Recent studies suggest a link between exercise-induced rhabdomyolysis and mutations of the ryanodine receptor (RYR1) associated with malignant hyperthermia (MH). We hypothesized that MH-susceptible mice (RYR1Y522S/wt) would exhibit greater anterior crural muscle [tibialis anterior (TA) and extensor digitorum longus (EDL) muscles] damage and strength deficits following the performance of a single or repeated bouts of eccentric contractions compared with wild-type (WT) mice. After a single injury bout, RYR1Y522S/wt mice produced more isometric torque than WT mice immediately and 3 and 7 days postinjury. Moreover, EDL muscle isometric specific force deficits were fully recovered for RYR1Y522S/wt but not WT mice 14 days postinjury. The percentage of fibers in TA muscle exhibiting signs of muscle damage 7 and 14 days postinjury were at least three times less in RYR1Y522S/wt than in WT mice. Uninjured and injured EDL muscle from RYR1Y522S/wt mice also displayed greater S-glutathionylation of RYR1 than that from WT mice. During the weekly injury bouts, torque production by RYR1Y522S/wt mice was fully recovered before the third and fourth injury bouts, whereas torque was still reduced for WT mice. Three days after multiple injury bouts, there were ∼50% fewer fibers exhibiting signs of muscle damage in RYR1Y522S/wt than in WT TA muscle. These findings indicate that the RYR1Y522S/wt mutation protects skeletal muscle from exercise-induced muscle injury and do not support a direct association between MH susceptibility and contraction-induced rhabdomyolysis when core temperature is maintained at lower physiological temperatures during exercise.
- contraction-induced muscle injury/damage
malignant hyperthermia (MH) is a pharmacogenetic disorder, whereupon administration of halogenated anesthetic to a genetically predisposed individual can induce a hypermetabolic episode. A MH episode can be characterized by increases in body temperature, skeletal muscle rigidity, hyperkalemia, metabolic acidosis, and rhabdomyolysis and, potentially, culminates in organ failure and death (38). Mutations associated with MH can involve the sarcoplasmic reticulum (SR) Ca2+ release channel (ryanodine receptor 1; RYR1) and promote an inordinate release of Ca2+ from the SR in response to halogenated anesthetics (e.g., halothane and isoflurane) and caffeine (3, 6).
Recently, a mouse model was generated with a mutation (Y522S) in RYR1 that has been associated with MH in humans (6, 16). Heterozygous expression of this mutation (RYR1Y522S/wt) has been shown previously to increase the sensitivity of RYR1 to halothane and caffeine in isolated soleus muscle and to electrical stimulation in myotubes, as well as to increase the resting cytosolic Ca2+ concentrations in myotubes and single soleus muscle fibers (6, 16). In addition, it has been found that thermal stress (41°C) induces muscle rigidity, sarcolemmal damage (evidenced by elevated plasma creatine kinase and K+), and death in RYR1Y522S/wt mice (6). Collectively, these findings indicate that the Y522S mutation results in a MH phenotype, disrupts cellular Ca2+ homeostasis, and supports the notion that RYR1 MH mutations increase susceptibility to exertional heat illness in humans.
Resistance exercise or eccentric contractions can help maintain or increase muscle mass and strength in healthy, aging, and some diseased populations (49). A typical response to an acute bout of eccentric contractions can include muscle fiber damage (26, 27), increased presence of muscle proteins in the blood, resting cytosolic Ca2+ perturbations (4, 21, 28, 46), mitochondrial disruption (14, 44), excitation-contraction (E-C) coupling failure (4, 21), and immediate and prolonged functional deficits (4, 20–22, 27, 29, 41). Usually, injured muscle undergoes a degenerative and regenerative process that repairs the injured muscle fibers and restores function on the order of days to weeks, depending on the age and health status of the individual and the severity of the injury. Performance of repeated bouts of eccentric contractions (i.e., resistance training) attenuates damage to the sarcolemma and force-bearing proteins (31). Consequently, recovery from a repeated bout of eccentric contractions occurs at a faster rate (23, 31). However, occasionally healthy trained or untrained individuals and diseased individuals exhibit a more severe breakdown of skeletal muscle in response to strenuous exercise (i.e., exercise-induced rhabdomyolysis) that can be life threatening (5, 7, 25, 32, 40, 43).
Case studies suggest that a link exists between exercise-induced rhabdomyolysis and RYR1 mutations associated with MH (11, 43). In these studies, individuals who had exhibited signs of exercise-induced rhabdomyolysis in the past were confirmed as MH susceptible through in vitro muscle contracture and genetic testing. The broad implication of these findings is that MH-susceptible individuals are at a heightened risk of experiencing exercise-induced rhabdomyolysis. Thus we sought to determine whether a single bout of resistance exercise or 4 wk of resistance training would induce rhabdomyolysis in MH-susceptible mice. Because we have recently reported enhanced oxidative and nitrosative stress of RYR1 in MH-susceptible mouse skeletal muscle (16), we also measured the level of S-glutathionylation of the RYR1 in injured and uninjured extensor digitorum longus (EDL) muscles. Specifically, we tested the hypothesis that strength deficits and myocellular damage would be exacerbated in anterior crural muscles of RYR1Y522S/wt mice after the performance of a single and multiple bouts of 150 eccentric contractions.
