Mice lacking calsequestrin-1 (CASQ1-null), a Ca2+-binding protein that modulates the activity of Ca2+ release in the skeletal muscle, exhibit lethal hypermetabolic episodes that resemble malignant hyperthermia in humans when exposed to halothane or heat stress.
Because oxidative species may play a critical role in malignant hyperthermia crises, we treated CASQ1-null mice with two antioxidants, N-acetylcysteine (NAC, Sigma-Aldrich, Italy; provided ad libitum in drinking water) and (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, Sigma-Aldrich; administered by intraperitoneal injection), before exposure to halothane (2%, 1 h) or heat (41°C, 1 h).
NAC and Trolox significantly protected CASQ1-null mice from lethal episodes, with mortality being 79% (n = 14), 25% (n = 16), and 20% (n = 5) during halothane exposure and 86% (n = 21), 29% (n = 21), and 33% (n = 6) during heat stress in untreated, NAC-treated, and Trolox-treated mice, respectively. During heat challenge, an increase in core temperature in CASQ1-null mice (42.3° ± 0.1°C, n=10) was significantly reduced by both NAC and Trolox (40.6° ± 0.3°C, n = 6 and 40.5° ± 0.2°C, n = 6). NAC treatment of CASQ1-null muscles/mice normalized caffeine sensitivity during in vitro contracture tests, Ca2+ transients in single fibers, and significantly reduced the percentage of fibers undergoing rhabdomyolysis (37.6 ± 2.5%, 38/101 fibers in 3 mice; 11.6 ± 1.1%, 21/186 fibers in 5 mice). The protective effect of antioxidant treatment likely resulted from mitigation of oxidative stress, because NAC reduced mitochondrial superoxide production, superoxide dismutase type-1 expression, and 3-nitrotyrosine expression, and increased both reduced glutathione and reduced glutathione/oxidized glutathione ratio.
These studies provide a deeper understanding of the mechanisms that underlie hyperthermic crises in CASQ1-deficient muscle and demonstrate that antioxidant pretreatment may prevent them.
Antioxidant pretreatment with N-acetylcysteine and Trolox protected calsequestrin-1-null mice from otherwise lethal episodes triggered by exposure to either halothane or heat stress. The results support evidence for a potential role of oxidative stress in life-threatening events of malignant hyperthermia and heat stress.
Malignant hyperthermia (MH) remains a rare yet potentially devastating event related to anesthetic exposure.
Most human MH susceptibility is linked to mutations in ryanodine receptor type-1(RYR1), the gene encoding the sarcoplasmic reticulum Ca2+-release channel of skeletal muscle.
Calsequestrin-1 (CASQ1) is a primary calcium-binding protein in the sarcoplasmic reticulum that interacts and modulates RYR1 calcium-release channel activity.
Mice lacking CASQ1 exhibit an MH-susceptible and environmental heat stress–susceptible phenotype.
Antioxidant pretreatment with N-acetylcysteine (Sigma-Aldrich, Italy) and Trolox (Sigma-Aldrich) protected calsequestrin-1-null mice from otherwise lethal episodes triggered by exposure to either halothane or heat stress. The results support evidence for a potential role of oxidative stress in life-threatening events of malignant hyperthermia and heat stress.
In humans, malignant hyperthermia susceptibility (MHS) is an autosomal dominantly inherited pharmacogenetic disorder most often linked to mutations in ryanodine receptor type-1 (RYR1), the gene that encodes the sarcoplasmic reticulum (SR) Ca2+-release channel of the skeletal muscle.1–3 Individuals are diagnosed as MHS if they have experienced a malignant hyperthermia (MH) crisis during anesthesia4,5 and/or score positive in a diagnostic in vitro caffeine–halothane contracture test.6,7 In some cases, life-threatening hyperthermic episodes have been reported in individuals in the absence of anesthetics during exposure to extreme environmental heat, physical exertion, or even during febrile illness.8 Hyperthermic crises triggered by high temperatures and humidity are collectively known as environmental heat stroke (EHS). EHS crises exhibit a clinical presentation similar, but not identical, to that of MH: increased internal temperature, rhabdomyolysis, hypermetabolism, cyanosis, and increased serum levels of potassium and creatine kinase (CK).9,10 Interestingly, some EHS patients have a family history of MH11 and an association between RYR1 variants and exertional- or stress-induced sudden death (or rhabdomyolysis) have been reported.12–14 Although hypothesized on occasion in the literature,10–12 the similar presentation and pathophysiologic mechanisms shared between MHS and EHS is still not widely recognized.
