Strenuous exercise triggers a life-threatening response in mice susceptible to malignant hyperthermia

Abstract

In humans, hyperthermic episodes can be triggered by halogenated anesthetics [malignant hyperthermia (MH) susceptibility] and by high temperature [environmental heat stroke (HS)]. Correlation between MH susceptibility and HS is supported by extensive work in mouse models that carry a mutation in ryanodine receptor type-1 (RYR1^Y522S/WT) and calsequestrin-1 knockout (CASQ1-null), 2 proteins that control Ca²⁺ release in skeletal muscle. As overheating episodes in humans have also been described during exertion, here we subjected RYR1^Y522S/WT and CASQ1-null mice to an exertional-stress protocol (incremental running on a treadmill at 34°C and 40% humidity). The mortality rate was 80 and 78.6% in RYR1^Y522S/WT and CASQ1-null mice, respectively, vs. 0% in wild-type mice. Lethal crises were characterized by hyperthermia and rhabdomyolysis, classic features of MH episodes. Of importance, pretreatment with azumolene, an analog of the drug used in humans to treat MH crises, reduced mortality to 0 and 12.5% in RYR1^Y522S/WT and CASQ1-null mice, respectively, thanks to a striking reduction of hyperthermia and rhabdomyolysis. At the molecular level, azumolene strongly prevented Ca²⁺-dependent activation of calpains and NF-κB by lowering myoplasmic Ca²⁺ concentration and nitro-oxidative stress, parameters that were elevated in RYR1^Y522S/WT and CASQ1-null mice. These results suggest that common molecular mechanisms underlie MH crises and exertional HS in mice.—Michelucci, A., Paolini, C., Boncompagni, S., Canato, M., Reggiani, C., Protasi, F. Strenuous exercise triggers a life-threatening response in mice susceptible to malignant hyperthermia.

Malignant hyperthermia (MH) susceptibility (MHS), which was identified and described for the first time in 1960 (1), is an inherited pharmacogenetic disorder characterized by a life-threatening hypermetabolic response to halogenated/volatile anesthetics (i.e., halothane or isofluorane) or to the depolarizing muscle relaxant succinylcholine (2, 3). The main clinical features of MH crises include skeletal muscle rigidity, increased oxygen consumption, hyperthermia, rhabdomyolysis (i.e., the rupture of muscle fibers), myoglobinuria, and increased plasma/serum levels of K⁺ and creatine kinase (CK), which could eventually lead to cardiac arrhythmia (and even arrest) or kidney failure. It is widely accepted that the triggering agents, which are commonly used during surgery interventions, trigger a sustained and uncontrolled release of Ca²⁺ from the sarcoplasmic reticulum (SR) of skeletal muscle fibers (4). Most MH cases have been associated with mutations in the RYR1 (ryanodine receptor 1) gene (5, 6), which encodes for a large protein of approximately 2200 kDa that forms the SR Ca²⁺ release channel involved in excitation-contraction (EC) coupling, the mechanism that in muscle allows the transduction of action potential into Ca²⁺ release from the SR (7, 8).

MH episodes may be life threatening if not corrected immediately by suspension of the triggering agent and administration of dantrolene, the only drug that is currently approved to treat acute MH crises in humans (9). Individuals are diagnosed as MH susceptible if they have experienced hyperthermic crises during anesthesia (10, 11) and/or score positive on a diagnostic in vitro contracture test (IVCT). IVCT is a procedure performed in biopsy samples that are exposed to increasing concentrations of halothane or caffeine, both agonists of the RYR1 channel, according to 2 standardized procedures from the European MH Group and the North American MH Group (12, 13).

To date, there are at least 35 known causative mutations found in the RYR1 gene that comprise approximately 70% of all MH-susceptible individuals, which suggests that MH could arise from mutations to proteins other than RYR1, possibly those that directly modulate RYR1 activity (14, 15). Indeed, in 2 families, representing 1% of all MH cases, MH has been linked to mutations in the gene that encodes for the α1-subunit of the dihydropyridine receptor, the voltage sensor of the T-tubule membrane, which directly controls the gating of RYR1 during EC coupling (16, 17).

Because outside of the operating room MH individuals are usually asymptomatic, susceptibility to MH is still viewed in the medical field only as a syndrome that is related to exposure to volatile anesthetics (18, 19). However, increasing evidence indicates that life-threatening hyperthermic crises—virtually identical to MH episodes induced by anesthesia (i.e., characterized by hyperthermia, rhabdomyolysis, etc.), can also occur in susceptible individuals during exposure to elevated environmental temperatures, febrile illness, or as a stress response to physical exertion (20–22), crises collectively known as environmental/exertional heat strokes (HS). Of importance, some patients with HS have a family history of MH (23) and an association between RYR1 variants, and exertional- or stress-induced rhabdomyolysis and sudden death have been reported (24–26).

The correlation between MH and HS is also supported by studies in animals. Indeed, in porcine stress syndrome, pigs that carry a point mutation in the RYR1 gene trigger MH episodes in response to halothane or during exposure to stressful conditions, such as elevated environmental temperature and/or emotional or physical stress (27, 28). Moreover, knock-in mice that carry gain-of-function mutations in the RYR1 gene that are causative of MHS in humans (R163C and Y522S) are susceptible to lethal overheating crises when exposed to either halogenated anesthetics or elevated temperature (29–31). Finally, calsequestrin-1-knockout (CASQ1-null) mice that lack calsequestrin-1 (32) also exhibit lethal anesthetic- and heat-induced hyperthermic episodes (33) that are similar to those in porcine stress syndrome and of RYR1-knock-in mice, which suggests that the Casq1 gene could in theory represent a likely candidate for MHS (34). However, a recent study failed to identify any mutations in the Casq1 coding region in 75 unrelated patients with MH within the North American population (35), whereas mutations in Casq1 have been identified in families affected by vacuolar myopathy (36, 37). Functional studies have allowed for the determination that MH/HS crises in RYR1^Y522S/WT and CASQ1-null mice are the result of increased resting Ca²⁺ levels at physiologic temperature (37°C) as a result of enhanced SR Ca²⁺ leak (29, 31, 33), and enhanced caffeine sensitivity of skeletal muscle to develop abnormal contractures during IVCT experiments (29, 33, 38). A key role during MH/HS crises is also played by excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (31), a hypothesis that was also confirmed in CASQ1-null mice where the use of antioxidant treatments prevented structural damage and reduced the mortality rate during exposure to halothane or heat (38, 39).

In the present work, we subjected RYR1^Y522S/WT and CASQ1-null mice to an exertional-stress (ES) protocol—incremental running on treadmill at 34°C and 40% humidity—to test whether strenuous exercise in challenging environmental conditions is a stimulus that, like anesthesia and heat, is able to induce lethal overheating episodes, and whether drugs that are commonly used to treat acute MH reactions in humans are also able to prevent lethal exertion-induced episodes.

