increased above 15°C, there was generally a progressive increase in the Po loss associated with the Ecc protocol. The Po loss did level off between 25 and 30°C before increasing sharply above 30°C. On the other hand, for the muscles that did the Iso protocol, there were no significant reductions in Po at <= 33.5°C. At 37°C, there was a significant Iso protocol-induced Po loss, but it was only one-fifth of that observed for the Ecc protocol.


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  		Fig. 3.   Effect of temperature on the maximal isometric tetanic force (Po) loss induced by the eccentric (Ecc) protocol and the isometric (Iso) protocol. See METHODS for details about Ecc and Iso protocols. Values with the same letter are not significantly different.

The temperature dependency of the loss in Po after the Ecc protocol did not mirror the temperature dependency for peak eccentric force or work done on the muscle. Figure 4 shows that peak eccentric force was actually highest for Ecc protocols done at 30°C. At this temperature, peak eccentric force was 13% higher than that observed at 37°C. The temperature dependency for work done on the muscle during the eccentric contractions was the same, with the work at 30°C being 14% higher than that at 37°C.


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  		Fig. 4.   Effect of temperature on the highest force recorded during the Iso and Ecc protocols. Values with the same letter are not significantly different.

A temperature dependency for force loss was also observed for one type of submaximal contraction (i.e., isometric twitch) but not another (i.e., caffeine-induced contraction). The effect of temperature on the isometric twitch force loss after the contraction protocols mirrored that observed for the Po loss. Isometric twitch force after the Ecc protocol was not significantly reduced at 15°C but was significantly reduced by 39% at 37°C. Twitch force was not significantly reduced after the Iso protocol at any temperature. On the other hand, there were no differences between the protocols in caffeine-induced force at any temperature (i.e., P = 0.18 for a protocol main effect and P = 0.34 for a protocol-temperature interaction). Figure 5 shows the forces elicited by exposure to 50 mM caffeine at 37°C after the Ecc and Iso protocols at the six temperatures.


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  		Fig. 5.   Effect of temperature during the Ecc and Iso protocols on the peak caffeine force measured at 37°C after the protocols. There were no differences between the Iso and Ecc protocols at any temperature (P >=  0.18).

Experiment 2: Importance of Muscle Temperature During Contraction vs. That In Between Contractions

Figure 6 shows the relative effect on the Ecc protocol-induced Po deficit of muscle temperature during contraction vs. the incubation temperature in between contractions. When the Ecc protocols were matched on peak eccentric force (i.e., the 4 rightmost bars in Fig. 6), there was no significant effect of incubation temperature on the Po deficit. In other words, for muscles doing eccentric contractions at 15 or 37°C, it did not matter what the temperature was in between the eccentric contractions. When muscles did maximal eccentric contractions at 37°C (i.e., the 2 leftmost bars in Fig. 6), there was a substantial protective effect if the temperature in between contractions was reduced to 15°C; the Po loss for the "37°C contractions:15°C incubation" condition was 25% less than that observed for the "constant 37°C" condition


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  		Fig. 6.   Effect of muscle temperature during contraction and during the incubation in between contractions on the Po loss induced by the Ecc protocol. The 2 leftmost bars represent data for muscles performing maximal eccentric contractions at 37°C (i.e., eliciting a peak eccentric force of 175% Po), whereas the 2 center bars also represent data for muscles performing eccentric contractions at 37°C but at a lower stimulation frequency (i.e., that eliciting a peak eccentric force of 147% Po). The 2 rightmost bars represent data for muscles performing maximal eccentric contractions at 15°C (i.e., eliciting a peak eccentric force of 147% Po). Values with the same letter are not significantly different.