Animal studies support the hypothesis of MHS and EHS being related disorders: in porcine stress syndrome,15,16 pigs carrying a point mutation in RYR1 undergo MH episodes in response to halothane exposure, but can also trigger during heat challenge or emotional/physical stress. In addition, knockin mice carrying point mutations in RYR1 linked to MHS in humans also exhibit heat- and anesthetic-induced MH episodes.17,18
We previously reported that male mice lacking calsequestrin-1 (CASQ1), the primary Ca2+-binding protein in the SR that interacts and modulates RYR1 Ca2+-release channel activity, exhibit lethal anesthetic- and heat-induced hyperthermic episodes19,20 similar to that observed in RYR1 knockin mice and MHS/EHS humans. Specifically, muscle fibers from CASQ1-knockout (CASQ1-null) mice exhibit impairments in Ca2+ handling consistent with their enhanced heat and anesthetic sensitivity: (a) resting Ca2+ levels are slightly increased at physiologic temperature (37°C) because of enhanced SR Ca2+ leak,19 (b) total SR Ca2+ store content is greatly reduced,21 and (c) SR depletion during repetitive stimulation is accelerated.22–24 However, a recent study failed to identify any mutations in the CASQ1 gene in MHS individuals within the North American population.25
Ca2+-dependent production of reactive oxygen/nitrogen species (ROS/RNS) has been proposed to play a crucial role in the cascade of events leading to enhanced SR Ca2+ leak, hypercontracture, and rhabdomyolysis in mice carrying a mutation in RYR1 linked to MHS in humans (RYR1Y522S/WT).26 In the current study, we tested the hypothesis that excessive ROS production also plays a central role in lethal hyperthermic crises observed in CASQ1-null mice. To test this hypothesis, we treated CASQ1-null mice with two different antioxidants (N-acetylcysteine [NAC] and Trolox) to determine whether these treatments reduced the incidence of lethal events triggered by halothane and heat stress.
Materials and Methods
In Vivo Experiments
CASQ1-null Mice and Antioxidant Treatment.
CASQ1-null mice were generated as described previously.21 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 experiments were conducted according to the Directive of the European Union 2010/63/UE and the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethical Committees of the University of Chieti (Chieti, Italy) and the University Committee on Animal Resources at the University of Rochester Medical Center (Rochester, USA). Four-month-old male CASQ1-null mice were randomly assigned to three experimental groups: control group (untreated CASQ1-null mice); CASQ1-null mice treated with NAC (Sigma-Aldrich, Italy), and CASQ1-null mice treated with (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, Sigma-Aldrich). For in vivo NAC treatment, CASQ1-null mice were provided ad libitum access to drinking water containing NAC 1% weight/volume (1% w/v) for 2 months (from 2 to 4 months of age). This dose of NAC for 2 months did not cause any noticeable negative side effects in CASQ1-null mice. Another cohort of CASQ1-null mice were treated with Trolox, dissolved in 1 M NaHCO3 water solution and administered by intraperitoneal injection daily for 1 month at a dose of approximately 45 μg/g of body weight.27 Control CASQ1-null mice received either daily NAC-free drinking water or intraperitoneal injection of 1 M NaHCO3 water solution without Trolox for 1 month. A group of wild type (WT) mice, randomly selected, were treated with NAC as an experimental control for NAC treatment for in vitro experiments. Samples size that we used in all in vivo experiments were based on the previous study.19
Heat-stress Protocol and Measurements of Internal Core Temperature.
The effects of heat challenge were studied in WT, untreated CASQ1-null, NAC-treated CASQ1-null, and Trolox-treated CASQ1-null mice. Animals were placed in a temperature-controlled environmental chamber in which the ambient temperature was maintained at 41°C (see Ref. 19 for more detail). Breathing and spasmodic contractions were visually monitored. Muscle rigidity was manually confirmed by limb resistance immediately after animal death. Surviving animals were returned to normal housing conditions and monitored for 24 h to assess possible delayed deaths. Internal temperature was measured using a rectal thermometer taped on the tails of the animals and recorded every 5 min throughout the duration of heat challenge (41°C, 1 h).
