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J Appl Physiol 85: 58-67, 1998;
8750-7587/98 $5.00
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Vol. 85, Issue 1, 58-67, July 1998

E-C coupling failure in mouse EDL muscle after in vivo eccentric contractions

Christopher P. Ingalls1, Gordon L. Warren1, Jay H. Williams2, Christopher W. Ward2, and R. B. Armstrong1

1 Muscle Biology Laboratory, Texas A&M University, College Station, Texas 77843-4243; and 2 Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic Institute, Blacksburg, Virginia 24061-0430

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The objectives of this research were to determine the contribution of excitation-contraction (E-C) coupling failure to the decrement in maximal isometric tetanic force (Po) in mouse extensor digitorum longus (EDL) muscles after eccentric contractions and to elucidate possible mechanisms. The left anterior crural muscles of female ICR mice (n = 164) were injured in vivo with 150 eccentric contractions. Po, caffeine-, 4-chloro-m-cresol-, and K+-induced contracture forces, sarcoplasmic reticulum (SR) Ca2+ release and uptake rates, and intracellular Ca2+ concentration ([Ca2+]i) were then measured in vitro in injured and contralateral control EDL muscles at various times after injury up to 14 days. On the basis of the disproportional reduction in Po (~51%) compared with caffeine-induced force (~11-21%), we estimate that E-C coupling failure can explain 57-75% of the Po decrement from 0 to 5 days postinjury. Comparable reductions in Po and K+-induced force (51%), and minor reductions (0-6%) in the maximal SR Ca2+ release rate, suggest that the E-C coupling defect site is located at the t tubule-SR interface immediately after injury. Confocal laser scanning microscopy indicated that resting [Ca2+]i was elevated and peak tetanic [Ca2+]i was reduced, whereas peak 4-chloro-m-cresol-induced [Ca2+]i was unchanged immediately after injury. By 3 days postinjury, 4-chloro-m-cresol-induced [Ca2+]i became depressed, probably because of decreased SR Ca2+ release and uptake rates (17-31%). These data indicate that the decrease in Po during the first several days after injury primarily stems from a failure in the E-C coupling process.

excitation-contraction; extensor digitorum longus; fluo 3; fura red; calcium-selective minielectrode

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

IT HAS GENERALLY BEEN ASSUMED that disruptions in force-generating and/or -transmitting structures are responsible for the immediate and prolonged reduction in maximal isometric tetanic force (Po) after eccentric contractions. However, several observations do not support this assumption. First, it is difficult to ascribe significant decreases in Po (~50%) to disruption in force-bearing structures when histological measurements indicate that only a small fraction (<10%) of the muscle fibers appears abnormal immediately after induction of the injury (15, 24). Second, there is no temporal relationship between reduced muscle function and the progression of the histopathology after eccentric contraction-induced injury (24, 25). Finally, and more importantly, we (32, 34) and others (3) have demonstrated that the reduction in Po immediately after a series of in vitro eccentric contractions stems primarily from a failure in the excitation-contraction (E-C) coupling process. E-C coupling is operationally defined as the sequence of events that starts with the passage of the action potential along the plasmalemma and ends with the release of Ca2+ from the sarcoplasmic reticulum (SR).

Although E-C coupling failure can explain most of the decrement in Po immediately after eccentric contraction-induced injury (3, 32, 34), the contribution of disrupted E-C coupling to the prolonged decrease in Po is not known. In addition, it is possible that the E-C coupling impairment observed in the previous studies is unique to in vitro injury models. Therefore, the first objective of this study was to determine whether E-C coupling failure occurs in muscles injured in vivo, and, if so, to estimate to what extent the failure contributes to the Po decrement during the 14 days after initiation of the injury.

Having confirmed that E-C coupling failure occurs after eccentric contraction-induced injury by using an in vivo model, we then attempted to determine the site and mechanism of the defect. The second objective of this study was to examine the functional integrity of the t tubule and SR Ca2+ release and uptake elements after eccentric contraction-induced muscle injury. Because increases in muscle intracellular Ca2+ concentration ([Ca2+]i) can reduce SR Ca2+ release (5, 7), the third objective was to measure [Ca2+]i at rest and during tetanic and 4-chloro-m-cresol-induced contractions after in vivo injury.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Experimental design. Two studies were designed to investigate the presence and relative contribution of E-C coupling failure to the Po decrement after eccentric contraction-induced injury. 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 (27). In the first study, relative changes in caffeine-induced contracture force and Po were examined before (Pre) injury and at 0, 1, 3, 5, and 14 days postinjury in injured and contralateral mouse extensor digitorum longus (EDL) muscles (n = 36). The above-mentioned time points were selected on the basis of a previous study using the same injury model (24). In the second study, we used a second drug, 4-chloro-m-cresol, to determine whether the E-C coupling failure findings were unique to the action of caffeine. 4-Chloro-m-cresol is thought to be a novel and specific activator of the SR Ca2+ release channel and has a 10-fold greater sensitivity than does caffeine (14). Relative changes in 4-chloro-m-cresol-induced contracture force and Po were examined at 0 and 3 days postinjury in injured and contralateral control EDL muscles (n = 16). Any observed reductions in caffeine- or 4-chloro-m-cresol-elicited forces would be interpreted as reflecting alterations in SR function, myofibrillar Ca2+ sensitivity, or force-generating and/or -transmitting elements. Greater percent reductions in Po compared with drug-induced forces after injury induction would reflect a failure in the E-C coupling process.