MATERIALS AND METHODS

RYR1^Y522S/WT and CASQ1-null mice

RYR1^Y522S/WT and CASQ1-null mice were generated as previously described (29, 32). All animals used in this study were males, as this gender is more susceptible than females to MH/HS-like crises when exposed to heat or halothane (33). Mice were housed in microisolator cages at 20°C in a 12-h light/dark cycle and provided free access to water and food. All functional, structural, and molecular analyses were carried out in extensor digitorum longus (EDL) muscles, with the exception of single-fiber experiments, which were performed in flexor digitorum brevis (FDB) muscles. All experiments on animals were conducted according to the Directive of the European Union 2010/63/UE and approved by the Committee on the Ethics of Animal Experiments of the University of Chieti (15/2011/CEISA/COM).

In vivo experiments

ES protocol and core temperature recordings

Four-month-old mice were subjected to an ES protocol carried out on a treadmill (Columbus Instruments, Columbus, OH, USA) that was placed in a climatic chamber in which the temperature and humidity were maintained at constant values of 34°C and 40%, respectively. In the 5 d before experiments, mice were trained for 10 min each day at room temperature at a speed of 10 m/min to become accustomed to running on the treadmill. A mild electrical stimulus (0.5 mA) was applied to mice that stepped off the treadmill to keep them exercising. During the ES protocol, speed was initially set at 5 m/min for 5 min and then increased as follows: 10 m/min for 10 min, 15 m/min for 10 min, and 20 m/min for 10 min. Finally, speed was increased for 1 m/min every 1 min to reach a final speed of 30 m/min. For mice that triggered an exertional HS during the ES protocol, running was stopped at the onset of lethal crises. During crises, mice stopped running, stood firm on the electrical grid (turned off) and began to manifest the typical symptoms of an MH crisis: impaired and spasmodic movements, tachypnea, tachycardia, difficulty in breathing, and whole-body contractions (all visual observations), which were followed by sudden death. For mice that survived the ES protocol, running was stopped when mice reached exhaustion, evaluated as the inability of the animal to maintain running speed despite prolonged contact (≥10 s) with the electric grid.

For measurements of core body temperature, the initial absolute internal temperature (t₀), measured at the beginning of the ES protocol as well as the temperature at the end (t_f) of the experiment (30th min, a nontriggering stress protocol), was recorded by using a rectal thermometer (4-channel thermometer TM-946; XS instruments, Modena, Italy).

Pretreatment with azumolene

Both RYR1^Y522S/WT and CASQ1-null mice were randomly assigned to 2 experimental groups that were subjected to the ES protocol: untreated and azumolene-treated groups. Azumolene (Santa Cruz Biotechnology, Dallas, TX, USA), a 30-fold more water soluble analog of dantrolene, was dissolved in DMSO, diluted in 0.9% NaCl injectable solution, and administrated 60 min before the ES protocol by intraperitoneal injection at the dose of 5 µg/g of body weight (40).

Assessment of rhabdomyolysis

Histologic examination

Immediately after the ES protocol, EDL muscles were carefully dissected and fixed at room temperature in 3.5% glutaraldehyde 0.1 M Na cacodylate buffer, pH 7.2, overnight. Small bundles of fixed fibers were postfixed in 2% OsO₄ in the same buffer for 2 h, then block-stained in aqueous saturated uranyl acetate. After dehydration, specimens were embedded in an epoxy resin (Epon 812; Electron Microscopy Sciences, Hatfield, PA, USA). Semithin (800 nm) sections were cut with a Leica Ultracut R Microtome (Leica Microsystems, Vienna, Austria) using a Diatome diamond knife (Diatome, Biel, Switzerland). After staining with Toluidine Blue dye, sections were viewed by using a Leica DMLB light-microscope (Leica Microsystems). The percentage of fibers that presented signs of rhabdomyolysis was determined as previously described (38). In brief, fibers were classified as follows: 1) apparently normal fibers, 2) fibers losing striation, and 3) fibers presenting contractures; data was presented as a percentage of all fibers evaluated.

Immunofluorescence analysis

EDL muscles were dissected from mice in basal conditions and immediately after ES protocol and fixed at room temperature with paraformaldehyde 2% for 2 h. Small bundles of fixed fibers were washed 3 times in phosphate buffered saline (PBS) buffer plus 1% bovine serum albumin (BSA; PBS/BSA) and incubated in blocking buffer (PBS/BSA plus 10% goat serum and 0.5% Triton X-100) for 1 h at room temperature followed by overnight incubation at 4°C with primary Abs (see below for Abs used and dilution). Bundles were then washed 3 times with PBS/BSA buffer and incubated with secondary Abs for 1 h at room temperature before being mounted on coverslips with antibleach medium (SlowFade Gold Antifade Mountant; Thermo Fisher Scientific, Waltham, MA, USA).

Primary Abs used were as follows: mouse monoclonal anti-RYR1/RYR3 34C (1:20; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA); and rabbit polyclonal anti-mitochondrial preprotein translocases of the outer membrane homolog 20, (TOM20) (1:50; Santa Cruz Biotechnology). Secondary Abs used were as follows: Cy5-labeled goat anti-mouse IgG (1:100); and Cy3-labeled goat anti-rabbit IgG (1:200) for double labeling (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Images were acquired by using a Zeiss LSM510 META laser-scanning confocal microscope system (Zeiss, Jena, Germany) that was equipped with a Zeiss Axiovert 200 inverted microscope and a Plan Neofluar oil-immersion objective (×63/1.3 NA). Negative controls for each immunostaining assay were performed by immunolabeling samples with only the secondary Abs.

Plasma and serum analyses

Plasma/serum markers of rhabdomyolysis (i.e., CK, K⁺, and Ca²⁺) were measured in control mice (not exposed to stress protocol) and in mice with a brief exposure (30 min) to a nontriggering ES challenge, as previously described (38). Blood samples were then processed for spectrophotometrical measurements using a Screen Touch Master spectrophotometer (Hospitex Diagnostic, Sesto Fiorentino, Italy).

Ex vivo and in vitro experiments

Contractile responses of EDL muscles

EDL muscles were dissected from hind limbs of mice and mounted in a myograph (Muscle Tester System, SI-H; World Precision Instruments, Heidelberg, Germany) between a force transducer (SI-H KG7B; World Precision Instruments) and a micromanipulator-controlled shaft in a small chamber and were continuously perfused with an oxygenated Krebs solution. Before the experimental protocols, stimulation conditions were optimized by increasing the muscle length until force development during tetanus was maximal. During this phase, the temperature was kept constant at 25°C. To evaluate the development of contractures that were induced by high-frequency tetanic stimulation, EDL muscles were kept at 30°C and were electrically stimulated for 20 min with a series of consecutive tetani (500-ms duration, 80 Hz for each tetanus) that was applied every 10 s (duty cycle, 0.05). Basal tension was measured every 5 min until the end of the experiment.