To determine the susceptibility to volatile halogenated anesthetics, WT, untreated CASQ1-null, and NAC- and Trolox-treated CASQ1-null mice were exposed to an air mixture containing halothane (Sigma-Aldrich) at concentrations sufficient to induce stage 3 anesthesia (2% halothane, with more added as necessary to induce and maintain this level of anesthesia) using an Isotec 3 evaporator (Datex-Ohmeda, GE Healthcare, USA). During halothane exposure, mice were kept in a chamber at constant temperature (32°C) to avoid a decrease in body temperature during anesthesia. Respiration was monitored visually. The maximum exposure time to halothane was 1 h, and surviving mice were then recovered by suspension of anesthetic.
Quantitative Plasma and Serum Analyses.
For quantitative assessment of markers of rhabdomyolysis in blood/serum, i.e., CK, potassium, and calcium, control CASQ1-null mice (not exposed to heat or halothane) and mice with brief (30 to 35 min) exposure to a nontriggering heat stress challenge were immediately anesthetized with diethyl ether before collecting blood samples through cardiac puncture. Mice were then subjected to thoracotomy, and 500 to 800 μl of blood was collected from the right ventricle with a 26-G needle. Approximately half of this volume was placed in vials containing lithium heparin to prevent blood clotting and centrifuged at 2,500g (4°C for 15 min) to isolate plasma. The other half of the blood was placed in a vial without anticoagulant, and serum was obtained by centrifugation at 4,000g (4°C for 20 min). CK (in serum) and K+ and Ca2+ (in plasma) levels were measured spectrophotometrically using a Screen Touch Master (Hospitex Diagnostic, Italy). All standards for measurements of CK, K+, and Ca2+ were purchased from Hospitex Diagnostic.
Grip Strength Test.
Strength developed by untreated/NAC-treated WT and untreated/NAC-treated CASQ1-null mice during instinctive grasp was measured as described previously.28 Briefly, mice were held by the tail and lowered to a metal grating connected to the shaft of a Shimpo Fgv 0.5X force transducer (Metrotec Group, Spain). Once the mouse had firmly grasped the grating, a steady, gentle pull was exerted on the tail. Measurements of peak force generated by each mouse using fore and hind limbs were repeated three times with appropriate intervals (at least 30 s) to avoid fatigue. Average peak force values were normalized to total body mass measured before each experiment.
Immediately after heat stress, extensor digitorum longus (EDL) muscles were carefully dissected from control CASQ1-null mice (n = 3 mice; n = 101 skeletal muscle fibers analyzed) and NAC-treated CASQ1-null mice (n = 4 mice; n = 186 skeletal muscle fibers analyzed). EDL muscles were also removed from WT mice (n = 3 mice; n = 97 skeletal muscle fibers analyzed) subjected to identical treatment after killing. After fixation of EDL muscles at room temperature (RT) in 3.5% glutaraldehyde, 0.1 M Na cacodylate buffer (Electron Microscopy Sciences (EMS), USA), pH 7.2 overnight, small bundles of fixed fibers were postfixed in 2% OsO4 (EMS) in the same buffer for 2 h, and then block stained in aqueous saturated uranyl acetate (EMS).29 After dehydration, specimens were embedded in an epoxy resin (Epon 812). For histologic analyses, semi-thin (800 nm) sections were cut with a Leica Ultracut R Microtome (Leica Microsystems, Austria) using a Diatome diamond knife (Diatome Ltd., Switzerland). After staining with toluidine blue dye, the sections were viewed using a Leica DMLB light microscope (Leica Microsystems).
Determinations of the number of damaged fibers from WT and treated/untreated CASQ1-null mice after exposure to heat stress were performed on histologic sections. Individual fibers were visually scored for rhabdomyolysis based on the presence of extended damage (i.e., contractures, disassembly of contractile elements, extended regions lacking striations). The number of fibers with such alterations is presented as a percentage of all fibers evaluated.
Ex Vivo and In Vitro Experiments
Contractile Responses and Caffeine Sensitivity of Isolated Intact EDL Muscles.
EDL muscles were dissected from hind limbs of untreated or NAC-treated WT and untreated or NAC-treated CASQ1-null mice and mounted in a myograph (Muscle Tester System, SI, Germany) between a force transducer (SI H KG7B, SI) and a micromanipulator- controlled shaft in a small chamber where oxygenated Krebs solution (Sigma-Aldrich) was continuously circulated. For experiments with EDL muscles obtained from both WT and CASQ1-null treated mice, experiments used a Krebs solution + 2.5 mM NAC. Contractile responses and caffeine sensitivity were studied as described previously.19 The temperature was kept constant at 25°C. Stimulation conditions were optimized, and muscle length was increased until force development during tetanus was maximal. To record responses to caffeine, muscles continuously stimulated at 0.2 Hz were 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). Experiments were performed on six WT EDL (three mice), five NAC-treated WT EDL (three mice), six CASQ1-null EDL (three mice), and eight CASQ1-null NAC-treated EDL (mice mice) muscles.