Three studies were designed to explore possible sites of E-C coupling failure. One study evaluated the functional integrity of the plasmalemmal and t-tubular membranes, whereas the other two studies examined SR function. Because depolarization of the t tubule activates voltage sensors (8) and promotes Ca2+ release from the SR (27), eccentric contractions may reduce Po by disrupting the plasmalemma and/or t tubules, hence reducing the extent of muscle excitation. The integrity of the plasmalemma and t tubule was examined by comparing percent reductions in Po with K+-induced contracture force immediately after the injury in injured and contralateral control muscles (n = 9). Elevated concentrations of extracellular K+ are known to produce muscle contracture by depolarizing t-tubular membranes adjacent to the voltage sensors (8). Smaller percent reductions in K+-elicited force compared with Po would suggest that disruptions in the plasmalemma or t-tubular system contribute to the Po decrement. However, proportional percent reductions in Po and K+-induced force would indicate a failure in the E-C coupling process at or below the level of the voltage sensor.

Consistent with observations of small decreases in caffeine-elicited forces (34), impaired SR function could also contribute to a small part of the Po reduction after eccentric contractions. To evaluate the integrity of the SR, Ca2+ release and uptake rates in injured and contralateral control EDL muscle homogenates were measured by using two different techniques. First, by using fura 2 and spectrofluorometry, the ability of the SR to sequester and release Ca2+ was measured Pre and at 0, 1, 3, 5, and 14 days postinjury (n = 41). Second, a Ca2+-selective minielectrode was used to measure SR Ca2+ uptake and release in crude homogenates at 0 and 3 days postinjury (n = 21). This latter technique was determined to be a more sensitive means of determining SR Ca2+ release and uptake rates.

Two studies were designed to determine [Ca2+]i at rest and during tetanic and 4-chloro-m-cresol-induced contractions after eccentric contraction-induced injury. First, the resting [Ca2+]i was compared between injured and contralateral control EDL muscles at 0, 3, and 6 h postinjury (n = 16). Because we have shown that phagocytic cell infiltration begins ~24 h after injury induction (24), it is likely that this inflammatory response will disrupt muscle membranes and elevate resting [Ca2+]i. Therefore, the experimental times were chosen in an effort to relate changes in [Ca2+]i to the contractions themselves and not to phagocytic cell infiltration. Second, E-C coupling impairment was evaluated by comparing peak [Ca2+]i during tetanic contractions with 4-chloro-m-cresol-induced contractures in EDL muscles at 0 and 3 days postinjury (n = 24). These times were selected on the basis of the SR Ca2+ release and uptake studies, which showed that SR function was relatively unaffected immediately after injury and was significantly depressed 3 days after injury initiation. Therefore, if the eccentric contraction-induced E-C coupling failure stems solely from a defect above the level of the SR Ca2+ release channel, then peak tetanic [Ca2+]i should be reduced whereas peak 4-chloro-m-cresol-induced [Ca2+]i should be unaffected. However, if the E-C coupling defect includes the SR Ca2+ release channel, then both the tetanic and 4-chloro-m-cresol-induced Ca2+ transients should be depressed.

Animals. Female ICR mice (n = 164), 8-12 wk old, were used in the study. Their mean body mass was 31.3 ± 2.7 (SD) g. The mice were housed in groups of 5-6 animals per cage, supplied with food and water ad libitum, and maintained in a room at 20-22°C with a 12-h photoperiod. For the in vivo injury induction, mice were anesthetized with 0.33 mg/kg fentanyl citrate, 16.7 mg/kg droperidol, and 5.0 mg/kg diazepam (16, 17). Mice were euthanized with an overdose of pentobarbital sodium. All animal care and use procedures were approved by the institutional animal care and use committee and met the guidelines set by the American Physiological Society.

In vivo muscle injury induction. The anterior crural muscles from the left hindlimbs of mice were injured by using 150 eccentric contractions as previously described (16, 24).

In vitro caffeine-, 4-chloro-m-cresol-, and K+-induced contracture experiments. After the in vivo injury protocol, EDL muscles were dissected free and studied at 37°C by using the in vitro preparation, as previously described (16, 24, 32). Isometric twitch and tetanic contractions were initiated at 7 and 8 min into the incubation. After the measurement of Po, 200 mM K+, 50 mM caffeine, or 10 mM 4-chloro-m-cresol were used to elicit contractures. Preliminary experiments indicated that concentrations of 4-chloro-m-cresol greater than 7.5 mM elicited maximal contracture force. 4-Chloro-m-cresol was prepared as a 1.0 M stock dissolved in ethanol. Caffeine (50 mM) was dissolved in the Krebs-Ringer solution. For the K+ contractures, the [K+] · [Cl-] product was kept constant in the change from normal Krebs-Ringer solution (34) to one containing 200 mM K+. The high-K+ solution substitutes 0 mM NaCl, 1.295 mM KCl, and 98.55 mM K2SO4 for 119 mM NaCl, 5 mM KCl, and 0 mM K2SO4 in the normal solution. We have found the 50 mosM increase in tonicity does not induce any force transients or have any short-term effects on force production.