The generation of RYR1Y522S/wt mice has been described in detail elsewhere (6). The actual mutated amino acid number in these mice is Y524S; however, the corresponding RYR1 mutation in humans (Y522S) will be used for nomenclature because these mice are meant to be a model for the human disease. Mice were bred at Georgia State University, and genotypes were confirmed using polymerase chain reaction (PCR) analysis. The mice were housed in groups of 5–10 animals per cage, supplied with food and water ad libitum, and maintained in a room at 20–22°C with a 12-h photoperiod. Mice were euthanized with an overdose of pentobarbital sodium (200 mg/kg). 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.
Two studies were performed to determine whether eccentric contractions induce rhabdomyolysis in RYR1Y522S/wt mice. In the first study, RYR1Y522S/wt and wild-type (WT) mice performed a single bout of 150 eccentric contractions with the left anterior crural muscles [tibialis anterior (TA) and EDL muscles]. A total of 38 RYR1Y522S/wt and 42 WT mice were used in the muscle function experiments of this study. We have shown that this eccentric contraction protocol causes a significant loss of maximum strength (∼50%), requiring a prolonged period (≤4 wk) of recovery time (20, 21). Isometric torque produced by this muscle group as a function of stimulation frequency (20–400 Hz) was measured in anesthetized mice before and immediately after the eccentric contraction bout. Peak torque was recorded for each of the 150 maximal eccentric contractions. The recovery of anterior crural muscle strength after injury was also evaluated in vivo by measuring isometric torque as a function of stimulation frequency (20–400 Hz) in different groups of mice at 3, 7, and 14 days after injury (RYR1Y522S/wt: n = 9, 6, and 12; WT: n = 11, 6, and 10, respectively). For each respective group of mice, the extent of myocellular damage was assessed in uninjured and injured TA muscles at 3, 7, and 14 days after injury. In addition, isometric force production of the EDL muscle as a function of stimulation frequency (10–300 Hz) was assessed in vitro in uninjured muscles as well as in muscles 3, 7, and 14 days after injury (RYR1Y522S/wt: n = 11, 9, 6, and 9; WT: n = 14, 11, 6, and 10, respectively). Another group of RYR1Y522S/wt (n = 21) and WT (n = 23) mice were used to assess oxidation (i.e., S-glutathionylation) of RYR1 in uninjured EDL muscle and in EDL muscle at 3 and 14 days after injury.
After observing no signs of rhabdomyolysis after one bout of eccentric contractions, we determined whether multiple bouts of eccentric contractions induce rhabdomyolysis in RYR1Y522S/wt mice. In the second study, 8 RYR1Y522S/wt and 7 WT mice performed 4 bouts of 150 eccentric contractions separated by 1 wk. Isometric torque produced by the anterior crural muscles as a function of stimulation frequency (20–400 Hz) was measured before and immediately after each eccentric contraction bout. Peak torque was recorded for each of the 150 maximal eccentric contractions during each bout. The recovery of anterior crural muscle strength after injury was evaluated in vivo by measuring isometric torque as a function of stimulation frequency (20–400 Hz) 3 days after injury. The extent of myocellular damage was assessed in injured TA muscles 3 days after injury. In addition, isometric force production of the EDL muscle as a function of stimulation frequency (10–300 Hz) was assessed in vitro in injured muscles 3 days after injury.
In Vivo Muscle Strength Analysis and Injury Induction
Contractile function (i.e., torque-frequency relationship) of the left anterior crural muscles was measured in vivo as previously described (20–22). After mice were anesthetized (0.3 mg/kg fentanyl citrate, 16.7 mg/kg droperidol, and 5.0 mg/kg diazepam), the left hindlimb was aseptically prepared. The mouse was then placed on a heated platform, and halogen lamps were directed at the torso of the mouse to maintain body temperature. The left knee was clamped, and the left was foot secured to an aluminum “shoe” that was attached to the shaft of an Aurora Scientific 300B servomotor. Sterilized needles were inserted through the skin for stimulation of the left common peroneal nerve. Stimulation voltage and needle electrode placement were optimized with 5–15 isometric contractions (200-ms train of 0.1-ms pulses at 300 Hz). Contractile function of the anterior crural muscles was assessed by measuring peak isometric torque as a function of stimulation frequency (20–400 Hz). Injury to the anterior crural muscles was induced by the performance of 150 eccentric contractions (38° angular movement at 2,000°/s starting from a 19° dorsiflexed position). Core body temperature was monitored continuously during all functional testing with a mouse rectal thermocouple.
Preliminary experiments indicated that some RYR1Y522S/wt mice experience hypermetabolic episodes (i.e., hyperventilation and increased core temperature) and death when core body temperature was maintained near 37°C while they were anesthetized. Therefore, to test the current hypothesis, the core temperature of RYR1Y522S/wt and WT mice was maintained at ∼35°C during all in vivo testing by manipulating the temperature setting on the heating platform or the distance the halogen lamp was from the torso of the animal. The average core body temperature during in vivo testing was 34.2 ± 0.3 and 34.6 ± 0.2°C for RYR1Y522S/wt and WT mice, respectively. At this lower temperature, no mice died due to hypermetabolic episodes.