Cytosolic Ca2+ Measurements in Intact FDB Muscle Fibers.
Single fibers were isolated from flexor digitorum brevis (FDB) muscles of untreated/NAC-treated WT and untreated/NAC-treated CASQ1-null mice according to a modified collagenase/protease method described previously.30 Briefly, FDB muscles were dissected from mice and incubated in 0.2% collagenase and 10% fetal bovine serum Tyrode solution (Sigma-Aldrich) at 37°C for 1 to 2 h. After digestion, collagenase was removed, and FDB fiber bundles were transferred to Tyrode solution (for 3 min), then 10% fetal bovine serum Tyrode solution for 15 min (to block collagenase activity), and finally washed for 3 min in Tyrode solution before trituration with Pasteur pipettes of decreasing diameter to disperse single myofibers. After dissociation, FDB myofibers were plated on laminin-coated glass coverslips positioned at the center of 35-mm dishes to allow their sedimentation and attachment. FDB fibers from untreated WT and CASQ1-null mice were incubated overnight in the culture medium. FDB fibers isolated from NAC-treated WT and CASQ1-null mice were incubated overnight with 2.5 mM NAC dissolved in the culture medium. Twenty-four hours after dissociation, fibers were incubated with 5 μM Fura-2 acetoxymethyl ester (Fura-2 AM; Invitrogen, Italy) in 125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM KH2PO4, 5.5 mM glucose, 1 mM CaCl2, 20 mM Hepes, and 1% bovine serum albumin, pH7.4 (incubation buffer) for 30 min at 37°C. After Fura-2 loading, fibers were washed twice for 10 min in the incubation buffer without bovine serum albumin. A minimum of 30 min was allowed for Fura-2 de-esterification before the fibers were imaged. Intracellular Ca2+ transients were recorded at 25°C using a dual-beam excitation fluorescence photometry setup (IonOptix Corp., USA), as described previously.21,22 Single muscle fibers were stimulated with two distinct frequency protocols. Five fibers from each dish were routinely monitored as follows: (1) after 10 min of steady-state pacing at 0.5 Hz, 10 successive Ca2+ transients at 1 Hz were recorded from each fiber; (2) two trains of high-frequency stimulation (60 Hz for 2 s) were delivered with a recovery time of 5 min between trains. Fura-2 ratios were calculated and analyzed using IonWizard software (IonOptix Corp.). Peak amplitude was calculated by subtracting basal fluorescence ratio values from peak ratio values. FDB fibers evaluated in these experiments were obtained from WT (three mice), NAC-treated WT (three mice), untreated CASQ1-null (three mice), and NAC-treated CASQ1-null (three mice) mice.
In Vivo Electroporation of Mitochondria-targeted cpYFP cDNA into Hind Limb Footpads of Anesthetized Mice.
Mitochondria-targeted (mt)-cpYFP was expressed in FDB muscles by electroporation as described previously.31 Briefly, 4- to 5-month-old WT and CASQ1-null mice were anesthetized by intraperitoneal injection of 100 mg/kg ketamine, 10 mg/kg xylazine, and 3 mg/kg acepromazine. Hind limb footpads of anesthetized mice were then injected subcutaneously with bovine hyaluronidase (7 μl/foot, 2μg/μl). One hour later, hind limb footpads were subcutaneously injected with 30 μg of mt-cpYFP cDNA (total volume10 μl in 71 mM NaCl) using a 30-gauge needle. The footpad was then electroporated (100 V/cm, 20 ms duration, and 20 pulses delivered at 1 Hz) using subcutaneous gold-plated electrodes placed perpendicular to the long axis of the muscle as described previously.31
FDB Fiber Isolation and mSOF Measurement/Analyses.