IVCT in intact EDL muscles

To determine the caffeine sensitivity of resting tension and the caffeine-dependent decay in twitch force, EDL muscles were subjected to a previously described protocol (38). Briefly, muscles were continuously stimulated with electrical stimulation (0.2 s at 0.2 Hz applied every 5 s; duty cycle, 0.04) at 25°C and exposed to increasing concentrations (2, 4, 6, 8, 10, 14, 18, and 22 mM) of caffeine, with changes made every 3 min (no wash between applications). To test the effect of azumolene, EDL muscles from RYR1^Y522S/WT and CASQ1-null mice were perfused with a Krebs solution that contained 50 µM of the compound.

Cytosolic Ca²⁺ measurements in isolated single FDB fibers

Caffeine-induced Ca²⁺ release was measured in single FDB fibers that were obtained according to a modified collagenase/protease method as previously described (38, 41). A minimum of 30 min was allowed for Fura-2 de-esterification before fibers were imaged. [Ca²⁺]_i transients were recorded at 25°C by using a dual-beam excitation fluorescence photometry setup (IonOptix, Westwood, MA, USA) as previously described (32, 42). Single fibers were subjected to a continuous low-frequency stimulation (0.5 Hz) protocol to evaluate myoplasmic Ca²⁺ transients at increasing caffeine concentrations. To test the effect of azumolene on caffeine-induced Ca²⁺ release, RYR1^Y522S/WT and CASQ1-null FDB fibers were incubated for 30 min with a Krebs solution that contained 50 µm azumolene before Ca²⁺ recordings.

Western blot analyses

EDL muscles were dissected from mice in basal conditions and after exertional stress, and processed for Western blot (WB) as previously described (38). Protein concentration was determined spectrophotometrically by using a modified Lowry method (43). Of total protein, 20–40 μg was resolved in 10% PAGE, transferred to nitrocellulose membrane, and blocked with 5% nonfat dry milk (EuroClone, Milan, Italy) in Tris-buffered saline 0.1% and Tween 20 (TBS-T; Sigma-Aldrich, St. Louis, MO, USA) for 1 h. Membranes were probed with the following primary Abs diluted in 5% nonfat dry milk in TBS-T overnight at 4°C: anti–3-nitrotyrosine (3-NT) Ab (mouse monoclonal 1:500; Millipore, Billerica, MA, USA); anti–phospho-NF-κB p65 Ser536 (p-p65 1:1000) and anti–NF-κB p65 subunit (p65) Ab (rabbit polyclonal 1:1000; both from Santa Cruz Biotechnology); anti–glyceraldehyde-3-phosphate dehydrogenase Ab (mouse monoclonal 1:5000; OriGene Technologies, Rockville, MD, USA), used as a loading control. Membranes were washed 3 times in TBS-T and incubated with mouse and rabbit secondary Abs (horseradish peroxidase conjugated, 1:10,000; Millipore) diluted in 5% nonfat dry milk in TBS-T for 1 at room temperature. Proteins were detected by ECL liquid (PerkinElmer, Waltham, MA, USA) and quantification was made by using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Immunofluorescence evaluation of NF-κB activation

EDL muscles were dissected from mice in basal conditions and immediately after ES protocol, fixed in paraformaldehyde 2% for 2 h, and processed for confocal microscopy (CM) acquisition as described above. To assess NF-κB activation (nuclear translocation), EDL bundles were incubated with rabbit monoclonal anti–phospho-NF-κB p65 subunit (Ser536; 1:100; Cell Signaling Technology, Danvers, MA, USA) and with an anti–Alexa Fluor 488 (1:200; Abcam, Cambridge, United Kingdom). Bundles were also labeled with anti–α-actinin sarcomeric (1:400; Sigma-Aldrich) and Cy3-labeled goat anti-rabbit IgG (1:200), and with DRAQ5 fluorescent DNA probe (1:1000; Abcam) for nuclei staining.

Calpain activity

Calpain activity was measured in muscle homogentates by chemiluminescence assay using a Calpain Protease Assay kit (Promega, Madison, WI, USA). The assay provides a proluminescent calpain substrate in a buffer system that is optimized for calpain activity and luciferase activity. During the assay, calpain cleavage of the substrate generates a glow-type luminescent signal produced by the luciferase reaction. In this homogeneous, coupled-enzyme format, the signal is proportional to the amount of calpain activity present in the sample. In this study, muscles homogenates were prepared at a concentration of 15 mg/ml 10 mM KH₂PO₄ buffer, pH 7.4, in 0.9% NaCl and processed according to manufacturer instructions. Results are presented as calpain activity expressed as relative luminescence unit per microgram of total proteins.

Statistical analyses

Statistical significance in experiments on exertion-induced mortality and the quantitative analysis of fibers that presented structural damages were evaluated by using 2-tailed Fisher’s exact test. Measurements of the time to onset of lethal crises were analyzed by using a 2-tailed unpaired Student’s t test. One-way ANOVA followed by post hoc Tukey’s test was used for statistical analyses of all other experiments, with the exception of those regarding the time courses of ex vivo experiments in which we used a repeated-measures ANOVA followed by post hoc Tukey’s test for pairwise comparisons. In all cases, differences were considered statistically significant at P < 0.05. A 2-tailed Fisher’s exact test was performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA), whereas 1-way ANOVA, repeated-measures ANOVA, and 2-tailed unpaired Student’s t tests were performed using Origin 8.0 (OriginLab, Northampton, MA, USA) software.

RESULTS

ES triggers lethal hyperthermic episodes in MH-susceptible mice

We evaluated the mortality rate of mice that were exposed to an ES protocol (i.e., strenuous exercise in challenging environmental conditions: 34°C and 40% humidity With this experimental setup, we sought to mimic the environmental conditions that are encountered in some periods of the year (i.e., summer) in some places on the earth, conditions in which heat or exertional strokes are more frequent (20). Whereas the ES protocol was well tolerated by wild-type (WT) animals, both RYR1^Y522S/WT and CASQ1-null mice showed a high rate of mortality—80 and 78% respectively (Fig. 1A and Supplemental Table S1). Two deaths in CASQ1-null mice were delayed (i.e., within 24 h after ES challenge). Of note, the crises that were observed during the ES protocol displayed clinical signs very similar to those experienced by the same animals when exposed to either halothane or environmental heat (i.e., whole-body contractures, difficulty in breathing, and impaired and spasmodic movements) (29, 33). As expected from previous studies, the phenotype was more severe in RYR1^Y522S/WT mice than in CASQ1-null mice. Indeed, the time to onset of lethal events in the RYR1^Y522S/WT mouse strain was significantly shorter than in CASQ1-null mice: 31.9 ± 2.6 min vs. 43.0 ± 1.1 min, respectively, on the average (Fig. 1B). To test whether the lethal episodes induced by ES protocol share common features with MH reactions induced by anesthetics and heat (29, 33), we treated mice with a single injection of azumolene (a dantrolene analog) 60 min before the ES protocol. This pretreatment was effective in preventing exertional strokes: the mortality rate was indeed (almost) completely abolished in both mouse models, with only 1 CASQ1-null mouse that died within 24 h after ES challenge (Fig. 1A and Supplemental Table S1).