Experiment 3: Effect of Muscle Temperature on Eccentric Contraction-Induced Loss of Cell Membrane Integrity and Influx of Extracellular Ca2+

The effects of temperature on the rates of LDH release and muscle accumulation of 45Ca2+ are shown in Fig. 7. LDH release rates were quite low for control muscles and the muscles that did the Ecc protocol at <= 25°C. However, at >25°C, LDH release during the Ecc protocol increased as an exponential function of temperature. The temperature dependency of 45Ca2+ accumulation by muscles during the Ecc protocol was similar to that observed for LDH release. At <= 25°C, there was no effect of temperature on 45Ca2+ accumulation by muscles that did the Ecc protocol, nor were the 45Ca2+ accumulation rates by these muscles different from those observed for control muscles done at 15°C. 45Ca2+ accumulation in muscles that did the Ecc protocol increased steadily as a function of temperature at >25°C. At 37°C, accumulation was 2.2- to 2.4-fold of that observed at <= 25°C. However, part of this temperature-dependent increase is simply an effect of temperature on normal, unstimulated muscle because 45Ca2+ accumulation in control muscles at 37°C was 76% greater than that observed at 15°C. The high Ca2+ accumulation in muscles that did the Ecc protocol at 37°C does not appear to have resulted in a calpain activation that contributed to that temperature's high Po deficit as exposure of muscles to calpain inhibitors (leupeptin, calpeptin, or E-64d) before and during the Ecc protocol had no effect on the Po deficit.


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  		Fig. 7.   Effect of temperature on the rates of lactate dehydrogenase (LDH) release (top) and muscle 45Ca2+ accumulation (bottom) induced by the Ecc protocol. Control muscles performing no contractions during the time of the protocol were also studied at 15 and 37°C. Values with the same letter are not significantly different.

Experiment 4: Effect of Temperature on the Rate of SR Ca2+ Uptake

Figure 8 shows the effect of temperature on SR Ca2+ uptake in homogenates of uninjured EDL and TA muscles. The curve indicates the best fit for the TA muscle data assuming a Q10 effect on SR Ca2+ uptake. The Q10 fit is quite good with a r2 of 0.991 and a predicted 2.35-fold increase in SR Ca2+ uptake for a 10°C increment in temperature. A Q10 fit was not determined for the EDL muscle data because those muscles had been pooled, and thus the EDL data points in Fig. 8 do not reflect replicate measures. Nevertheless, it is clear that the results for the EDL muscles are nearly identical to those for the TA muscles. In summary, these results do not support our hypothesis that the SR Ca2+ uptake rate would level off or begin to fall as temperature increased above 30°C.


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  		Fig. 8.   Effect of temperature on the rate of SR Ca2+ uptake by tibialis anterior (TA) muscles and by the 6 extensor digitorum longus (EDL) muscles that were pooled together. The curve indicates the best fit for the TA muscle data assuming a Q10 effect. All TA muscle SR Ca2+ uptake values are significantly different from each other.

Experiment 5: Effect of Muscle Temperature on Eccentric Contraction-Induced Changes in Muscle Fiber Resting [Ca2+]i

The changes in muscle fiber resting [Ca2+]i during the Ecc protocol at 15 and 25°C are shown in Fig. 9. Resting [Ca2+]i before any eccentric contractions for the two temperatures was estimated at 141-147 nM, values typical of those reported in the literature for skeletal muscle (18). In contrast to our prediction, there were no differences between the two temperatures in the [Ca2+]i increase over the protocol (i.e., P = 0.56 for a temperature main effect and P = 0.93 for a temperature-time interaction). By the end of the Ecc protocol, [Ca2+]i for both temperatures was estimated to have increased by 31-34%. In control muscles, [Ca2+]i shows no tendency to increase over 15-20 min.


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  		Fig. 9.   Changes in muscle fiber resting free cytosolic [Ca2+] ([Ca2+]i) during Ecc protocols done at 15 and 25°C. [Ca2+]i increased during the protocol at both temperatures (P <=  0.0001), but there were no differences between the 2 temperatures (P >=  0.56). The arrows indicate the timing of the eccentric contractions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, there was a strong effect of temperature on the strength loss caused by a bout of eccentric contractions. In contrast, there was no effect of temperature on the Iso protocol-induced strength changes except at >33.5°C. A Q10 function describes the strength loss:temperature relationship for eccentric contractions reasonably well with an r2 of 0.78 and a Q10 coefficient of 2.12.