One week after in vivo electroporation of mt-cp YFP, FDB fibers were acutely isolated from FDB muscle electroporated with mt-cpYFP cDNA by enzymatic digestion in 0.1% collagenase A, 45 min at 37°C, and plated on glass bottom dishes in a rodent Ringer’s solution (146 mM NaCl, 5 mM KCl, 1 mM MgCl, 2 mM CaCl2, 10 mM HEPES, pH 7.4). Mitochondrial superoxide flash (mSOF) productivity was measured as described previously31 with or without preincubation in 2.5 mM NAC for 30 min. Mt-cpYFP was excited using a 488-nm laser and detected at 515/30-nm emission. Images were acquired using a Nikon Eclipse C1 Plus Confocal microscope (Nikon Instruments Inc., USA) equipped with a Superfluor 60X (1.4 NA) oil immersion objective. Automated detection and analysis of individual mSOF events during time lapse x, y imaging was performed using “Flash Collector,” a MATLAB-based image analysis program described in detail previously.31 Flash frequency and amplitude were expressed as the number of events/1,000 μm2·100 s and ΔF/F0, respectively. Output data from Flash Collector were processed for statistical analyses using Microsoft Excel and SigmaPlot (Systat Software Inc, USA) software suites.
Western Blot Analyses.
EDL muscles were dissected from untreated WT (n = 7), NAC-treated WT (n = 5), untreated CASQ1-null (n = 8), and NAC-treated CASQ1-null mice (n = 8) and then rapidly frozen in liquid nitrogen. EDL muscles were homogenized in 3% sodium dodecyl sulphate (Sigma-Aldrich) and 1 mM EGTA (Sigma-Aldrich) buffer using a mechanical homogenizer and then centrifuged for 15 min at 900g at RT. The supernatant was collected, and protein concentrations were determined spectrophotometrically using a modified Lowry method. Equal amounts of total protein (20 to 40 μg) were resolved in 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membrane. Blots were visualized with Ponceau stain (Sigma-Aldrich) to confirm successful transfer and then blocked with 5% nonfat dry milk (EuroClone, Italy) in Tris-buffered saline 0.1% and Tween 20 (TBS-T, Sigma-Aldrich) for 1 h. Membranes were then probed with primary antibody diluted in 5% nonfat dry milk in TBS-T overnight at 4°C: antisuperoxide dismutase type-1 (SOD1) antibody (rabbit polyclonal 1:2000, Santa Cruz Biotechnology, Inc., USA) and anti-3-nitrotyrosine (3-NT) antibody (mouse monoclonal 1:500, Merck Millipore, Italy) to detect expression of SOD1 and nitrated proteins, respectively; anti-α-actinin antibody (mouse monoclonal 1:5000, Sigma-Aldrich) and antiglyceraldehyde-3-phosphate dehydrogenase antibody (mouse monoclonal 1:5000, OriGene Technologies, Inc., USA) were used as loading controls for SOD1 and 3-NT, respectively. After incubation with primary antibodies, membranes were washed three times (10 min for each step) with TBS-T and then incubated with secondary antibodies (1:10,000, horseradish peroxidase–conjugated, Merck Millipore) diluted in 5% nonfat dry milk in TBS-T for 1 h at RT. Finally, membranes were washed three times in TBS-T as described for primary antibodies. The proteins were detected by enhanced chemiluminescent liquid (Perkin-Elmer, USA). The amount of protein on the immunoblots was quantified using Image J software (National Institutes of Health, USA).
Four-month-old untreated WT (n = 5), NAC-treated WT (n = 5), untreated CASQ1-null (n = 6), and NAC-treated CASQ1-null (n = 5) mice were killed and hind limb muscles were removed. Hind limb muscles were homogenized, and total reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were measured according to Rahman et al.32 Briefly, 0.1 g of tissue from hind limb muscles was homogenized in 1 ml of extraction buffer (0.1% Triton X-100 and 0.6% sulfosalicylic acid in 0.1 M potassium phosphate buffer with 5 mM EDTA disodium salt, pH 7.3) on ice three times using a MICCRA D-1 Homogenizer (ART Prozess- & Labortechnik GmbH & Co., Germany). After homogenization, 10% trichloroacetic acid was added to the solution, and the homogenized tissue sample was centrifuged at 8,000g for 10 min at 2° to 4°C to obtain a supernatant containing GSH. The sample was then stored at −80°C until further use. Intracellular total GSH and GSSG levels were measured as described previously.32 The assay used for measurement of total GSH and GSSG is based on the reaction of GSH (directly or through conversion of GSSG to GSH by 2-vinylpyridine) with 5,5′-dithiobis(2-nitrobenzoic acid), which produces the 2-nitro-5-thiobenzoate anion chromophore measured at 412 nm. The rate of 2-nitro-5-thiobenzoate anion formation (change in absorbance per minute) is proportional to the concentration of GSH in the sample. The assay was performed in 96-well plates (96 Well Tissue Culture Test Plate; Spl Life Sciences, Korea) using an Absorbance Microplate Reader SpectraMAX 190 (Molecular Devices, USA). Data were normalized to a GSH standard curve with the GSH concentration in the samples determined from a linear regression from the GSH standard curve.32 All reagents for these experiments were purchased from Sigma-Aldrich.