Figure 1.

Mortality rate and core temperature of mice during the ES protocol. A) Incidence of sudden (black) and delayed (gray) deaths during ES protocol (see also Supplemental Table S1). B) Time to onset of lethal crises in MH-susceptible mice (measured from the beginning of the ES protocol). C, D) Changes in absolute (C) and relative (ΔT) (D) core temperature during ES protocol, measured at the beginning (t₀) and at the end (t_f) of the experiment (see also Supplemental Table S2). Data in B–D are given as means ± sem. Azu, azumolene; Y522S, RYR1^Y522S/WT. *P < 0.05 (compared with azumolene-treated mice; C, D).

As typical MH crises in humans are characterized by an uncontrolled rise in body temperature, namely hyperthermia (20), we measured the core temperature at the beginning (t₀) and at the end (t_f) of the experiment in all mice that were subjected to the ES protocol. Core temperature, as reported in Fig. 1C, D as both absolute and relative temperature, increased in all animals during the ES protocol, including WT mice (ΔT = 2.9 ± 0.3°C), but more significantly in RYR1^Y522S/WT and CASQ1-null mice (ΔT = +5.4 ± 0.3°C and +5.0 ± 0.2°C, respectively; Supplemental Table S2). Of importance, pretreatment with azumolene was effective in reducing the rise in core temperature in both RYR1^Y522S/ and CASQ1-null mice (ΔT = +3.8 ± 0.3°C and ΔT = +3.4 ± 0.2°C, respectively; Fig. 1C, D and Supplemental Table S2).

ES causes rhabdomyolysis in muscle of MH-susceptible mice

Rupture of skeletal muscle fibers (i.e., rhabdomyolysis) is a typical clinical sign of MH and HS episodes, and causes the release of various molecules into the blood stream (23, 25, 44, 45). Here, we assessed the structural damage in skeletal muscle before and after the ES protocol by: 1) qualitative evaluation of CM images obtained on small bundles of EDL fibers labeled with primary Abs against RYR1, marking the position of calcium release units, and against the TOM20, marking the position of mitochondria (Fig. 2A–E and Supplemental Fig. S1); 2) measuring plasma and serum levels of CK, K⁺, and Ca²⁺ (Fig. 2F–H and Supplemental Fig. S1); and 3) quantifying the percentage of damaged fibers in histologic sections (Fig. 3). In mice that were not exposed to the ES protocol, immunolabeling of EDL fibers indicated that cross-striation is well preserved in WT and CASQ1-null mice, whereas some disarray is present in RYR1^Y522S/WT samples (Supplemental Fig. S1A–C). Abnormalities in adult RYR1^Y522S/WT fibers were previously reported (i.e., the presence of unstructured and contracture cores in approximately one third of fibers (46). Following ES protocol, most fibers from RYR1^Y522S/WT and CASQ1-null mice showed a severe disarray of both mitochondrial and EC coupling apparatuses (visible as compromised cross striation), whereas structure was less affected in WT mice (Fig. 2A–C). Structural damage involved mainly the formation of large contractures (Fig. 2B, asterisks) and loss of striation (Fig. 2C, dashed oval).

Figure 2.

Immunofluorescence of EDL fibers and blood levels of CK, K⁺, and Ca²⁺ after the ES protocol. A–E) Double immunolabeling of EDL muscle fibers with Abs against RYR1 (red) and TOM20 (green) in samples from WT (A), RYR1^Y522S/WT (Y522S; B), and CASQ1-null (C) mice and azumolene (Azu)-treated Y522S (D) and CASQ1-null (E) mice. Asterisks (B) and dashed oval mark (C) indicate contractures and a fiber losing cross-striation, respectively. F–H) Blood levels of CK in serum (F) and K⁺ (G) and Ca²⁺ (H) in plasma, collected from the 5 groups of animals after ES protocol (see also Supplemental Fig. S1 for immunofluorescence and blood analysis in control conditions). Data are given as means ± sem. Scale bars, 10 µm (insets, 5 µm). *P < 0.05.

Figure 3.

Quantitative analysis in histologic sections of EDL fibers presenting structural damage following the ES protocol. A–C) Histologic examination of EDL muscles after ES protocol allowed classification of fibers in 3 main classes: fibers with no apparent sign of damage (A); fibers losing striation (dashed oval; B); and fibers with contractures (asterisks; C). D) Quantitative analysis showing the percentage of EDL fibers presenting the different features classified in A–C. White, fibers with no apparent damage; gray, fibers losing striation; black, fibers with contractures (also see Supplemental Table S3). Azu, azumolene; Y522S, RYR1^Y522S/WT. Scale bar, 10 µm. *P < 0.05.

Blood analysis supported the presence of muscle damage after the ES protocol. Indeed, although no major differences were found between different groups of mice not exposed to the ES protocol, a slight but significant increase in CK levels was reported in samples from RYR1^Y522S/WT mice (Supplemental Fig. S1D–F). After the ES protocol, plasma and serum levels of CK, K⁺, and Ca²⁺ were all increased in untreated RYR1^{Y522S /WT} and CASQ1-null mice compared with WT mice (Fig. 2F–H). Data collected from mice that were pretreated with azumolene confirmed the findings obtained in CM acquisitions, showing a clear protective effect of azumolene also on rhabdomyolysis. Indeed, structural damage was prevented in RYR1^{Y522S /WT} and CASQ1-null mice (Fig. 2D, E), and blood levels of CK, K⁺, and Ca²⁺ were decreased to levels more similar to those of WT mice subjected to an identical ES protocol (Fig. 2F–H).