In Vitro Analysis of EDL Muscle
EDL muscles from RYR1Y522S/wt and WT mice were dissected free and studied at 30°C using an in vitro preparation as previously described (20–22). This temperature was chosen because preliminary experiments with the EDL muscle (data not shown) and previously published results of the soleus and diaphragm muscles from RYR1Y522S/wt mice (6, 16) indicated that at 35°C, resting tension is not stable. In the present study, resting tension was stable and similar for RYR1Y522S/WT and WT mice throughout the in vitro testing at 30°C. EDL muscles were mounted in a chamber containing a Krebs-Ringer bicarbonate buffer (pH 7.4) with (in mM) 144 Na+, 126.5 Cl−, 6 K+, 1 Mg2+, 1 SO42−, 1 PO43−, 25 HCO3−, 1.25 Ca2+, 0.17 leucine, 0.10 isoleucine, 0.20 valine, and 10 glucose, with 10 μg/ml gentamicin sulfate and 0.10 U/ml insulin (the buffer was equilibrated with 95% O2-5% CO2 gas). The distal tendon was attached by silk suture and cyanoacrylate adhesive to a fixed support, and the proximal tendon was attached to the lever arm of a servomotor system (Aurora Scientific 300B). Optimal physiological muscle length (Lo) in the chamber was set with a series of twitch contractions (0.2-ms pulse at 150 V). Next, peak isometric force as a function of stimulation frequency (10–300 Hz) was measured during isometric contractions (200-ms trains of 0.2-ms pulses) with 3 min between contractions. Caffeine sensitivity was assessed by measuring changes in baseline force during exposure to increasing caffeine concentrations (1, 2, 4, 8, 16, and 50 mM) and twitch contractions at a rate of 0.2 Hz. Force (N/cm2) produced by the EDL muscle was normalized to physiological cross-sectional area (PCSA) as described previously (27).
Cryosections (10 μm) were obtained from three levels (proximal, middle, and distal) of the TA muscle and stained with hematoxylin and eosin to estimate the extent of muscle damage caused by exercise. All myofibers were counted in the muscle cross sections from the three levels using BioQuant Nova Prime (v6.90.10) software and averaged to estimate total muscle myofiber number. Discolored myofibers containing three or more internal nuclei were classified as active degenerating myofibers (i.e., injured), whereas myofibers containing one or two internal nuclei with normal staining were used as a marker of previous damage and/or regeneration (9, 24, 30, 34, 50). We have previously used similar methods to estimate the extent of muscle damage after injury (27).
Western blotting of EDL muscle homogenates was performed and analyzed using methodology similar to that described by Aracena et al. (2). Nitrocellulose membranes were probed with a mouse anti-glutathione antibody (1:10,000; Virogen). Membranes were then stripped with Li-COR membrane stripping solution, following the manufacturer's instructions, and reprobed with a mouse anti-RYR1 antibody (1:10,000; Affinity Bioreagents). The ratio of glutathione to RYR1 fluorescence emission was determined for comparative analyses.
Individual factorial ANOVAs, ANOVAs with repeated measures, and t-tests were used to determine statistical differences in isometric and eccentric strength, caffeine contracture force and sensitivity, body weight, skeletal muscle wet weight, and myofiber number, damage, regeneration, and RYR1 S-glutathionylation. In the event of a significant interaction, simple main-effects analysis and Bonferroni's post hoc tests were conducted. An α level of 0.05 was used for all analyses. Values are means ± SE.
Characteristics of RYR1Y522S/wt and WT mice are listed in Table 1. In general, uninjured body and muscle wet weights were similar between groups of mice. Thus uninjured TA and EDL muscle wet weight-to-body weight ratios were similar between RYR1Y522S/wt and WT mice (e.g., TA body wt: 1.9 ± 0.06 vs. 1.9 ± 0.04 g). However, because body weight changes during recovery from injury, absolute torque (N·mm) was expressed relative to body weight (N·mm·kg−1) for comparative analyses.
Previous research with myotubes expressing the RYR1Y522S/wt mutation has demonstrated disturbances in Ca2+ homeostasis, enhanced sensitivity to voltage-gated stimulation, and reduced peak Ca2+ transients, indicating that the mutation could alter torque and force-producing capacity of uninjured RYR1Y522S/wt muscles (6, 16). In the present study, maximal isometric torque of the anterior crural muscles (primarily TA muscle) was measured in vivo across a range of stimulation frequencies (20–400 Hz). In general, RYR1Y522S/wt mice produced less peak isometric tetanic torque (N·mm·kg−1) but more unfused tetanic torque than WT mice before injury was induced. Compared with that from WT mice, anterior crural muscle strength from RYR1Y522S/wt mice was greater at some low frequencies (60 and 80 Hz) and lesser at some high frequencies (350 and 400 Hz, and tended to be greater at 200–300 Hz, P ≈ 0.10–0.06) (Fig. 1A). To further examine the effect of the RYR1Y522S/wt mutation on intrinsic function, isometric torque at each stimulation frequency was normalized to peak tetanic torque. This normalization emphasized a leftward shift in the torque-frequency curve for the RYR1Y522S/wt mice (i.e., greater torque was produced by the RYR1Y522S/wt mice at 60–250 Hz) (Fig. 1B).
During the preinjury torque-frequency test, we observed that the twitch (20 Hz) and tetanic (300 Hz) contractile responses of the RYR1Y522S/wt mice were distinguishably altered from the normal response exhibited by the WT mice. Representative twitch and tetanic torque curves are displayed in Fig. 1, C and D. Neither “peak” twitch torque at 20 Hz (Table 2) nor twitch torque measured during the 1st pulse of the 20-Hz contraction (20.8 ± 1.4 vs. 22.3 ± 1.3 N·mm·kg−1; P = 0.31) were different between RYR1Y522S/wt and WT mice. However, the twitch torque response of the WT mice displayed an 11.1 ± 2.3% decrease, whereas the RYR1Y522S/wt mice exhibited a 14.0 ± 2.4% increase in torque from the 1st to 5th twitch. In addition, the rates of peak tetanic torque production (+dP/dt) and relaxation (−dP/dt) were ∼18 and ∼36% slower, respectively, and the half time to relaxation was ∼38% longer for the RYR1Y522S/wt mice compared with that for the WT mice (Table 2).