The strength loss associated with eccentric contractions is believed to be caused by one or more mechanical factors (e.g., excessive stress, strain, and/or work done on the muscle) (5, 20, 29). However, the temperature effect on the Ecc protocol-induced strength loss does not appear to be mediated via temperature-dependent changes in these factors. First, the temperature-dependent strength loss cannot be attributed to a greater strain at the higher temperatures, because muscles at all temperatures were stretched over the same range (i.e., from 0.9 to 1.1 Lo). Also, there were no differences in Lo among the muscles tested at the six temperatures. Second, peak eccentric forces and work done on the muscles were highest when Ecc protocols were done at 30°C, with both measures declining as temperature rose from 30 to 37°C. However, the strength loss for Ecc protocol muscles at 30°C was half that observed at 37°C. In addition, in experiment 2 when the protocols were matched for peak eccentric force and work done on the muscle, the Po loss at 37°C was 6.4-fold greater than that at 15°C. Thus these observations argue against temperature-dependent increases in strain, stress, and/or work done on the muscle accounting for the temperature-dependent strength losses.

Because the isolated muscle depends on oxygen delivery via diffusion from its bathing medium, one must consider the possibility that a hypoxic muscle core at the warmer temperatures caused or potentiated the temperature-dependent strength loss. Our previous data argue against this possibility. First, using Krogh cylinder modeling and our previous measures of basal oxygen consumption in the mouse EDL muscle (33), we conservatively estimate the minimum PO2 in the isolated EDL muscle at 37°C to be 230 Torr (i.e., well above that in arterial blood). Second, we have reported the ATP content in 70% larger muscles injured using a similar protocol at 37°C to be the same as that in muscles taken directly from the animal (29). Third, the long-term stability of our isolated mouse EDL muscle preparation at 37°C argues against a hypoxic muscle (31). Finally, if the isometric force loss at higher temperatures had resulted from a compromised metabolism, then the force loss for control muscles that did isometric contractions should have exceeded that observed for muscles that did eccentric contractions. It is well accepted that isometric contractions require a greater energy expenditure than do eccentric contractions (14).

We attribute the temperature-dependent strength loss after eccentric contractions to a progressively greater failure of one or more steps in the E-C coupling pathway as temperature increases from 15 to 37°C. This conclusion is based on two observations. First, the caffeine-induced force measured after the Ecc protocol was unaffected by temperature, despite the strong temperature effect on the isometric force loss. Second, there were no differences in caffeine-induced force between the Ecc and Iso protocols at any temperature, and the Iso protocols resulted in minimal reductions in isometric force. Thus there was no evidence that a disruption and/or degradation of force-bearing structures within the muscle could account for any strength loss at any temperature. If a greater disruption and/or degradation of force-bearing structures had occurred at higher temperatures, then the caffeine-induced force should have been reduced in proportion to the extent of disruption and/or degradation (32). However, a reduced caffeine-induced force was not observed in any condition. The caffeine-induced force data also indicate that the SR in injured muscles could release Ca2+ normally if provided with an appropriate stimulus. These results point to the failure of an E-C coupling step before the release of Ca2+ by the SR.

In contrast to previous studies that provided direct evidence for E-C coupling failure after eccentric contractions (1, 2, 16), we relied in this study on the caffeine-induced force data as our sole, indirect indicator of E-C coupling failure, and there may be valid concerns about having done so. The peak caffeine-induced force was approximately one-fourth of Po, and thus the submaximal nature of the caffeine contraction may have led to erroneous conclusions about E-C coupling failure during maximal tetanic stimulation. However, we do not think this is the case for the following reason. Isometric twitch force is also submaximal and was less than the caffeine-elicited force at all temperatures. However, the reductions in isometric twitch force after the Ecc and Iso protocols showed a temperature dependence nearly identical to that of the Po reductions. If the lack of difference between the two protocols in caffeine-elicited force had been due to the contraction's submaximal nature, then we should not have observed a large difference between the two protocols in the twitch force reduction.