In histologic sections, we quantified the percentage of fibers that were affected by structural damage after the ES protocol (Fig. 3 and Supplemental Table S3). In this analysis, EDL fibers were classified in 3 main groups that presented the typical features encountered after the ES protocol: 1) fibers with no apparent damage (Fig. 3A); 2) fibers losing striation (Fig. 3B, dashed oval); and 3) fibers with evident contractures (Fig. 3C, asterisks). After the ES protocol, fibers that were losing striation were also frequent in WT muscles (∼41%; gray in Fig. 3D). The main difference between WT and both MH-susceptible mice was the percentage of fibers that presented evident contractures. Indeed, whereas fibers that presented contractures were rare in WT mice (12%; black in Fig. 3D), in RYR1^Y522S/WT and CASQ1-null EDL muscles, 92 and 40%, respectively, of fibers presented large areas in which sarcomeres were abnormally shortened (Fig. 3C, D; see Supplemental Table S3 for additional detail). To this quantitative assessment, we should add that the extent of the fiber area that was affected by loss of striation and contractures was usually confined to small regions in WT, but was more extended in MH-susceptible animals. The protective effect of azumolene was also confirmed by histologic analysis. Indeed, pretreatment of mice significantly reduced the percentage of fibers that presented structural damage in both RYR1^Y522S/WT (from 92 to 22% of fibers with contractures) and CASQ1-null mice (from 39 to 10% of fibers with contractures; Fig. 3D and Supplemental Table S3). Of note, after treatment with azumolene, the number of fibers with contractures in both RYR1^Y522S/WT and CASQ1-null mice was similar to that observed in WT mice.

EDL muscles and FDB fibers from MH-susceptible mice show increased responsiveness to both electrical stimulation and caffeine

EDL muscles that were isolated from WT, RYR1^Y522S/WT, and CASQ1-null mice were subjected to a high-frequency tetanic stimulation protocol (120 × 500 ms, 80-Hz tetani applied every 10 s; duty cycle, 0.05; 20 min) that was designed to reproduce the exertion of muscle ex vivo (Fig. 4A–C). When exposed to this prolonged protocol, WT muscles only showed a slight increase in basal tension toward the middle of the experiment (after approximately 10 min from start; Fig. 4A, B). Conversely, EDL muscles that were excised from RYR1^Y522S/WT and CASQ1-null mice showed a greater rise in basal tension that started earlier than in WT mice (approximately 3 and 5 min from start, respectively), which resulted in the development of muscle contractures in the second part of the experiment (Fig. 4A, B). Azumolene added in the Krebs solution at the concentration of 50 µM was effective in reducing this increase in basal tension during the stimulation, significantly in RYR1^Y522S/WT and completely in CASQ1-null muscles (Fig. 4A, B). The bar plot in Fig. 4C shows the specific basal tension at the end of the experiments (i.e., 20 min).

Figure 4.

Responsiveness of EDL muscles and FDB fibers to electrical stimulation and caffeine. A, B) Average basal tension during high-frequency (80 Hz) electrical stimulation in intact EDL muscles from RYR1^Y522S/WT (Y522S; A) and CASQ1-null (B) mice in the absence or presence of 50 µM azumolene (Azu). C) Specific basal tension (mN/mm²) recorded at the end of the experiment (i.e., 20 min). D, E) Average basal tension during an IVCT performed exposing EDL muscles from Y522S (D) and CASQ1-null (E) mice to increasing caffeine concentrations in the absence or presence of 50 µM Azu. F) Specific basal tension (mN/mm²) recorded at the end of the experiments (i.e., 22 mM of caffeine). G, H) Average Fura-2 fluorescence ratio curves, normalized to 0 mM of caffeine, recorded in single FDB fibers from Y522S (G) and CASQ1-null (H) mice in the absence or presence of 50 µM Azu. I) Myoplasmic Ca²⁺ levels recorded at the end of the experiment (i.e., 10 mM of caffeine). Data are given as means ± sem. *P < 0.05 vs. WT and/or Azu-treated samples, as inidcated.

Caffeine, an RYR agonist, is commonly used in IVCT, the gold standard for the diagnosis of MH susceptibility in humans (12, 13). Here, we tested the contractile response of EDL muscles (Fig. 4D–F) and measured the myoplasmic Ca²⁺ levels in single FDB fibers (Fig. 4G–I) as a response to increasing caffeine concentrations. Both RYR1^Y522S/WT and CASQ1-null EDL muscles displayed a greater caffeine sensitivity than WT, as shown by the development of tension at lower caffeine concentrations (Fig. 4D, E). We also analyzed the caffeine-dependent decay of twitch force, which was recorded by applying depolarizing pulses of 0.2 Hz every 5 s (Supplemental Fig. S2). Whereas WT EDL muscles showed a clear potentiation in the first part of the experiments, followed by minimal decay, in RYR1^Y522S/WT and CASQ1-null muscles, twitch tension displayed a progressive drop that started at the beginning of the experiments (Supplemental Fig. S2). In the presence of 50 µM azumolene, both the increase in caffeine sensitivity of EDL resting tension (Fig. 4D, E) and the caffeine-dependent decline in twitch force (Supplemental Fig. S2) were significantly rescued in both RYR1^Y522S/WT and CASQ1-null EDL muscles. The bar plot in Fig. 4F shows the specific basal tension at the end of the experiments (i.e., 22 mM caffeine).

As excessive basal tension and the development of full contractures (Fig. 4A–F) are indicative of abnormalities in [Ca²⁺]_i handling, we also measured the caffeine dependence of SR Ca²⁺ release in enzymatically dissociated single FDB fibers, loaded with the ratiometric Ca²⁺ dye Fura-2 (Fig. 4G–I). An increase in myoplasmic Ca²⁺ levels occurred in all specimens when caffeine concentration increased from 0 to 10 mM, although this increase was markedly more pronounced in both RYR1^Y522S/WT and CASQ1-null FDB fibers than in WT (Fig. 4G, H). As expected from the IVCT experiments in EDLs (Fig. 4D–F), azumolene strongly reduced the caffeine dependence of SR Ca²⁺ release in both RYR1^Y522S/WT and CASQ1-null FDB fibers (Fig. 4G, H). The bar plot in Fig. 4I shows the myoplasmic Ca²⁺ levels at the end of the experiment (i.e., 10 mM caffeine).

During the ES protocol, calpain activity is elevated in muscles from MH-susceptible mice

As excessive myoplasmic Ca²⁺ concentration activates calpains, a class of Ca²⁺-dependent nonlysosomal proteases that cleave a variety of substrates, including several sarcomeric proteins (47, 48), we evaluated whether calpain-mediated degradation could contribute to the ultrastructural alterations observed in RYR1^Y522S/WT and CASQ1-null muscle fibers during exertion-induced rhabdomyolysis. We evaluated total calpain activity by using a luminescence assay in muscle homogenates from control animals (WT, RYR1^Y522S/WT, and CASQ1-null mice in basal conditions; Fig. 5A), mice that were subjected to the ES protocol (Fig. 5B), and MH-susceptible mice that were pretreated with azumolene and subjected to the ES protocol (Fig. 5B). Already in basal conditions, total calpain activity (normalized on the total protein concentration) was ∼2-fold higher in muscles from RYR1^Y522S/WT and CASQ1-null mice than in muscles from WT specimens (Fig. 5A). After ES protocol, although calpain activity also increased in WT, muscles from both RYR1^Y522S/WT and CASQ1-null mice displayed a greater elevation (Fig. 5B). Consistent with the ability of azumolene to reduce the levels of myoplasmic Ca²⁺ concentration (Fig. 4G–I), total calpain activity was significantly decreased by pretreatment of MH-susceptible mice before the ES protocol (Fig. 5B).