In addition to assessing functional capacity of the anterior crural muscles in vivo, we measured specific isometric force (N/cm2) produced by EDL muscle in vitro at 30°C across a range of stimulation frequencies (10–300 Hz). During the 10-Hz stimulation, twitch force did not show signs of augmentation as it did in vivo at 20 Hz for the RYR1Y522S/wt mice (data not shown). Uninjured EDL muscle of RYR1Y522S/wt mice produced less peak isometric specific force at 10 Hz (approximately −9%) and during tetanic contractions (150–300 Hz; approximately −9%) (Fig. 1E and Table 2). Normalizing specific force at each frequency to peak tetanic specific force also demonstrated a leftward shift in the force-frequency curve for the RYR1Y522S/wt mice, as it did in vivo (Fig. 1F). The rate of and half time to relaxation during peak tetanic contractions were ∼25% slower and ∼10% longer for RYR1Y522S/wt than for WT mice, respectively (Table 2). Similar differences in −dP/dt (RYR1Y522S/wt vs. WT: −4.4 ± 0.3 vs. −5.6 ± 0.5 N/s) and half time to relaxation (18 ± 1 vs. 16 ± 1 ms) also were observed during twitch contractions. Normalizing −dP/dt and half time to relaxation values by peak force values did not remove these differences. The rate of force production (+dP/dt) during twitch (RYR1Y522S/wt vs. WT mice: 12.9 ± 0.6 vs. 13.9 ± 0.7 N/s) or peak tetanic contractions (Table 2) was not different between groups of mice.
Body and muscle wet weight.
Body weight and TA muscle and EDL muscle wet weights were affected in both RYR1Y522S/wt and WT mice during the days after muscle injury induction (Table 1). RYR1Y522S/wt and WT body weights were increased 14 days after injury induction compared with uninjured values. TA and EDL muscle wet weight was not differentially affected between genotypes by muscle injury; however, both muscle wet weights were generally altered in the days after injury. Relative to uninjured TA muscle wet weights, wet weight was increased by ∼9% at 3 days after injury, decreased ∼13% at 7 days after injury, and as similar to uninjured values at 14 days after injury. EDL muscle wet weight was reduced by ∼9% at both 7 and 14 days after injury compared with uninjured and 3-day postinjury wet weights.
In vivo muscle injury induction.
The anterior crural muscles of RYR1Y522S/wt and WT mice performed 150 maximal eccentric contractions, in vivo, to induce muscle injury. Peak eccentric torque measured for the 1st (RYR1Y522S/wt vs. WT: 203.0 ± 4.0 vs. 206.7 ± 3.8 N·mm·kg−1) and every 10th contraction thereafter was similar between RYR1Y522S/wt and WT mice. Therefore, there was no difference between RYR1Y522S/wt and WT mice in total torque produced, calculated as the sum of the torque produced during the 1st and every 10th contraction thereafter of the eccentric protocol (2,113.3 ± 39.1 vs. 2,117.7 ± 40.0 N·mm·kg−1), or the percent decline in peak torque from the 1st to 150th contraction (−48.8 ± 1.5 vs. −49.9 ± 0.6%).
In vivo strength deficits and recovery.
We had observed previously that immediate strength deficits associated with the eccentric contraction protocol used in this study are due to muscle injury and not fatigue (48). Immediately after the eccentric contraction protocol, torque was reduced at all frequencies for RYR1Y522S/wt (−41 to −91%) and WT mice (−53 to −92%) compared with preinjury values (Fig. 2). However, RYR1Y522S/wt produced significantly more torque than WT mice at low, moderate, and high frequencies at this time (40–200 Hz) and therefore had comparatively less torque deficits, or injury, immediately after the injury protocol (e.g., RYR1Y522S/wt vs. WT at 200 Hz: −50.8 ± 1.5 vs. −58.8 ± 1.0%).
At 3 and 7 days after the eccentric protocol, RYR1Y522S/wt mice continued to produce more isometric torque than WT mice at low, moderate, and high stimulation frequencies (Fig. 2). In addition, the strength deficits from preinjury torque values were significantly less for RYR1Y522S/wt than WT mice at all frequencies 3 days after injury and at low (20 Hz) and high (125–400 Hz) frequencies 7 days after injury. For example, at 200 Hz, isometric torque was reduced by 44.0 ± 3.8 and 12.2 ± 5.0% for RYR1Y522S/wt mice and by 64.1 ± 1.6 and 47.2 ± 2.9% for WT mice 3 and 7 days after injury, respectively. The greater torque production by RYR1Y522S/wt mice indicates an intrinsic resistance and potentially enhanced recovery during the first week after the injury protocol.
Fourteen days after the induction of muscle injury, strength at low to moderate stimulation frequencies (20–80 Hz) had recovered to preinjury values for both RYR1Y522S/wt and WT mice (Fig. 2). RYR1Y522S/wt mice produced greater torque at some low frequencies (60 and 80 Hz) than WT mice, as they did before injury was induced. At high frequencies, isometric strength was still reduced for both groups of mice, but to a similar degree (e.g., RYR1Y522S/wt vs. WT at 200 Hz: −13.6 ± 2.7 vs. −15.6 ± 2.7%).