Four-month-old male WT and CASQ1-null mice were randomly assigned to one of three different experimental groups: control group (untreated mice); WT and CASQ1-null mice treated with NAC; and CASQ1-null mice treated with Trolox.
The following experiments were performed blinded: caffeine in vitro contracture tests (performed at the University of Padova by Marta Canato and Carlo Reggiani); quantitative histologic analysis of fiber damage (performed by Cecilia Paolini); and measurements of GSH and GSSG (performed by Dr. De Marco).
Statistical significance in experiments on halothane- and heat-induced mortality (fig. 1) was evaluated using a two-tailed Fisher exact test. One-way ANOVA followed by Tukey post hoc test was used for statistical analyses of all other experiments except for measurements of time-to-lethal- crises (presented in fig. 2) in which statistical significance was determined using an un-paired Student t test (two tailed). We performed a two-way repeated measures ANOVA followed by Tukey post hoc test for the pairwise comparisons, for experiments in figures 2 and 3 in which we measured a dependent variable over multiple time points. In all cases, differences were considered statistically significant at P < 0.05. We performed Fisher exact test using GraphPad Prism 5 (GraphPad Software, USA), whereas both one-way ANOVA and two-way repeated measures ANOVA and Student t tests were performed using Origin 8.0 software (OriginLab Corporation, USA).
Antioxidant Treatment Protects CASQ1-null Mice from Heat- and Halothane-induced Sudden Death
We treated two different groups of CASQ1-null mice with two potent antioxidant compounds. One group was treated with NAC, a precursor of GSH, provided ad libitum in drinking water at a final concentration of 1% w/v for 60 days (from 2 to 4 months of age). A second group of CASQ1-null mice was treated with Trolox, a water soluble analog of vitamin E that exhibits both radical scavenging and antioxidant activities, administered daily for 4 weeks by intraperitoneal injection, before heat (41°C, 1 h) or halothane (2%, 1 h at 32°C) exposure. Both heat and halothane exposure were well tolerated in all WT animals but was lethal in approximately 80% of untreated CASQ1-null mice (fig. 1; table 1). However, both NAC and Trolox treatment significantly reduced heat- and halothane-induced mortality in CASQ1-null mice. The incidence of death in untreated, NAC-treated, and Trolox-treated CASQ1-null mice was, respectively, 86% (18/21, n = 21), 29% (6/21, n = 21), and 33% (2/6, n = 6) for heat stress and 79% (11/14, n = 14), 25% (4/16, n = 16), and 20% (1/5, n = 5) for halothane sensitivity.
Antioxidants Reduce Hyperthermia during Exposure to High Environmental Temperatures
We monitored rectal temperatures in WT, untreated CASQ1-null, and NAC- and Trolox-treated CASQ1-null mice during heat challenge (60 min 41°C; fig. 2A; table 2). The time-dependent increase in core temperature observed in untreated CASQ1-null mice was significantly greater than that of WT animals. However, the change in core temperature (fig. 2B) of both NAC- and Trolox-treated CASQ1-null mice was significantly lower than that of untreated CASQ1-null mice (40.6° ± 0.3°, 40.5° ± 0.3°, and 42.3 ± 0.1°C, respectively), but not WT mice (40.1° ± 0.1°C). In addition, in the NAC-treated CASQ1-null mice that did not survive heat challenge (4/21; see table 1), the time to the onset of lethal crises was significantly prolonged (fig. 2C), consistent with some degree of protection by NAC also in these animals.
NAC Treatment of CASQ1-null Mice Normalizes the Threshold of Caffeine-induced Contracture
EDL muscles from CASQ1-null mice exhibit an increased contractile responsiveness to low concentrations of caffeine compared with that observed for EDL muscles from WT mice (fig. 3A). In addition, twitch force of EDL muscles from CASQ1-null mice, but not WT mice, declines progressively during exposure to increasing caffeine concentrations (fig. 3B). Both the increase in caffeine sensitivity of resting tension and caffeine-dependent decline in twitch force observed for EDL muscles from CASQ1-null muscles was ameliorated by NAC treatment (also present in the Krebs solution during the experiment, at the concentration of 2.5 mM). Specifically, the threshold for caffeine-induced resting contracture for EDL muscles from NAC-treated CASQ1-null muscles was significantly higher with respect to EDL muscles from untreated CASQ1-null and very close to that of EDL muscles from WT mice (fig. 3A). In addition, the increased caffeine-dependent decline in twitch amplitude in EDL muscles from CASQ1-null mice was partially counteracted by NAC treatment (fig. 3B). Finally, NAC treatment normalized both resting basal contracture (fig. 3C) and twitch tension (fig. 3D) of EDL muscles from CASQ1-null mice during exposure to 22 mM caffeine. EDL muscles from WT mice treated with NAC did not show significant differences with untreated WT in caffeine sensitivity of resting tension, caffeine-dependent decline in twitch force, and in both specific basal and twitch tension at 22 mM of caffeine.