If E-C coupling failure after eccentric contractions was in fact exacerbated at higher temperatures, the explanation for the temperature dependence is not clear. Because relatively small increases in [Ca2+]i have been found to impair SR Ca2+ release, a possible explanation is that a progressively greater loss of intracellular Ca2+ homeostasis occurs as temperature rises. Lamb and co-workers (19) reported that exposure of mechanically skinned fibers to 2.5 µM Ca2+ for 60 s caused a 46% reduction in depolarization-induced force. Also, using voltage-clamped fibers, Delbono (9) reported SR Ca2+ release to be reduced by 50% when the [Ca2+]i immediately before a contraction was raised to 0.25 µM. From these data and the observations that fiber [Ca2+]i can be elevated after injurious eccentric contractions (1, 2, 16, 21), we hypothesized that there was a progressive loss of intracellular Ca2+ homeostasis as temperature increased and that this exacerbated the E-C coupling failure.

In the present study, as temperature increased, there was, however, little evidence for an increased loss of intracellular Ca2+ homeostasis after the Ecc protocol. First, as temperature rose over the 15 to 25°C range, there was no progressive loss of cell membrane integrity or a greater influx of extracellular Ca2+ during the Ecc protocol. LDH release rates were identical for the Ecc protocols done at 15, 20, and 25°C, as were the muscle 45Ca2+ accumulation rates (Fig. 7). Second, the rate of SR Ca2+ uptake went up 2.6-fold over the 15 to 25°C range (Fig. 8) and thus would not be expected to contribute to an impaired intracellular Ca2+ homeostasis. Finally, the [Ca2+]i increase that occurred over the Ecc protocol at 25°C was nearly identical to that observed at 15°C (Fig. 9), thus arguing against a greater loss of intracellular Ca2+ homeostasis as temperature increased from 15 to 25°C. However, we cannot rule out that a given elevation in [Ca2+]i at 25°C might have a greater effect than would the same elevation at 15°C.

On the other hand, as temperature increased from 25 to 37°C, there was some but not strong evidence for an increased loss of intracellular Ca2+ homeostasis following the Ecc protocol. As temperature increased from 25 to 37°C, there was a progressive loss of cell membrane integrity as reflected by the 33-fold increase in LDH release (Fig. 7). This loss of cell membrane integrity presumably contributed to the 120% increase in 45Ca2+ accumulation observed over that range. Unfortunately, our attempt at measuring fiber [Ca2+]i during the Ecc protocol at 37°C was not successful, most likely because of efflux of the Ca2+-sensitive fluorescent dyes through the damaged cell membrane. Thus we have no direct evidence that intracellular Ca2+ homeostasis worsened over the 25-37°C range. The tripling of the SR Ca2+ uptake rate over the 25-37°C range certainly would not contribute to a worsening of intracellular Ca2+ homeostasis (Fig. 8).

The present study does provide some insight into the mechanism of the strength loss after eccentric contractions. In experiment 2, we made use of rapid changes in muscle temperature during the Ecc protocol in an attempt to tease out the nature of the mechanism of the strength loss. When the protocols were matched on peak eccentric force and work done on the muscle (i.e., the 4 rightmost bars in Fig. 6), it was found that only the temperature at which the eccentric contractions were done affected the magnitude of the strength loss. The muscle's temperature in between contractions did not significantly affect the strength loss. These data suggest that the eccentric contraction-induced strength loss is due to a failing component within the muscle whose integrity is temperature dependent. The integrity of this failing component must decline as temperature rises over the 15 to 37°C range.