In vitro force deficits and recovery.
Isometric force-producing capacity of injured EDL muscle from RYR1Y522S/wt and WT mice was tested, in vitro, 3, 7, and 14 days after the eccentric contraction protocol. Compared with uninjured muscle, RYR1Y522S/wt EDL muscle specific isometric force (N/cm2) was recovered at low frequencies (10 and 20 Hz) by 7 days and at all stimulation frequencies (10–300 Hz) by 14 days after injury induction. Fourteen days after injury induction, WT EDL muscle specific isometric force produced at low stimulation frequencies (10–60 Hz) was similar to uninjured values but was still reduced (∼15%) at moderate to high stimulation frequencies (100–300 Hz). Specific force did not recover to uninjured values at any frequency 3 or 7 days (except 10 and 20 Hz for RYR1Y522S/wt mice) after the in vivo eccentric protocol for either group of mice. In addition, specific force was not different between RYR1Y522S/wt and WT mice (all frequencies) 3, 7, or 14 days after the eccentric protocol.
Increased caffeine sensitivity is a well-established marker of MH susceptibility. It has previously been reported that RYR1Y522S/wt soleus muscle has increased caffeine sensitivity (6). In the present study, caffeine sensitivity (EC50) was greater for uninjured RYR1Y522S/wt EDL muscle (9.4 ± 0.7 mM) than for WT EDL muscle (28.1 ± 1.7 mM); peak caffeine contracture force was also higher in uninjured RYR1Y522S/wt EDL muscle (7.3 ± 0.3 N/cm2) than in WT EDL muscle (4.7 ± 0.5 N/cm2). In uninjured and injured muscle, RYR1Y22S/wt EDL muscle produced more contracture force than WT EDL muscle at all caffeine concentrations (1–50 mM). At each postinjury time point, RYR1Y522S/wt EDL muscle maintained a lesser EC50 and greater peak contracture force than WT muscle (Fig. 3). However, caffeine sensitivity and peak caffeine contracture force were altered similarly after injury in both groups of mice, since EC50 was increased at all postinjury days and peak caffeine contracture force was reduced only at 3 days after injury compared with uninjured values (Fig. 3). Therefore, the RYR1Y522S/wt EDL muscle exhibits enhanced caffeine sensitivity consistent with previous experiments with this model (6) and an MH-susceptible phenotype.
Histological analysis of muscle damage.
In uninjured muscle tissue, a low level of muscle degeneration and regeneration was present (Figs. 4 and 5). However, in uninjured RYR1Y522S/wt TA muscle, the percentage of fibers exhibiting active degeneration and regeneration (i.e., central nuclei) were ∼60 and ∼110% greater than in uninjured WT TA muscle, respectively (Fig. 5).
TA muscles were dissected from RYR1Y522S/wt and WT mice 3, 7, and 14 days after the injury protocol for histological analysis of muscle damage (Figs. 4 and 5). The total number of fibers counted in TA muscles from both groups of mice was similar each day after injury (RYR1Y522S/wt vs. WT: ∼2,285 ± 56 vs. 2,105 ± 77 fibers) and did not significantly change from their respective uninjured value (2,239 ± 72 vs. 2,307 ± 67 fibers). There were, however, differences between RYR1Y522S/wt and WT mice in the percentage of total fibers that exhibited signs of active degeneration and regeneration during the days after injury (Fig. 5). The percentage of fibers exhibiting active degeneration were 38, 84, and 69% less in RYR1Y522S/wt TA muscle than WT muscle at 3, 7, and 14 days after injury, respectively. Seven and 14 days after injury, the percentage of fibers actively regenerating were 69 and 72% less in RYR1Y522S/wt TA muscle than in WT muscle. These findings are consistent with RYR1Y522S/wt mice incurring less injury or damage from the eccentric contraction injury protocol.
The level of oxidative stress, evidenced by an enhanced S-glutathionylation and S-nitrosylation of RYR1, has previously been found to be greater in skeletal muscle from RYR1Y522S/wt than from WT mice (16). In the present study, S-glutathionylation of RYR1 was determined in uninjured and injured EDL muscle homogenates (Fig. 6). In uninjured muscle, S-glutathionylation of RYR1 was ∼70% greater for RYR1Y522S/wt mice than for WT mice. Three and 14 days after the induction of muscle injury, RYR1 S-glutathionylation was ∼60 and ∼140% greater in RYR1Y55S/wt muscle compared with values in WT muscle at each respective time point. Compared with their respective uninjured values, RYR1 S-glutathionylation increased ∼50% in WT muscle and tended to increase ∼40% in RYR1Y522S/wt muscle (P = 0.093) 3 days after injury. In addition, 14 days after injury, RYR1 S-glutathionylation was increased by ∼83% in RYR1Y522S/wt muscle and tended to increase by ∼30% in WT muscle (P = 0.091). These findings indicate that RYR1 is oxidized to a greater extent in RYR1Y522S/wt than in WT EDL muscle both before and after injury and that performing eccentric contractions increases RYR1 S-glutathionylation in RYR1Y522S/wt and WT skeletal muscle.
Body and muscle wet weight.