Figure 5.

Total calpain activity in EDL muscle homogenates in basal condition and after the ES protocol. Calpain activity, expressed as relative luminescence units normalized to the total protein content (µg), measured in muscle homogenates from mice in basal conditions (A) and from mice subjected to the ES protocol untreated or pretreated with azumolene (B). Azu, azumolene; Y522S, RYR1^Y522S/WT. Data are given as means ± sem. *P < 0.05.

Levels of 3-NT, a marker of oxidative stress, are elevated during the ES protocol in muscles from MH-susceptible mice

Previous studies in RYR1^Y522S/WT and CASQ1-null mice demonstrated that, during an MH crisis triggered by exposure to either halothane or heat, ROS and RNS had a pivotal role in promoting a feed-forward mechanism that led to excessive SR Ca²⁺ release, hypercontracture, and rhabdomyolysis of muscle fibers (31, 38). To verify whether nitro-oxidative stress was also elevated during lethal crises induced by physical exertion, we evaluated the levels of 3-NT, a product of nitration of tyrosine residues in proteins mediated by RNS (49). Measurements of 3-NT expression levels were performed in homogenates from EDL muscles of control animals (WT, RYR1^Y522S/WT, and CASQ1-null mice in basal conditions), mice subjected to the ES protocol, and MH-susceptible mice that were pretreated with azumolene and subjected to the ES protocol (Fig. 6). WB analyses revealed that, already in basal conditions, the levels of 3-NT were significantly higher in the muscles of MH-susceptible mice (both RYR1^Y522S/WT and CASQ1-null) than in WT (Fig. 6A, B). After strenuous exercise, 3-NT levels increased in all specimens (including WT), but elevation in MH-susceptible mice reaches higher values compared with WT control mice (Fig. 6A, C). Pretreatment with azumolene in MH-susceptible mice was effective in controlling the increase in 3-NT levels during exposure to the ES protocol (Fig. 6A, C).

Figure 6.

3-NT levels in EDL muscles in basal condition and after ES protocol. A) Representative immunoblot showing expression levels of 3-NT in EDL muscles from control mice (Ctrl, basal conditions), mice that were subjected to the ES protocol (ES), and MH-susceptible mice that were pretreated with azumolene (Azu) before exposure to the ES protocol. B, C) Relative band densities expressed as 3-NT/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio in basal conditions (B) and after exposure to the ES protocol in mice untreated or pretreated with Azu (C). Y522S, RYR1^Y522S/WT. Data are given as means ± sem. *P < 0.05.

Exposure to ES protocol induced NF-κB activation

NF-κBs are a family of transcription factors (5 members) that regulate expression of several genes that are involved in proinflammatory response and cell survival, which can be activated by different stimuli, including biomechanical and oxidative stresses (50, 51). The most common NF-κB form in cells is the p65/p50 heterodimer that normally resides in the cytosol in an inactive form bound to inhibitory proteins, but that, once activated, allows phosphorylation of the p65 subunit and nuclear translocation of the p65/p50 complex. Here, we measured the activation of the NF-κB p65 subunit (p65) in EDL muscles of WT, RYR1^Y522S/WT, and CASQ1-null mice in control animals (i.e., basal conditions), mice that were subjected to the ES protocol, and MH-susceptible mice that were pretreated with azumolene and subjected to the ES protocol. Specifically, we measured the expression levels of phospho-p65 (p-p65), the activated form, in WB experiments (Fig. 7), and evaluated its translocation from the myoplasm to the nucleus by immunofluorescence combined with CM acquisitions (Supplemental Fig. S3). WB analysis indicated that, already in basal conditions, the amount of p-p65, normalized to total p65, was significantly higher in muscles of both RYR1^Y522S/WT and CASQ1-null mice compared with WT (Fig. 7A, B). After ES protocol, levels of p-p65 increased in all specimens (including WT), with levels remaining significantly greater in MH-susceptible mice (Fig. 7A, C). Pretreatment with azumolene significantly reduced the amount of p-p65 in MH-susceptible mice to levels similar to those of WT mice exposed to the same ES protocol (Fig. 7A, C). WB results are supported by data collected in CM (Supplemental Fig. S3), showing that EDL fibers from RYR1^Y522S/WT and CASQ1-null mice exhibited higher nuclear fluorescence signal levels of p-p65 than did WT muscle fibers, either in basal conditions (Supplemental Fig. S3A) or after the ES protocol (Supplemental Fig. S3B). Of interest, nuclear p-p65 signal was significantly reduced in samples from MH-susceptible animals that were pretreated with azumolene (Supplemental Fig. S3C).

Figure 7.

NF-κB p65 subunit (p65) activation in EDL muscles in basal condition and after ES protocol. A) Representative immunoblot showing expression levels of phospho-p65 (p-p65) and total p65 in EDL muscles from control mice (Ctrl, basal conditions), mice that were subjected to the ES protocol (ES), and MH-susceptible mice that were pretreated with azumolene (Azu) before exposure to the ES protocol (samples for these experiments were from mice also used in Fig. 6B, C). B, C) Relative band densities, expressed as p-p65/p65 ratio in basal conditions (B) and after exposure to the ES protocol in mice untreated or pretreated with Azu (C). Y522S, RYR1^Y522S/WT. Data are given as means ± sem. *P < 0.05.

DISCUSSION

Life-threatening HS episodes, triggered by either environmental heat or physical exertion, are characterized by hyperthermia and rhabdomyolysis (20–23, 25), a pathophysiologic framework also typical of anesthetic-induced MH crises. These similarities have suggested, already several years ago, that MH and HS may be related syndromes triggered by different stimuli (52). However, this correlation is not widely recognized. Studies conducted in animal models have demonstrated that hyperthermic crises, which share common molecular mechanisms, are triggered by both anesthetics and environmental heat (27–30, 33). Here, we tested in RYR1^Y522S/WT and CASQ1-null mice (mouse models of anesthetic- and heat-induced MH crises) whether strenuous physical exertion may also represent a stimulus capable of triggering lethal episodes in these mice, and whether overheating episodes triggered by exertion share common molecular mechanisms with classic anesthetic-induced MH crises.