SR Ca2+ release and uptake rates. SR Ca2+ release and uptake rates were measured from a muscle homogenate fraction by using a modification of the fluorometric assay described by Kandarian et al. (19). EDL muscles were homogenized in 20 volumes of an isolation buffer (20 mM HEPES, 250 mM sucrose, 0.2% sodium azide, and 0.2 mM phenylmethylsulfonyl fluoride, pH 7.5). The homogenate was centrifuged at 1,600 g for 10 min; the supernatant was frozen in liquid N2 and stored at -80°C. Protein content of the supernatant was determined by the Bradford method, as modified by Bio-Rad. Ca2+ uptake and release were measured by using a Jasco CAF-110 fluorometer, and fura 2 served as the extravesicular free Ca2+ concentration ([Ca2+]) indicator. The ratio of the fluorescence (i.e., >500 nm) due to excitation at 340 and 380 nm was sampled by computer and converted into free [Ca2+], as described by Grynkiewicz et al. (11). The incubation buffer consisted of (in mM) 100 KCl, 7.5 pyrophosphate, 20 HEPES, and 1.0 MgCl2, as well as 1 µM fura 2 and 2.5 µM free Ca2+ (pH 7.0 and 37°C). Two hundred micrograms of homogenate protein were suspended in 1 ml of buffer. Ca2+ uptake was initiated by the addition of 10 µl of 100 mM Na2ATP. Three minutes after ATP was added, Ca2+ release was initiated by the addition of 10 µl of 500 µM AgNO3. Ag+ is thought to promote Ca2+ release by binding to thiols on the SR Ca2+ release channel (1). The rates of SR Ca2+ uptake and release were determined as the steepest negative (uptake) and positive (release) slopes of the extravesicular free [Ca2+] vs. time curve and were normalized to the supernatant protein content.

Using rat gastrocnemius muscle homogenates, we have verified that this technique can be used to measure Ca2+ release and uptake by the SR. We found that the peak rates of uptake and release were 1.35 ± 0.15 and 1.89 ± 0.08 µmol · mg protein-1 · min-1, respectively, with coefficients of variation at <= 7% (n = 8). Ca2+ uptake and release rates in homogenates correlated very well with those recorded in SR vesicles obtained from the same muscle (r = 0.94 and 0.96, respectively). In addition, we found that the uptake rate was not affected by the inclusion of 0.2% sodium azide, which disrupts mitochondrial Ca2+ transport, in the incubation buffer (1.40 ± 0.11 µmol · mg protein-1 · min-1, n = 6). In addition, 20 µM cyclopiazonic acid, which inhibits the Ca2+-ATPase, completely blocks Ca2+ uptake. Qualitatively similar results were obtained by using homogenates from mouse tibialis anterior and EDL muscles.

SR Ca2+ release and uptake rates were also measured from whole EDL muscle homogenates by using a Ca2+-selective minielectrode. Ca2+-selective mini- and reference electrodes were made as described by Baudet et al. (4) and Sigel and Affolter (30). EDL muscles were homogenized and processed as described previously, but without centrifugation. Two aliquots (80 µl) were removed and frozen in liquid N2 and then stored at -80°C. Six Ca2+-EGTA standards with 1 mM free Mg2+ (Molecular Probes) ranging from 250 nM to 10.4 µM of free [Ca2+] were used for calibration of the minielectrode. Rates of SR Ca2+ release and uptake were determined as described previously, except that the assay medium also included 10 mM NaN3 and Ca2+ release was initiated by adding 10 µl of 500 µM AgNO3 immediately after [Ca2+] had decreased to 1.0 µM (Fig. 1).


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  		Fig. 1.   Representative record from which maximal rates of sarcoplasmic reticulum (SR) Ca2+ uptake and release were determined by using Ca2+-selective minielectrode. Data shown are from a control extensor digitorum longus (EDL) muscle. [Ca2+], Ca2+ concentration.

Using mouse tibialis anterior muscles, we have verified that this technique is suitable for measuring Ca2+ release and uptake rates in crude homogenates. Cyclopiazonic acid (40 µM) completely blocked Ca2+ uptake in crude homogenates. Although Ag+-induced Ca2+ release was only reduced 55% by 100 µM ruthenium red, it was completely blocked by 4 mM dithiothreitol. There were no effects of freezing the homogenates on the rates of uptake and release. This method proved to be a more reliable measurement than the fluorometric method. The mean deviation between duplicates for release and uptake were 2.7 and 4.4%, respectively, for the electrode method vs. 12.7 and 14.3%, respectively, for the fluorometric method. The SR Ca2+ release and uptake rates in control muscles agreed very well between the two techniques. By using a SR fraction protein content-to-wet weight ratio of 0.0846 attained in the fluorometric study, the SR Ca2+ uptake and release rates measured by using the minielectrode were estimated to be 13% higher (2.24 vs. 1.99 nmol · mg protein-1 · min-1) and 25% lower (4.33 vs. 5.74 nmol · mg protein-1 · min-1), respectively, than with the fluorometric technique. Both the fluorometric and minielectrode techniques offer a simple and reliable means of measuring SR Ca2+ release and uptake rates in muscle homogenates.