Body weights were similar between genotypes before the 4 wk of eccentric training began (RYR1Y522S/wt and WT: 23.1 ± 1.0 and 23.3 ± 0.8 g) and did not significantly change over the course of the training (i.e., average body weight: 23.5 ± 0.7 and 23.7 ± 0.8 g). Body weight was also similar between genotypes 3 days after the final eccentric training bout (24.2 ± 0.7 and 23.7 ± 0.8 g). In addition, 3 days after the final training bout, both TA and EDL muscle wet weight were similar between RYR1Y552S/wt (TA and EDL: 45.8 ± 1.8 and 10.6 ± 0.3 mg) and WT mice (48.3 ± 1.3 and 10.4 ± 0.2 mg).
In vivo eccentric torque.
RYR1Y522S/wt and WT mice performed a bout of 150 eccentric contractions every 7 days for 4 wk to determine whether multiple injurious bouts of exercise would induce rhabdomyolysis in MH-susceptible mice. In general, the injury stimulus was similar for each group of mice. In both groups, peak eccentric torque for the first contraction was reduced during the 2nd, 3rd, and 4th bouts compared with the 1st bout; the greatest reduction occurred during the 2nd bout, whereas peak eccentric torque began to recover during the 3rd and 4th bouts (Fig. 7). Peak eccentric torque for the first contraction of the 1st, 3rd, and 4th bout was similar between RYR1Y522S/wt and WT mice, as was the total eccentric torque produced during these bouts (Fig. 7). However, during the 2nd bout, peak eccentric torque for the first contraction and total eccentric torque produced were greater for RYR1Y522S/wt than for WT mice (165.7 ± 10.5 vs. 126.0 ± 11.2 N·mm·kg−1 and 1,984.1 ± 136.7 vs. 1,453.5 ± 97.5 N·mm·kg−1, respectively). The decline of peak eccentric torque from the 1st to 150th contraction was not different between genotypes for any eccentric bout, although compared with the 1st bout, the decline was significantly attenuated for both groups of mice from the 2nd bout onward (i.e., peak eccentric torque was reduced by ∼50% during the 1st bout but only by ∼35% during subsequent bouts). These findings indicate that the injury stimulus was at least no less for RYR1Y522S/wt mice than for WT mice, since the capacity to produce eccentric torque was not uniquely compromised in RYR1Y522S/wt mice.
In vivo isometric torque.
Isometric torque produced by the anterior crural muscles was measured immediately before and after each bout of eccentric contractions as well as 3 days after the final bout. Consistent with the findings in study 1, RYR1Y522S/wt mice exhibited lesser strength deficits than WT mice after the 1st eccentric bout (Fig. 8). Although the relative strength deficits, calculated by dividing immediate postinjury torque by preinjury torque for each bout at each frequency, were similar after the 2nd eccentric bout, RYR1Y522S/wt mice had lesser strength deficits at nearly all frequencies after the 3rd and 4th bouts. For example, following the 4th eccentric bout, RYR1Y522S/wt mice experienced an ∼45 and 30% reduction in torque at 100 (unfused) and 300 Hz (fused), respectively, whereas WT mice had an ∼65 and 35% torque deficit at 100 and 300 Hz, respectively. Therefore, it appears that even after multiple bouts of eccentric contractions, RYR1Y522S/wt mice are less susceptible to muscle injury than WT mice.
Study 1 indicated that RYR1Y522S/wt mice recover anterior crural muscle function faster than WT mice after a single bout of eccentric contractions. By comparing the isometric contraction torques as a function of stimulation frequency before each injury bout, the ability of mouse anterior crural muscle to recover from weekly sessions of eccentric contraction-induced injury was assessed in study 2 (Fig. 8A). Seven days after the 1st injury bout, torque was reduced for both groups of mice at most frequencies, but RYR1Y522S/wt mice produced greater torque at low to moderate frequencies (40–80 Hz) and tended to produce greater torque at higher frequencies (P = 0.06–0.10) than WT mice. Seven days after the 2nd and 3rd injury bouts (i.e., before injury bouts 3 and 4), RYR1Y522S/wt mice were fully recovered to uninjured values, whereas torque produced by WT mice was still reduced at moderate to high frequencies (100–300 Hz). These results indicate that RYR1Y522S/wt mice recover faster than WT mice following weekly bouts of eccentric contractions.
Three days after the 4th and final injury bout, anterior crural muscle isometric torque as a function of stimulation frequency was not different between RYR1Y522S/wt and WT mice (200 Hz: 88.2 ± 4.4 vs. 84.7 ± 9.4 N·mm·kg−1). However, both groups of mice showed some adaptation to the injury stimulus, since the torque deficits appeared to be reduced compared with the torque deficits observed 3 days after a single injury bout (i.e., single vs. multiple: approximately −50 vs. −20% at 200 Hz).
In vitro EDL muscle specific isometric and caffeine contracture force.
EDL muscle specific isometric force as a function of stimulation frequency was not different between RYR1Y522S/wt and WT mice 3 days after the 4th injury bout (200 Hz: 17.8 ± 0.6 vs. 18.0 ± 0.8 N/cm2). Compared with their respective uninjured EDL specific force values, neither group of mice had recovered back to baseline values, although there was marked improvement at all stimulation frequencies for both groups of mice 3 days following the 4th injury bout compared with 3 days after a single injury bout (e.g., single vs. multiple: 7.67 ± 0.57 vs. 17.9 ± 0.48 N/cm2 at 200 Hz).