Main findings

When exposed to the ES protocol, both RYR1^Y522S/WT and CASQ1-null mice show a mortality rate of approximately 80%, similar to that observed when the same mice were exposed to halothane (the triggering agent in humans) or heat (Fig. 1A) (29, 33, 38). The exertion-induced lethal episodes exhibit a clinical presentation similar to that observed during classic anesthetic-induced MH episodes in humans: difficulty in breathing, impaired and spasmodic movements, diffuse skeletal muscle rigidity (visual observations), hyperthermia (Fig. 1C, D), and, finally, rhabdomyolysis (Figs. 2 and 3), all features indicative of abnormal skeletal muscle function. Whether cardiac muscle, which expresses low levels of RYR1 (53), could be a contributor to sudden death in MH-susceptible mice (at least in RYR1^Y522S/WT mice) deserves further investigation. However, the current view in the MH field is that both anesthetic-induced MH and exertion-induced HS (either in humans or animal models) are the result of rhabdomyolysis of skeletal fibers, which causes an abnormal increase in ions (i.e., K⁺) and proteins (i.e., CK and myoglobin) in the bloodstream that then may determine cardiac arrhythmia (and even arrest) and/or kidney failure (3, 15, 24, 25, 44, 45).

Although the incidence of lethal events induced by physical exertion is similar in the 2 strains of mice, the time to onset of lethal crisis (Fig. 1B), the blood markers of rhabdomyolysis (Fig. 2F–H), and the number of fibers that present structural damage (Fig. 3D) all indicate that the phenotype of RYR1^Y522S/WT mice is more severe than that of CASQ1-null mice. Of importance, pretreatment of MH-susceptible mice with azumolene before the ES protocol, an analog of dantrolene, the only drug clinically available to treat acute MH episodes in humans (9), abolished (almost) completely the sudden deaths of MH-susceptible mice by reducing hyperthermia and muscle damage (Figs. 1–3). The ability of azumolene to prevent exertional strokes in vivo is likely mediated by normalization of [Ca²⁺]_i levels. Indeed, during IVCT performed on intact EDL muscles or single FDB fibers (exposed to a high-frequency electrical stimulation or to increasing caffeine concentrations), azumolene strongly reduces the increase in [Ca²⁺]_i and the consequent development of contractures in samples from RYR1^Y522S/WT and CASQ1-null mice (Fig. 4).

Possible molecular mechanisms underlying rhabdomyolysis during strenuous exercise

In Fig. 8, we propose a mechanistic model of the possible cellular and molecular events that lead to rhabdomyolysis of skeletal muscle fibers during MH/HS crises on the basis of previous findings and the present work. Muscle fibers from both RYR1^Y522S/WT and CASQ1-null mice display an excessive leak of Ca²⁺ from the SR already in basal conditions, namely, without exposure to environmental triggers (29, 33). This Ca²⁺ leak in RYR1^Y522S/WT mice is the direct result of the gain-of-function point mutation (a substitution of a tyrosine with a serine in position 522), whereas in CASQ1-null mice, hyperactivity of RYR1 would likely be the consequence of a lack of CASQ1 inhibition on the RYR1 open state (54). How halogenated anesthetics, environmental heat, and exertion exacerbate SR Ca²⁺ leak during MH reactions remains to be determined. It is possible that halogenated anesthetics directly influence the opening probability of RYR1 (55), whereas environmental heat and exertion could increase the opening probability of RYR1 by thermodynamic modification of the channel conformation.

Figure 8.

Schematic model illustrating the possible molecular pathways underlying muscle fiber rhabdomyolysis during ES. During exposure to strenuous exercise, excessive SR Ca²⁺ release (Fig. 4) and overproduction of oxidative species (Fig. 6) likely promote a feed-forward mechanism (31), which causes the generation of abnormal muscle tension (Fig. 4) and damage/rhabdomyolysis of muscle fibers (Figs. 2 and 3), possibly as the consequence of enhanced activation of calpains and NF-κB pathways (Figs. 5 and 7).

Whereas excessive SR Ca²⁺ leak is widely recognized as the initial event in MH reactions, other important factors may come into play during the cascade of events that lead to rhabdomyolysis of fibers:

1. Several publications point to excessive oxidative stress as being a main contributor factor (31, 38, 39, 56), as enhanced production of ROS and RNS during MH crises, at least in RYR1^Y522S/WT mice, results in a dangerous feed-forward cycle that involves RYR1 S-nitrosylation/glutathionylation, which, in turn, increases the opening probability of the channel (31). In line with these findings, we showed that treatment with antioxidants successfully prevented lethal crises in CASQ1-null mice (38). In the present work, we have shown that levels of 3-NT were significantly elevated in muscles of MH-susceptible mice, both in basal conditions and after ES (Fig. 6). The excessive oxidative stress could be the result of a chronic hypermetabolic state of skeletal fibers, possibly resulting from an increased ATP demand caused by hyperactive Ca²⁺ pumps that need to remove excessive myoplasmic Ca²⁺ (by reuptake into the SR or extrusion in the extracellular space).

2. Ca²⁺ influx from the extracellular space could also play a role in the complex events that lead to MH crises. Indeed, IVCT, the diagnostic method used to assess/confirm MHS, does not work in the absence of extracellular Ca²⁺ (57–59), which suggests that Ca²⁺ influx could contribute to muscle contracture and rhabdomyolysis. Of note, recent evidence suggests that store-operated calcium entry (SOCE), the mechanism that allows Ca²⁺ influx after depletion of intracellular stores (i.e., SR), is significantly enhanced in human skeletal fibers from patients with MH (60) and also in fibers from both RYR1^Y522S/WT and CASQ1-null mice (61). We also reported that in CASQ1-null mice, SR undergoes deep depletion during high-frequency stimulation (42), a stimulus that, in principle, could activate entry of extracellular Ca²⁺ during ES. As we have no data on SR depletion in RYR1^Y522S/WT fibers, further investigation is required to determine the possible role of SOCE in hyperthermic episodes of RYR1^Y522S/WT and CASQ1-null mice.

Azumolene prevents ES crises: how?

The mechanism by which dantrolene prevents MH reactions is still controversial. Many studies have suggested that dantrolene has a direct effect on the release of Ca²⁺ from SR (62, 63), likely by binding to a specific RYR1 site, 590–609 aa (64), which may stabilize the closed state of the channel (65, 66). Other studies have suggested that dantrolene blocks excitation-coupled Ca²⁺ entry while failing to observe a direct effect of dantrolene on RYR1 in double-lipid bilayers (67). The therapeutic action of dantrolene and its analog azumolene could also arise by their capability to inhibit Ca²⁺ influx via the SOCE pathway (68, 69). However, RYR1-coupled SOCE inhibition by azumolene does not occur when the drug is applied after caffeine/ryanodine treatment or before thapsigargin-induced store depletion (69), which strongly suggests that the inhibitory effect of azumolene (and, by extension, of dantrolene) on SOCE may indeed result from its ability to bind the closed state of RYR1 (66). Finally, other studies concluded that azumolene is not a SOCE inhibitor, because azumolene failed to inhibit both the magnitude and activation rate of SOCE currents in myotubes from WT and MH mice (i.e., RYR1^Y524S/WT and double CASQ-null) (61).