Measurement of [Ca2+]i at rest and during tetanic- and 4-chloro-m-cresol-induced transients by using confocal laser scanning microscopy (CLSM). Measurement of [Ca2+]i was based on the ratiometric confocal approach using the visible wavelength Ca2+-sensitive dyes fluo 3 and fura red (23). The acetoxymethyl ester forms of fluo 3 and fura red (Molecular Probes) were prepared as 3 mM stock solutions in DMSO and stored at -20°C. EDL muscles were incubated with 20 µM fluo 3 and 20 µM fura red in an oxygenated modified Krebs-Ringer solution (no amino acids or protein) containing 3 mg/ml of Pluronic F127 for 30 min (resting [Ca2+]i study) or 60 min ([Ca2+]i transients study) at room temperature. The superfused muscles were observed by using a NORAN Odyssey XL CLSM on a Zeiss Axioskop upright microscope and a 50-mW argon-krypton laser. The dyes were excited by using the 488-nm line, and the fluorescence in the two channels was collected by using 515- to 545-nm (i.e., primarily that of fluo 3) and 635- to 685-nm (i.e., primarily that of fura red) band-pass filters. A Zeiss Achroplan ×10 water-immersion objective (numerical aperture of 0.30) was used to acquire the optical sections. EDL muscles were mounted in a superfused preparation (Krebs-Ringer solution) for acquisition of longitudinal optical sections (35). The distal end of muscle was attached to a fixed post, whereas the proximal end was attached to a micrometer that allowed for adjustment of resting muscle length. Sarcomere length was set to ~2.6 µm for the resting [Ca2+]i and 3.1 µm for the [Ca2+]i transient studies by using an eyepiece reticle and a Zeiss Achroplan ×40 water-immersion objective (numerical aperture of 0.75).

For the resting [Ca2+]i study, the entire ventral side of the EDL muscle was systematically imaged by using a rectangular-shaped grid system that approximated the size of a mouse EDL muscle. Because a given optical section within the grid system contained 4-12 muscle fibers, fluorescence originated not only from the cytosol of the fibers but also from other cell types (e.g., fibroblast, endothelial cells, neurons, and so on). Considering that there is spatial heterogeneity of the injury between and within muscle fibers (33), this approach was used in lieu of manually tracing regions of interest within a given fiber because it is more objective and does not exclude any part of the muscle fiber. After background subtraction, the fluo 3 channel was divided by the fura red channel to obtain an eight-bit ratio image of the resting [Ca2+]i. The number of voxels vs. dye ratio was generated and smoothed by using a 51-point Savitzky-Golay algorithm for each muscle. A [Ca2+]i calibration was applied to the eight-bit gray-scale voxel intensity (see below). The number of voxels displaying a given [Ca2+]i was converted into the volume of EDL muscle by multiplying the pixel area by the z-axis full width one-half maximum (i.e., 32.1 µm) for the configured setup. Typical CLSM fluo 3, fura red, and ratio images of a normal EDL muscle are shown in Fig. 2.


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  		Fig. 2.   Typical confocal laser scanning microscopic images showing a control EDL muscle. A: fluorescence from fluo 3 channel. B: fluorescence from fura red channel. C: image of fluo 3-to-fura red ratio.

[Ca2+]i transients were measured from a predefined anatomic location on the ventral side of the EDL muscle near the proximal end. To minimize motion artifact, the EDL muscle was sandwiched between a Plexiglas platform and a dipping cone attached to the ×10 water-immersion objective. After 20 min of perfusion with Krebs-Ringer solution (20°C), a tetanic contraction (350-ms train with 0.2-ms pulses at 100 Hz) was performed, and the fluorescence from the fluo 3 and fura red channels was sampled at a rate of 30 Hz with two-frame averaging. Ten minutes after the tetanic contraction, a subfusion tetanus (20 Hz) was performed (data not shown). Ten minutes after the second tetanus, the EDL was exposed to 10 mM 4-chloro-m-cresol. 4-Chloro-m-cresol was used to activate the SR Ca2+ release channel because, unlike caffeine, it promoted Ca2+ release at 20°C. The change in fluorescence of the fluo 3 and fura red channels was recorded over the next 5 min. [Ca2+]i during Po and 4-chloro-m-cresol contractions was assessed by measuring changes in [Ca2+]i in the entire image field and in individual muscle fibers. Typical CLSM images acquired from the fluo 3 channel before and during tetanic stimulation and during 4-chloro-m-cresol incubation are shown in Fig. 3.


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  		Fig. 3.   Typical confocal laser scanning microscopic images showing fluorescence in fluo 3 channel just before tetanic stimulation (A), during tetanic contraction (B), and during subsequent 4-chloro-m-cresol-induced activation (C).

In vitro [Ca2+]i calibration methods were used to estimate [Ca2+]i. In the resting [Ca2+]i study, intracellular fluo 3 and fura red concentrations were determined as described by Kao (20), except that we found 10 µM beta -escin to be preferable to digitonin. The intracellular concentrations of fluo 3 and fura red were determined to be 1.1 and 1.0 µM, respectively. Fluo 3 and fura red salts at these concentrations were then added to the [Ca2+] standards, which were prepared on the basis of the methods of McGuigan et al. (26). These standards contained 50 mg/ml of aldolase to better approximate the in vivo dissociation constant of the dyes (12, 21). The standard solutions were loaded into capillary tubes (50 µm ID, 80 µm OD, Vitro Dynamics) and imaged by using the same settings and conditions as during the EDL muscle measurements. Background fluorescence was subtracted from the fluo 3 and fura red channels, and fluo 3-to-fura red ratio (R) images of the capillary were acquired. Dye ratio values were converted to [Ca2+]i by using the Hill equation {R = Rmax [Ca2+]iN/(Ca50N + [Ca2+]iN)} to fit the dye ratio-[Ca2+] relationship (r2 = 0.96-0.98), where N is a constant and Ca50 is half-maximal Ca2+ activity. In the [Ca2+]i transient study, a simpler in vitro calibration procedure was used, in part, because the 4-chloro-m-cresol treatment negated the possibility of conducting calibrations on each muscle. Intracellular fluo 3 and fura red concentrations were determined to be 2.9 and 3.0 µM, respectively, in a separate experiment; the dye salts were then added to Molecular Probes [Ca2+]i standards (C-3721) and imaged as described previously.