RYR1Y522S/wt mice produced significantly more EDL muscle caffeine contracture force than WT mice at all caffeine concentrations (0–50 mM) 3 days after the final injury bout. The magnitude of force and differences between groups of mice are similar to the values reported in study 1 for uninjured muscle (Fig. 3).
Three days after the 4th and final injury bout, injured TA muscles were dissected for histological analysis of muscle degeneration and regeneration. Although the percentage of fibers actively degenerating was similar between RYR1Y522S/wt and WT mice (0.2 ± .03 vs. 0.3 ± 0.1%), the percentage of fibers actively regenerating, as evidenced by central nuclei, was significantly greater in WT than RYR1Y522S/wt TA muscle (30.1 ± 4.5 vs. 12.0 ± 2.1%). On average, more than 2,000 fibers per cross section were included in this analysis (RYR1Y522S/wt vs. WT: 2,051 ± 33.8 vs. 2,706 ± 83.7 fibers).
Malignant hyperthermia is an inheritable skeletal muscle disorder in which a mutation to the SR Ca2+ release channel (RYR1) can result in an inordinate release of Ca2+ (3) that initially causes hypermetabolism and rhabdomyolysis but may ultimately lead to organ failure and death (38). An MH episode may be induced by administration of halogenated anesthetics, making surgery dangerous if the patient is unaware that he or she is MH susceptible (38). There appears to be an association between exertional heat illness and exercise-induced rhabdomyolysis with MH, suggesting that thermal and mechanical stress are also MH triggers (33, 43). This association is substantiated by case studies wherein MH-susceptible individuals experienced MH-like symptoms (e.g., rhabdomyolysis) after exercise performed in thermally neutral or stressful environments (e.g., 11, 19, 25, 39, 43). The present study examined the interaction between MH susceptibility and exercise (mechanical stress)-induced rhabdomyolysis by having MH-susceptible mice (RYR1Y522S/wt) perform a single and multiple bouts of eccentric contractions under environmental conditions that did not promote hyperthermia.
RYR1Y522S/wt mice were injured less and recovered faster than WT mice after single and multiple injury bouts. Evidence for this includes RYR1Y522S/wt mice having 1) lesser isometric strength deficits immediately and 3 and 7 days after a single injury bout (Fig. 2); 2) a full recovery of EDL muscle specific force (100–300 Hz) by 14 days after a single injury bout, while WT mice were still reduced (∼17% at 200 Hz); 3) markedly less fiber degeneration and regeneration in the 2 wk after a single injury bout (Figs. 4 and 5); 4) lesser immediate strength deficits at some stimulation frequencies after the 1st and 2nd injury bouts and at nearly all frequencies after the 3rd and 4th injury bouts (Fig. 8); 5) a full recovery of isometric strength before the 3rd and 4th injury bouts, while WT strength was still reduced (Fig. 8A); and 6) markedly less muscle damage incurred over the four bouts of eccentric training than WT mice. After a single injury bout, the initial functional deficits (e.g., 53–92% deficit of isometric torque), the rate of functional recovery (e.g., ∼16% peak isometric torque deficit 14 days after injury), and the degree of muscle damage (Figs. 4 and 5) for the WT mice are all consistent with previous studies using the same injury model (20, 21, 27). On the basis of these results, it does not appear that MH-susceptible mice with the RYR1Y522S/wt mutation are prone to severe rhabdomyolysis in response to a single or multiple bouts of 150 eccentric contractions when core body temperature is maintained at ∼35°C.
Failure to associate MH susceptibility with exercise-induced rhabdomyolysis in this study does not appear to be related to the specific RYR1 mutation expressed (i.e., Y522S). MH has been described as a heterogeneous genetic trait with variable phenotype, attributable to the multiple RYR1 mutations associated with MH susceptibility in humans and the inconsistent penetrance of the myopathy among family members (11). It has been proposed that the MH phenotype variability could be due to secondary genetic mutations acting in tandem with the primary RYR1 mutation (37) or epigenetic allele silencing (52, 53). Importantly, MH-susceptible individuals who express a variety of RYR1 mutations have been reported to experience exercise-induced rhabdomyolysis that was not associated with exertional heat exposure (43), suggesting that sensitivity to mechanical stress in MH-susceptible individuals may not be mutation specific.
Alternatively, failure to observe exercise-induced rhabdomyolysis in MH-susceptible mice may be the result of the lower core body temperature (∼33–36°c) at which the in vivo eccentric contraction experiments were performed. There are reports of rhabdomyolysis occurring after MH-susceptible individuals exercised in thermally stressful conditions (11, 25, 33), suggesting that the combination of mechanical and thermal stress may lead to MH-related rhabdomyolysis. Prior research with RYR1Y522S/wt mice has demonstrated that thermal stress (41°C) induces skeletal muscle membrane damage and death (6). A mechanism explaining the association between MH susceptibility and thermal sensitivity in RYR1Y522S/wt mice has recently been elucidated: The RYR1Y522S/wt mutation induces a constitutive RYR1 Ca2+ leak that enhances production of free radical oxygen and nitrogen species. S-nitrosylation of RYR1 increases the sensitivity of the channel to thermal stress, promoting further RYR1 Ca2+ release that institutes a vicious cycle of SR Ca2+ release (16). To this effect, we observed that RYR1Y522S/wt mice were likely to exhibit signs of hypermetabolism (e.g., increased respiration rate) and die during or soon after in vivo injury induction if core body temperature rose above ∼37°C (data not shown). We believe that the hypermetabolism in these mice was solely in response to overheating of the mice and not to muscle damage, since RYR1Y522S/wt mice were likely to become hypermetabolic and die after performing a small number of isometric contractions at ∼38°C (data not shown). By maintaining core body temperature below ∼36°C in the present study, we presumably isolated the effects of mechanical stress on MH-susceptible mice but may have removed any coordinated action between mechanical and thermal stress in inducing rhabdomyolysis.