NAC Treatment Prevents Rhabdomyolysis in Muscle of CASQ1-null Mice following Heat Challenge
To assess the efficacy of NAC in preventing rhabdomyolysis, EDL muscles from WT and untreated/treated CASQ1-null mice were quickly dissected at the end of heat challenge (or immediately after a lethal crisis). After fixation, the examination of histologic sections showed in figure 4, A–C revealed that 37.6 ± 2.5% (38/101, n = 3 mice) of fibers were severely damaged in three EDL muscles from three triggered CASQ1-null mice (fig. 4B). Similar alterations were only very rarely observed in surviving WT (6.2 ± 1.4%, 6/97, n = 3 mice) and CASQ1-null mice (11.6 ± 1.1%, 21/186, n = 5 mice) treated with NAC (fig. 4, A and C). The decreased incidence of fiber damage between untreated CASQ1-null mice and both WT and NAC-treated CASQ1-null mice was significant (fig. 4D). Consistent with the development of rhabdomyolysis after heat challenge, serum CK levels and plasma K+ and Ca2+ concentrations were significantly increased in untreated CASQ1-null mice, but not in either WT or NAC-treated CASQ1-null mice (fig. 4, E–G). This reduction in blood CK, K+, and Ca2+ levels in NAC-treated CASQ1-null mice was consistent with the significantly reduced incidence of rhabdomyolysis assessed by histologic analysis (fig. 4D). Conversely, no differences in CK, K+, or Ca2+ levels were detected between the different groups (WT, untreated CASQ1-null mice, and NAC-treated CASQ1-null mice) under resting, nonchallenged conditions (data not shown).
NAC Treatment Rescues Ca2+ Transients and Improves Muscle Function in CASQ1-null Mice
We determined the effect of NAC treatment on myoplasmic Ca2+ levels at rest and during prolonged high-frequency stimulation (60 Hz, 2 s) in FDB fibers loaded with the ratiometric Ca2+ dye, Fura-2 (fig. 5, A–D). At RT (25°C), resting Fura-2 ratios were not different in fibers from untreated WT, NAC-treated WT, untreated CASQ1-null, and NAC-treated CASQ1-null mice (data not shown). Although high myoplasmic Ca2+ levels in fibers from WT mice were maintained throughout prolonged high-frequency stimulation (fig. 5A), a significant decay was found in fibers from CASQ1-null mice (fig. 5C). However, treatment of CASQ1-null mice with NAC reduced the Ca2+ transient decay during prolonged high-frequency stimulation to a level comparable with that of fibers from WT mice (fig. 5, C and D). In FDB muscle fibers from NAC-treated WT mice (fig. 5A), the Ca2+ transient decay was slightly, but significantly, reduced with respect to that observed for untreated WT fibers (fig. 5, B and E). In addition, grip strength of CASQ1-null mice was significantly increased by NAC treatment. Figure 5F shows that although grip strength decreased in untreated CASQ1-null mice from 2 to 4 months of age, NAC treatment enhanced grip strength of CASQ1-null mice to a level closer to that of age-matched WT mice (fig. 5, F and G), an effect not observed in NAC-treated WT mice.
NAC Treatment Reduces Mitochondrial Superoxide Flash Production in FDB Fibers of CASQ1-null Mice
To determine the effect of NAC on oxidative stress in the skeletal muscle of CASQ1-null mice, we measured mSOF activity in dissociated FDB muscle fibers. NAC treatment significantly reduced mSOF frequency in FDB fibers from both WT and CASQ1-null mice at 20° and 37°C (fig. 6, A–D), whereas a temperature-dependent increase in mSOF frequency persisted in fibers from both WT and CASQ1-null mice (fig. 6C). Importantly, NAC treatment reduced mSOF frequency in CASQ1-null fibers at 37°C to a level similar to that observed for fibers from untreated WT mice (fig. 6D, red dotted line). Conversely, mSOF amplitude was unchanged except for a minor decrease in fibers from NAC-treated CASQ1-null mice at 20°C (fig. 6E).