Statistical analyses. Analyses of the in vivo injury induction, in vitro muscle mechanics, and SR Ca2+ release and uptake experiments were conducted by using time (e.g., Pre, 0, 1, 3, 5, and 14 days)-by-condition (e.g., injured or control) ANOVA, with repeated measures on the condition factor. Because the analysis of fluo 3-to-fura red ratios and [Ca2+] values resulted in the same statistical conclusions in both the resting and tetanic [Ca2+] studies, only the estimated [Ca2+] data are presented. Changes in the distribution of [Ca2+]i between injured and contralateral control muscles were determined by chi 2 analyses. Changes in the [Ca2+]i during tetanic transients were analyzed by using condition (injured and control)-by-time (0.06, 0.13, 0.20,...0.47 s) ANOVAs with repeated measures on the time factor; t-tests were used for the 4-chloro-m-cresol-induced [Ca2+]i transients. If assumptions of normality or equal variance were violated, one-way Kruskal-Wallis ANOVA or Mann-Whitney rank sum test was employed. When significant differences were detected, Student-Newman-Keuls and Dunn's post hoc tests were applied for parametric and nonparametric tests, respectively. All statistical testing was performed by using Jandel SigmaStat 1.0 and 2.0 (San Rafael, CA). An alpha  level of 0.05 was used for all statistical tests. Values presented in RESULTS are means ± SE.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In vivo injury protocol. The peak torque during the preinjury isometric tetanic contraction averaged 3.15 ± 0.04 N · mm. The peak torque during the first eccentric contraction equaled 6.30 ± 0.065 N · mm, or 202% of the torque during the preinjury isometric contraction. The peak torque during the postinjury isometric tetanic contraction equaled 1.48 ± 0.03 N · mm, 53% lower than that of the preinjury isometric contraction.

In vitro EDL muscle Po. The reductions in Po after injury induction have been combined at the appropriate time points from the caffeine, 4-chloro-m-cresol, and K+ studies and are presented in Fig. 4. The mean Po for all the control muscles was 419 ± 6 mN. There was a 50.5 ± 2.3% decrement in Po immediately after the eccentric contraction protocol. The Po decrement remained unchanged for 5 days, with the mean decrease over that time period equaling 50.9 ± 1.5%. The absolute Po for the injured muscle was still significantly different from that of the contralateral control at 14 days postinjury (-12.6 ± 2.9%). In summary, the eccentric contraction protocol resulted in an immediate and prolonged reduction in Po. Recovery of Po started sometime after 5 days and was still incomplete at 14 days.


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  		Fig. 4.   Percent reductions in maximal isometric tetanic force (Po) and caffeine-induced force in injured EDL muscles compared with contralateral control muscles after eccentric contraction-induced injury. Inset: ratio of caffeine-induced force to Po after injury induction. Solid bars, contralateral control muscles; open bars, injured muscles; a-d, means are significantly different (P <=  0.05). Pre, before injury.

Time course of E-C coupling failure after eccentric contraction-induced muscle injury. The purpose of the caffeine- and 4-chloro-m-cresol-induced force studies was to determine the presence and the contribution of the E-C coupling failure to the Po decrement in the EDL muscle over 14 days after injury induction. Because caffeine and 4-chloro-m-cresol act at the level of the SR to cause Ca2+ release, comparing Po and drug-induced forces between injured and contralateral control muscles provides insight into the mechanisms responsible for the Po decrement. The mean caffeine-induced force for all the control muscles was 108 ± 1 mN. This relatively low contracture force compared with our previous work on soleus muscle (~27 vs. ~74% of Po) presumably stems primarily from the lesser sensitivity of fast-twitch muscle to caffeine (18). There was a 14.8 ± 1.6% decrement in caffeine-induced force immediately after the eccentric contraction protocol (Fig. 4). The caffeine-induced force decrement remained unchanged for 5 days, with the mean decrease over that time period equaling 15.5 ± 1.7%. The percent reductions in Po were ~3.3-fold greater than were the percent decreases in caffeine-induced force from 0 to 5 days (Fig. 4). Thus we estimate that the E-C coupling failure can explain at least 75% of the Po decrement immediately after the eccentric contraction protocol. The contribution of E-C coupling failure to the Po decrement appears to diminish over time (Fig. 4, inset).

The mean 4-chloro-m-cresol-induced force for the contralateral control muscles was 57 ± 1 mN. 4-Chloro-m-cresol-induced force had declined by 12.0 ± 1.8 and 15.0 ± 2.0% at 0 and 3 days after injury, respectively. In a comparison of the percent decrements in Po and 4-chloro-m-cresol-induced forces, E-C coupling failure was estimated to explain at least 75 and 71% of the Po decrement at 0 and 3 days after injury, respectively. In summary, results from the caffeine and 4-chloro-m-cresol studies indicate that the E-C coupling process is significantly impaired from immediately after to at least 5 days after injury induction.

Possible sites of E-C coupling impairment. The purpose of the K+-induced force study was to determine whether the site of E-C coupling disruption lies above, at, or below the level of the voltage sensor. The mean K+-induced force for the contralateral control muscles was 22 ± 3 mN. K+-induced contracture force was reduced by 51.2 ± 5.8% immediately after eccentric contraction-induced injury, which was not different (P = 0.10) from the percent reduction in Po (i.e., 46.2 ± 2.6%) for this group of muscles. The proportional percent reductions in Po and K+-induced force suggest that the E-C coupling failure occurs at or below the level of the voltage sensor.