Less apparent TA muscle damage and functional deficits exhibited by RYR1Y522S/wt mice do not appear to stem from a weaker injury stimulus. The magnitude of strength loss associated with the performance of eccentric contractions is determined primarily by peak eccentric force (29, 45). Anterior crural muscles from RYR1Y522S/wt and WT mice produced similar peak eccentric torque during all eccentric contraction bouts, with the exception of the 2nd bout of study 2, where RYR1Y522S/wt mice actually produced greater total peak torque than WT mice. In addition, the relative decrease in peak eccentric torque over each injury bout was nearly identical between the two groups of mice (Fig. 7).
Greater functional capacity of RYR1Y522S/wt than WT anterior crural muscles after injury may be related to the differential level of RYR1 oxidation observed between genotypes (Fig. 6; Ref. 16). Studies in which RYR1 is oxidized in vitro via application of redox reactive agents (e.g., hydrogen peroxide) have indicated that RYR1 oxidation increases RYR1 open probability, sensitivity to activators (e.g., caffeine), and Ca2+-induced Ca2+ release (CICR) while decreasing the sensitivity of the channel to inhibition by Mg2+ (1, 13, 17, 35, 51). It was shown previously that RYR1Y522S/wt mice exhibit an intrinsically enhanced oxidation of RYR1, evidenced by a greater level of S-glutathionylation and S-nitrosylation of the SR Ca2+ release channel, which increased RYR1 open probability and enhanced Ca2+-induced Ca2+ release in these mice (16). In addition, we demonstrated in the present study that injured RYR1Y522S/wt EDL muscle exhibited a greater level of RYR1 S-glutathionylation than WT muscle and that performing eccentric contractions increased RYR1 S-glutathionylation in EDL muscle of both groups of mice for a prolonged period of time (Fig. 6). It is intriguing to postulate that an enhanced CICR resulting from an increased oxidation of the SR Ca2+ release channel augments skeletal muscle force production following muscle injury. In support, Posterino et al. (36) reported that application of hydrogen peroxide to mechanically skinned muscle fibers enhanced CICR and, when E-C coupling was disturbed (i.e., the t system was only partially depolarized), augmented twitch force. Because the injury model used in this study disrupts E-C coupling through the first week and potentially up to 14 days after injury (20, 21, 47, 48), greater RYR1 oxidation (and presumably enhanced CICR) in RYR1Y522S/wt than WT muscle could explain the variance in force-producing capacity after injury.
The decreased vulnerability to and potentially faster recovery from muscle injury of the RYR1Y522S/wt mice also could be related to previous findings of an increased resting cytosolic Ca2+ concentration ([Ca2+]) in RYR1Y522S/wt skeletal muscle (16). It is possible that the elevated cytosolic [Ca2+] in uninjured RYR1Y522S/wt muscle activates muscle fiber cytokine production (e.g., IL-1 and IL-6) (15, 18), which has been implicated in playing a role in the rise in temperature during an MH episode (33) but also may be involved in mediating an acute immune response in muscle tissue (12, 42). Alternatively, an elevated resting cytosolic [Ca2+] could increase endogenous protease activity (e.g., calpains and/or proteasome) (8, 10). Either a low level immune response (i.e., phagocytic activity) or an increased proteolytic activity in RYR1Y522S/wt muscle could remove some stress-susceptible components in the muscle before injury and thereby attenuate damage to force-bearing structures and immediate strength deficits (47). The finding that uninjured RYR1Y522S/wt mice exhibit greater histological signs of muscle degeneration and regeneration than WT mice (Figs. 4 and 5) suggests that there may indeed be a greater intrinsic level of protein turnover and cell remodeling in these mice.
MH susceptibility conferred by the RYR1 Y522S mutation was not associated with rhabdomyolysis after a single or multiple bouts of eccentric contractions performed under environmental conditions that did not promote hyperthermia. However, these findings do not rule out a possible interaction between thermal and mechanical stress in inducing rhabdomyolysis in MH-susceptible individuals. Further testing is required to confirm that the exercise-induced rhabdomyolysis observed in MH-susceptible individuals is mutation specific. Contrary to our hypothesis, the RYR1Y522S/wt mutation reduced the vulnerability to eccentric contraction-induced muscle injury. Previous research with RYR1Y522S/wt mice has demonstrated that the mutation elevates resting cytosolic [Ca2+], which in turn enhances oxidation of RYR1, resulting in increased channel open probability and CICR in uninjured muscle (16). In the present study, we have demonstrated that oxidation of RYR1 is greater in RYR1Y522S/wt than WT muscle during the days and weeks after muscle injury induction and that performing eccentric contractions increases RYR1 oxidation in muscle from both groups of mice. On the basis of these data, it appears that a portion of the greater force-producing capacity of RYR1Y522S/wt mice after injury may be due to CICR augmentation of force. Determining how and to what extent RYR1 redox modifications alter SR Ca2+ release and force production in injured muscle warrants further investigation.
This work is supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-41802 (to S. L. Hamilton).
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