Results from the present study show that 50 µM azumolene is able to reduce the occurrence of contractures during electrical stimulation or caffeine IVCT in intact EDL muscles from RYR1^Y522S/WT and CASQ1-null mice (Fig. 4A–F). Consistent with IVCT experiments, the presence of azumolene also limits the abnormal rise in myoplasmic Ca²⁺ concentrations in single FDB fibers from MH-susceptible mice (Fig. 4G–I). Although azumolene has a potent effect in reducing the excessive accumulation of Ca²⁺ in the myoplasm, these data do not provide conclusive evidence for the molecular action of azumolene. However, one interesting observation comes from the time course of the caffeine-dependent decay of twitch force: whereas WT EDL muscles show a clear potentiation in the first part of the experiments followed by minimal decay, both RYR1^Y522S/WT and CASQ1-null muscles display a fast drop of twitch force from the beginning of the protocol (Supplemental Fig. S2). This behavior could be explained by a rapid caffeine-induced SR Ca²⁺ depletion, consistent with previous observations in CASQ1-null FDB fibers showing a fast electrically induced SR depletion (42). As azumolene significantly reduces the caffeine-dependent decay of twitch force (Supplemental Fig. S2), we could speculate that this drug prevents SR depletion by directly acting on RYR1, as proposed in Fig. 8.

Dysfunctional Ca²⁺ handling, oxidative stress, and fiber damage: which cellular pathways are involved?

How excessive Ca²⁺ leak (and/or entry) and oxidative stress during MH episodes lead to structural damage and disarray of contractile elements is still unclear. Early studies that proposed that excessive increase of [Ca²⁺]_i concentration is responsible for muscle damage, mitochondrial swelling, and degeneration of myofibrils are present in the literature (70). Several studies have demonstrated that calpains, one of the most important Ca²⁺-dependent proteolytic pathways in skeletal muscle fibers, is involved in a large number of physiologic and pathologic processes in skeletal muscle (71–73). For example, in dystrophic fibers (74, 75), the dysregulated activation of the calpain proteolytic pathway could be the result of the excessive myoplasmic Ca²⁺ concentration. Consistent with these results, in muscles from both RYR1^Y522S/WT and CASQ1-null mice, [Ca²⁺]_i levels are elevated and calpain activity is significantly higher than in WT, either in basal condition or after ES protocol (Fig. 5). In the mechanistic model shown in Fig. 8, we proposed that both the Ca²⁺ ions released from SR and those coming from the extracellular space (possibly via SOCE) may trigger an uncontrolled increase in calpain activity that, in turn, by degrading specific sarcomeric proteins, could be responsible for the disruption of the myofibrillar architecture. In support of the idea that Ca²⁺-dependent activation of calpains could be involved in the series of cellular events that underlie rhabdomyolysis during MH reactions, we also show that pretreating RYR1^Y522S/WT and CASQ1-null animals with azumolene, which prevents the abnormal increase of myoplasmic Ca²⁺ levels (Fig. 4G–I), total calpain activity was brought back to WT values (Fig. 5). Calpain-dependent proteolytic pathways could also be activated by other means, as excessive oxidative stress could lead to oxidative modification of muscle proteins, unfolding, and enhanced proteolytic degradation rate (76, 77).

Confirming the strict link between impaired Ca²⁺ handling, oxidative stress, skeletal muscle protein degradation, and rhabdomyolysis during exertional HS, our data also show that in muscle fibers from both RYR1^Y522S/WT and CASQ1-null mice, the NF-κB p65 subunit is significantly more activated than in WT, either in basal condition or after ES protocol (Fig. 7 and Supplemental Fig. S3A, B). This transcription factor is known to regulate the expression of several genes that are involved in the proinflammatory response and cell survival, which can be activated by different stimuli, including exposure to proinflammatory cytokines and biomechanical and oxidative stresses (50, 51), all factors that are elevated during exertional stress. NF-κB has been previously proposed to play a pivotal role also in Duchenne muscular dystrophy, a myopathy in which both [Ca²⁺]_i concentration and oxidative stress are elevated (78–80), as in our MH animal models. Our results also show that reduction of myoplasmic Ca²⁺ levels and oxidative stress in azumolene-treated animals also significantly reduced NF-κB activation (Fig. 7 and Supplemental Fig. S3C).

CONCLUSIONS

Our study indicates that strenuous physical exertion triggers lethal episodes in MH-susceptible mice and that these episodes share common features with MH episodes triggered by anesthetics and heat (i.e., hyperthermia and rhabdomyolysis). The fact that azumolene prevents both anesthetic- and exertion-induced episodes in RYR1^Y522S/WT and CASQ1-null mice represents a further indication that common pathophysiologic mechanisms underlie both disorders, at least in mice. Our findings also provide additional elements of discussion: MH-susceptible individuals could, in principle, be considered at risk for exertion-induced episodes; and drugs that are commonly used to treat classic anesthetic MH reactions (i.e., dantrolene) could be considered as possible pharmacologic interventions for acute HS, a class of disorders that currently has no cure.

AUTHOR CONTRIBUTIONS

F. Protasi conceived of and directed the study; A. Michelucci, C. Paolini, S. Boncompagni, M. Canato, and C. Reggiani performed the experimental work and data analysis; and A. Michelucci, S. Boncompagni, and F. Protasi wrote the manuscript.

Glossary

3-NT

3-nitrotyrosine

BSA

bovine serum albumin

CASQ1

calsequestrin type-1

CASQ1-null

CASQ1-knockout

CK

creatine kinase

CM

confocal microscopy

EC

excitation-contraction

EDL

extensor digitorum longis

ES

exertional-stress

FDB

flexor digitorum brevis

HS

heat stroke

IVCT

in vitro contracture test

MHS

malignant hyperthermia susceptibility

PBS

phosphate buffered saline

RNS

reactive nitrogen species

ROS

reactive oxygen species

RYR

ryanodine receptor

SOCE

store-operated calcium entry

SR

sarcoplasmic reticulum

TBS-T

Tris-buffered saline 0.1% and Tween 20

TOM20

translocase of the outer mitochondrial membrane homolog 20

WB

Western blot

WT

wild type