The objective of the two SR Ca2+ release and uptake studies was to determine whether the ability of the SR to sequester and release Ca2+ was impaired during the 2 wk after injury. The effect of eccentric contraction-induced injury on SR Ca2+ release by using the fluorometric method is shown in Fig. 5. No significant difference between injured and contralateral control EDL muscles was detected for the Pre, 0-, 1-, and 14-day groups. However, eccentric contractions resulted in 19.5 and 23.6% reductions in SR Ca2+ release rates at 3 and 5 days postinjury, respectively. With the use of the Ca2+-selective minielectrode, normalized SR Ca2+ release rates were also significantly reduced by 19.5% at 3 days postinjury (Table 1). However, this method, which provided greater sensitivity than did the fluorometric technique, also revealed a significant 6.1% reduction in SR Ca2+ release rate immediately after the injury.


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  		Fig. 5.   Fluorometrically determined maximal rates of SR Ca2+ release and uptake in EDL muscles after eccentric contraction-induced injury. Dashed and dotted lines, mean (±SE) SR Ca2+ release and uptake rates for contralateral control muscles, respectively. * Significantly different from respective contralateral control value (P <=  0.05).

                              
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  		Table 1.   Normalized and total SR Ca2+ release and uptake rates measured with Ca2+-selective minielectrode

The effect of injury on SR Ca2+ uptake by using the fluorometric method is also shown in Fig. 5. No significant differences between injured and contralateral control EDL muscles were found for the Pre, 0-, 1-, and 14-day groups. Eccentric contractions resulted in 31.0 and 29.4% decreases in rates of Ca2+ uptake at 3 and 5 days postinjury, respectively. With the use of the Ca2+-selective minielectrode, normalized SR Ca2+ uptake rates were significantly reduced at both 0 and 3 days postinjury (11.6 and 16.8%, respectively; Table 1). In summary, modest reductions in rates of SR Ca2+ release and uptake occur immediately after the injury, and the magnitude of the reductions increases over the next 5 days; SR function appears normal by 14 days postinjury.

Resting [Ca2+]i distribution. The purpose of this study was to determine whether eccentric contractions result in elevated [Ca2+]i during the first 6 h after injury. Changes in the distribution of resting [Ca2+]i at 0, 3, and 6 h after injury induction are shown in Fig. 6. Although the majority of the Ca2+ in the distribution of resting [Ca2+]i comes from the cytosol of muscle fibers, the wide distribution of [Ca2+]i is explained by the simultaneous imaging of multiple muscle fibers and cell types. There were significant (P < 0.0001) shifts toward higher [Ca2+]i in the injured muscles at all three time points. Compared with the contralateral control muscle, the median [Ca2+]i in the injured EDL muscles was elevated 28% at 0 h (19 vs. 24 nM), 42% at 3 h (17 vs. 24 nM), and 4% at 6 h (19 vs. 20 nM) postinjury. Thus, although the eccentric contraction protocol elevated resting [Ca2+]i during the first 6 h after injury induction, the magnitude of the increase was small compared with the potential ~100-fold increase in [Ca2+]i during contractile activation.


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  		Fig. 6.   Resting distribution of intracellular Ca2+ concentration ([Ca2+]i) in EDL muscles immediately (A), 3 h (B), and 6 h (C) after injury induction. Injured muscles exhibited significant shifts in distribution at all 3 time points (P < 0.0001).

[Ca2+]i transients. The purpose of this study was to evaluate E-C coupling disruption by comparing peak [Ca2+]i during Po and 4-chloro-m-cresol-induced contractions. The statistical relationships were the same between the full-image and single-fiber analyses for the 100-Hz tetanic contraction. Therefore, only the single-fiber image-analysis data are presented. Mean data from single-fiber image analysis of Po and 4-chloro-m-cresol-induced [Ca2+]i transients at 0 and 3 days postinjury are shown in Fig. 7. Despite the somewhat arbitrary nature of the in vitro [Ca2+]i calibration procedure, the tetanic [Ca2+]i values observed in the control muscles in the present study (~450-800 nM) compare favorably with those reported by others (3, 6) studying single fibers of mouse muscle (~300-800 nM). There were no differences (P = 0.08) between control and injured 4-chloro-m-cresol-induced peak [Ca2+]i immediately after injury induction. However, injured muscles displayed reduced peak [Ca2+]i during the tetanic contraction (P < 0.001). In contrast to the 0-day group, injured muscles at 3 days exhibited decreases in both Po (P < 0.001) and 4-chloro-m-cresol-induced (P = 0.004) peak [Ca2+]i. Thus, immediately after injury initiation, the ability of the injured muscle SR to release Ca2+ was normal when stimulated by 4-chloro-m-cresol, but SR Ca2+ release was impaired when activated via electrical stimulation. However, at 3 days postinjury, there was a decreased ability to release Ca2+, as evidenced by a reduced 4-chloro-m-cresol response.