We consider it unlikely that a secondary event or sequence of events involving temperature-modulated enzymatic degradation can explain most of the strength loss. If it did, then reducing the temperature in between eccentric contractions from 37 to 15°C should have greatly blunted the strength loss. It did not, nor did raising the temperature from 15 to 37°C in between contractions exacerbate the strength loss. Enzyme-catalyzed reactions typically have Q10 coefficients in the 2-3 range, and thus a 37 to 15°C transition should have reduced the rate of a "degradative" reaction during the rest periods by 80-90%. Admittedly, muscle temperature did not change instantaneously in experiment 2 on changing from one recirculating bath to another (see METHODS). It is possible that during the brief period of time at higher muscle temperatures during the rest periods, an enzyme-dependent degradative pathway could have completed its function.

When maximal eccentric contractions were done at 37°C, reducing the temperature in between contractions to 15°C did confer some protection against strength loss (i.e., the 2 leftmost bars on Fig. 6). Under these conditions, the strength loss was 25% less than that occurring when muscles were at 37°C continuously. Thus these data argue for an enzyme-dependent degradative pathway contributing to the strength loss, but perhaps only after cellular homeostasis has been disrupted to a greater extent. It is unknown what enzyme-dependent degradative pathway might be involved. A calpain-mediated degradative process seems unlikely because application of inhibitors had no effect on the strength loss. It is possible that the enzyme-dependent pathway could be one resulting in production of reactive oxygen species and subsequently causing lipid peroxidation of cell membranes. This hypothesis would be compatible with our observations in experiment 3 of a temperature-dependent loss of cell membrane integrity.

The identity of the temperature-dependent failing component that results in the eccentric contraction-induced strength loss is unknown. There are several indications that it may be one that undergoes a phase transition in the 25-35°C range. First, there was a leveling off of the Po loss between 25 and 30°C, but the Po loss increased rapidly as temperature rose above 30°C. Second, both LDH release and muscle 45Ca accumulation were temperature independent at <= 25°C, but at higher temperatures, the two measures showed an exponential relation to temperature. This latter observation suggests that the plasmalemma and/or t-tubules may undergo a phase transition at 25-30°C that has a deleterious effect. However, it would be pure speculation to suggest that such a phase transition is causally related to the strength loss. Many membranes undergo phase transitions in the 20-40°C range with the exact transition temperature depending on the phospholipid composition of the membrane (17). We did attempt to identify a membrane in mouse EDL muscle undergoing a phase transition in this temperature range. Using differential scanning calorimetry, we ran analyses of muscle homogenates and SR membrane fractions, but the results were negative because of what we believe was a lack of instrument sensitivity (data not shown).

In conclusion, the data from the present study indicate a strong effect of temperature on the isometric strength loss induced by eccentric contractions in the mouse EDL muscle. We attribute the temperature-dependent strength loss to an exacerbation of E-C coupling failure as temperature increases over the 15 to 37°C range. It is uncertain whether the temperature-dependent strength loss results from a progressive loss of intracellular Ca2+ homeostasis. The data suggest that this is most likely not the case as temperature increases over the 15 to 25°C range. However, as muscle temperature rises above 25°C, there is a progressive loss of cell membrane integrity and an increasing influx of extracellular Ca2+. It is not known if [Ca2+]i increases as a result. The cause of the eccentric contraction-induced strength loss appears to be attributable to the failure of a component whose integrity decreases as temperature increases. An enzyme-mediated degradative pathway does not appear to play a major role in the strength loss. Finally, this study's findings may have important implications in the clinic and on the athletic field as our data tend to discount the pervasive idea that "warming up" prior to exercise protects against muscle injury.


    FOOTNOTES

Address for reprint requests and other correspondence: G. L. Warren, Dept. of Physical Therapy, University Plaza, Georgia State Univ., Atlanta, GA 30303-3083 (E-mail: phtglw{at}langate.gsu.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.

First published December 21, 2001;10.1152/ajpregu.00671.2001

Received 9 November 2001; accepted in final form 18 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 282(4):R1122-R1132
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