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  		Fig. 7.   Changes in [Ca2+]i during Po and 4-chloro-m-cresol-induced contractions immediately (A) and 3 days (B) after injury induction. Means at 1,200 s are [Ca2+]i transients induced by 4-chloro-m-cresol. [Ca2+]i during relaxation is not shown. Inset: typical record of %changes in fluorescence in fluo 3 (black-lozenge ) and fura red (star ) channels during activation. * Significant difference between injured and control EDL muscles (P < 0.05).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Contribution of E-C coupling failure to the Po decrement after eccentric contraction-induced injury. The results of the caffeine- and 4-chloro-m-cresol-induced force studies indicate that E-C coupling impairment is responsible for the majority of the reduction in Po for at least 5 days after initiation of eccentric contraction-induced injury. On the basis of the disproportionate reduction in Po compared with drug-induced contracture force, we estimate that at least 75% of the reduction in Po is due to E-C coupling impairment immediately after the injury. Moreover, the estimates of the contribution of E-C coupling failure to the decrements in Po showed remarkable agreement between the caffeine (75.3%) and 4-chloro-m-cresol (75.1%) studies immediately after injury. These estimates are conservatively low because they do not take into account the E-C coupling failure attributable to SR dysfunction. These results also agree with previous in vitro soleus muscle (34) and single-fiber (3, 32) analyses, which indicated that E-C coupling impairment played the primary role in force reduction immediately after eccentric contractions. Although the contribution of E-C coupling failure to the decreased Po gradually declined with recovery, it still accounted for at least 57% of the force reduction at 5 days.

The estimate of the E-C coupling failure contribution to the Po decrement after injury is based on three assumptions about the caffeine- and 4-chloro-m-cresol-induced force studies. First, injured and control muscles similar in size and fiber type distribution will be activated to the same extent on exposure to the same caffeine (or 4-chloro-m-cresol) concentration. Second, any drug's effect on myofibrillar Ca2+ sensitivity affects injured and contralateral control muscles similarly. Third, the decrement in Po stems from an injury that is not restricted to one part of the EDL muscle. Because the caffeine and 4-chloro-m-cresol contracture forces represent a fraction of Po (~27 and ~12%, respectively) in normal mouse EDL muscle, it appears that the drugs are unable to maximally activate all fibers simultaneously and may not ever fully activate the more deeply located fibers. Therefore, if the injury was localized to the interior of the muscle, and caffeine and 4-chloro-m-cresol were unable to activate these deeper fibers, then the drug-induced force would overestimate the functional capacity of the injured muscle. However, two observations from the present studies suggest that the injury is not localized to interior myofibers. First, the fact that eccentric contractions caused similar relative decreases in Po and K+-induced forces suggests that the superficial fibers are injured. Second, the finding that peak tetanic [Ca2+]i is reduced in superficial fibers at 0 and 3 days after eccentric contractions also indicates these fibers are injured.

The reduction in peak [Ca2+]i during tetanic contractions immediately and 3 days after induction of injury (Fig. 7) supports the conclusion that an inability to activate the contractile apparatus is the primary mechanism for the decrement in Po after eccentric contractions. The exact contribution of reduced tetanic [Ca2+]i to the eccentric contraction-induced decrements in Po cannot be determined from the present study. However, if one assumes that the tetanic [Ca2+]i is on the steep portion of the force-[Ca2+]i curve, then the 25-36% decrease in [Ca2+]i in the injured muscle could translate into relatively larger Po decrements. Balnave and Allen (3) also demonstrated that eccentric contraction-induced decrements in Po were associated with significant reductions in peak tetanic [Ca2+]i in mouse single muscle fibers. In contrast, Morgan et al. (28) did not observe reduced peak tetanic [Ca2+]i after eccentric contractions in single frog muscle fibers. However, a number of differences could account for the different findings, such as species, temperature, and level of muscle activation. In the study by Morgan et al., it also appears that the degree of muscle damage incurred was minimal on the basis of the small Po decrements (~13%).

E-C coupling failure site. The fact that the E-C coupling mechanism between the t tubule and SR is not completely understood in healthy skeletal muscle compounds the problem of determining the E-C coupling failure mechanism in muscles injured by eccentric contractions. Results from the present and previous studies (13, 34) indicate that plasmalemma and t tubules can transmit action potentials immediately after initiation of the injury. Microelectrode results showed that membrane potential was unaltered immediately after induction of injury in vitro (34). We have also found no change in the root mean square of electromyographic signals from injured mouse tibialis anterior muscles despite a 38% reduction in in vivo torque production (13), suggesting that there was no impairment of the plasmalemma to conduct action potentials. Therefore, these previous results and the present finding of proportional percent reductions in K+-induced contracture force and Po immediately after injury suggest that the E-C coupling defect is located at or below the level of the voltage sensor in the t tubule.

The results from the caffeine- and 4-chloro-m-cresol-induced force studies indicate that most of the Po decrement stems from a failure in the E-C coupling process above the level of the SR Ca2+ release channel from 0 to 5 days after induction of injury. The SR Ca2+ release and uptake and [Ca2+]i transient results immediately after induction of the injury support this interpretation. The magnitude of the reductions in the maximal rate of SR Ca2+ release (0-6%) is too small to account for the decrements in Po (~51%) immediately after injury. Significant attenuation in peak tetanic [Ca2+]i when there is no difference in peak 4-chloro-m-cresol-induced [Ca2+]i (Fig. 7A) also suggests that the defect in the E-C coupling pathway lies above the level of the SR Ca2+ release channel immediately after the injury. Therefore, the locus of the E-C coupling defect presumably is at the t tubule-SR Ca2+ release channel interface because the functional integrity of the plasmalemma/t tubules and the SR and force-bearing elements is relatively intact immediately after eccentric contraction-induced injury.

The deterioration over time of both peak 4-chloro-m-cresol-induced [Ca2+]i and SR Ca2+ release and uptake rates suggests that impaired SR Ca2+ release and uptake may contribute to the reductions in Po at later times (i.e., 3-5 days) after induction of injury. However, the observation that the rate of SR Ca2+ release and uptake decreased during the 5 days after injury (Fig. 5), whereas reductions in Po remained unchanged (Fig. 4), suggests that the progressive impairment of SR function was inconsequential in terms of force production. There were no significant correlations between percent decrements in Po and SR Ca2+ release and uptake rates during 14 days after initiation of the injury (r = 0.15-0.36; P >=  0.49). The reduction in SR function probably occurred in fibers that already had attenuated force production and that were undergoing degeneration.

Possible mechanisms of eccentric contraction-induced E-C coupling failure. Eccentric contractions could modify proteins associated with the t tubule voltage sensor and SR Ca2+ release channel through the allosteric effects of ions or metabolites, physical disruption, and/or proteolytic degradation. A number of ions and metabolic by-products are known to modulate Ca2+ release by acting on the t tubule voltage sensor complex, SR Ca2+ release channel, or both (31). For example, elevated levels of Mg2+, H+, and Pi, and reduced levels of ATP within the myoplasm, are known to inhibit SR Ca2+ release (31). Elevated [Ca2+]i can attenuate depolarization-induced force production without disrupting the functional integrity of the t tubule, the ability of the SR to release Ca2+, or the capacity to produce maximal force in skinned fibers (22). In contrast to the relatively high [Ca2+] (e.g., 2.5-100 µM) used by Lamb et al. (22), several other studies have shown that only very small increases in resting [Ca2+]i (e.g., 0.10-1.0 µM) are needed to reduce the rate of SR Ca2+ release in skeletal muscle fibers (5, 7). The small changes in resting [Ca2+]i observed in the present study agree with those reported by Balnave and Allen (3), who found that resting [Ca2+]i increased from 20 to 40 nM immediately after injury and remained elevated for the duration of the experiment (i.e., 30 nM at 1 h) in mouse single muscle fibers.

Several observations suggest that Ca2+-mediated degradation of one or more proteins involved in regulating SR Ca2+ release may be responsible for the prolonged E-C coupling failure after eccentric contraction-induced injury. First, the temperature and pH dependence of Ca2+-induced E-C coupling disruption described by Lamb and coworkers (22) suggests that the mechanism is enzymatic. Second, Ca2+-activated neutral proteases are known to degrade specific proteins associated with the SR (9, 29). Third, although increased [Ca2+]i can disrupt the E-C coupling process without degrading the alpha 1-subunit of the voltage sensor, triadin, or ryanodine receptor protein, protease (i.e., Ca2+-dependent and some lysosomal) inhibition by leupeptin blocked E-C coupling failure during exposure to 2.5 µM [Ca2+] (22). Therefore, proteolysis of some key triadic proteins may be responsible for disruption of E-C coupling when skeletal muscle fibers are exposed to relatively high resting [Ca2+]i. Whether the small eccentric contraction-induced increases in resting [Ca2+]i observed in the present and previous (3) studies disrupt the E-C coupling process by initiating proteolytic degradation of triad proteins remains unknown and awaits further investigation.

Other mechanisms contributing to the Po decrement. When the small reductions in caffeine- and 4-chloro-m-cresol-induced contracture force (i.e., ~15%) are considered in light of the smaller decrements in SR function (i.e., at least at <3 days), it appears that factors outside the E-C coupling pathway contribute to a small part of the Po decrement. Possible mechanisms contributing to the decrement in Po include 1) altered myofibrillar Ca2+ sensitivity; 2) mechanical disruptions in force-generating and/or -transmitting structures; and 3) proteolytic degradation of contractile or force-transmitting proteins.

Increased myoplasmic levels of certain metabolites (e.g., H+, Pi) are known to decrease the sensitivity of the myofilaments to Ca2+ (10). Pi has been shown to be chronically elevated after injury (2), and we have shown that contractile activity after injury is associated with an increased anaerobic metabolism (35). Therefore, it is possible that acidosis and/or elevated Pi could reduce myofibrillar Ca2+ sensitivity and decrease contractile force.

Our laboratory has previously reported that eccentric contractions performed in vitro resulted in reduced maximal Ca2+-activated force in skinned EDL muscle fibers (32, 35). These reductions in maximal force could be due to altered cross-bridge cycling or altered ability of the fiber to transmit force. The reduction in fiber force was twofold greater than that of fiber maximal actomyosin ATPase activity (35); this is suggestive of a disruption in force-transmitting elements.

It has been shown that significant reductions in actin and myosin heavy chain content do not occur until 5 days after injury induction (16). Therefore, although myofibrillar protein content does not contribute to the Po decrement during the first several days after the injury, significant reductions in protein content at 5 and 14 days may explain some of the force loss at these times.

Conclusions. E-C coupling failure is the primary mechanism for the immediate and prolonged decrease in Po during the first 5 days after eccentric contraction-induced injury of mouse EDL muscle. Although SR function is impaired somewhat after injury induction, it appears that immediately after initiation of injury the primary site for the E-C coupling failure lies at the interface of the t tubule and the SR Ca2+ release channel. Future studies should directly test the hypothesis that the elevated resting [Ca2+]i that results from eccentric contraction-induced injury is a contributing mechanism.

    ACKNOWLEDGEMENTS

This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-42761.

    FOOTNOTES

Address for reprint requests: C. P. Ingalls, Muscle Biology Laboratory, Dept. of Health and Kinesiology, Texas A&M Univ., College Station, TX 77843-4243 (E-mail:Ingalls{at}unix.tamu.edu).

Received 6 June 1997; accepted in final form 26 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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