NAC Treatment Reduces Superoxide Dismutase Type-1 Expression, 3-Nitrotyrosylation, and Oxidative Stress
To verify the efficacy of NAC treatment to reduce oxidative stress, we measured (a) expression levels of SOD1, one of three enzymes that catalyze the dismutation of superoxide (O2−) into O2 and H2O2,33 the first step in the elimination of ROS; (b) levels of 3-NT, a product of nitration of tyrosine residues of proteins mediated by RNS such as peroxynitrite anion and nitrogen dioxide, which are indicators of oxidative stress and oxidative protein damage34 ; (c) global amount of GSH, a molecule synthesized from amino acids that is capable of reducing disulfide bonds formed within cytoplasmic proteins to cysteines by serving as an electron donor; and (d) the ratio of GSH and GSSG, a recognized biomarker of oxidative stress.35 Western blot analyses, performed on homogenates from EDL muscles of untreated WT, NAC-treated WT, untreated CASQ1-null, and NAC-treated CASQ1-null mice revealed that SOD1 expression in CASQ1-null mice, significantly increased compared with WT mice, was normalized to values close to that of WT mice after treatment with NAC (fig. 7, A and B). The increased expression of SOD1 in CASQ1-null mice likely reflects a compensatory response to the excessive production of superoxide within mitochondria (see fig. 6), a finding in agreement with the observation that SOD1 expression and activity are increased under conditions of high oxidative stress.36–39 In the same samples, we also measured 3-NT levels, a recognized biomarker of oxidative stress, cell damage, and inflammation. As shown in figure 7, C and D, EDL muscles from untreated CASQ1-null mice display a significant increase in 3-NT compared with that of EDL muscles from untreated WT and NAC-treated CASQ1-null mice. Thus, NAC treatment of CASQ1-null mice reduced 3-NT to a level comparable with that of untreated WT mice.
Because GSH and GSSG levels reflect the overall cellular redox state, we quantified the amount of GSH and GSH/GSSG ratio in hind limb muscle homogenates from untreated/NAC-treated WT and untreated/NAC-treated CASQ1-null mice. Although both GSH (nmol/g of tissue, fig. 7E) and the GSH/GSSG ratio were significantly reduced (~57 and ~46%, respectively; fig. 7, E and F) in muscles from CASQ1-null mice compared with that of WT mice, treatment of CASQ1-null mice with NAC restored both GSH levels and the GSH/GSSG ratio to values similar to that of WT, consistent with NAC treatment reducing oxidative stress.
Ablation of CASQ1 in mice results in increased susceptibility of CASQ1-knockout animals to lethal heat- and halothane-induced hyperthermic episodes.19,20,40 This phenotype closely resembles to that observed in porcine stress syndrome,15,16 knockin mice carrying MHS mutations in RYR1,17,18 and some cases of RYR1-linked MH and EHS in humans.11–13 Herein, we tested the hypothesis that excessive oxidative stress contributes to lethal hyperthermic episodes in male CASQ1-null mice.
In our studies, antioxidant treatment (with either NAC or Trolox) reverses several pathophysiologic phenomena that underlie hyperthermic crises in CASQ1-null mice: (a) increased core temperature (fig. 2A), (b) enhanced caffeine sensitivity of the skeletal muscle to develop contractures (fig. 3, A and C), and (c) increased muscle fiber damage and rhabdomyolysis (fig. 4, A–D). Most importantly, antioxidant treatment prevented lethal heat- and halothane-induced episodes (fig. 1), possibly by reducing mitochondrial superoxide production and oxidative stress (figs. 6 and 7). These results support recent evidence that excessive production of oxidative species is a key event in the onset of these life-threatening crises.26
Molecular Mechanisms Underlying Lethal Hyperthermic Episodes: Role of Oxidative Stress
MHS is linked to RYR1 mutations that enhance SR Ca2+ leak and the sensitivity of the SR Ca2+ release process to activation.41 However, this may be an oversimplified view of the complex events that underlie MH/EHS responses as the recent findings provide additional information that must also be taken into account.
Only a subset of MHS individuals have been linked to a specific gene mutation: 60% to 70% to RYR141,42 and approximately 1% to the dihydropyridine receptor α1S-subunit.43,44 Mutations in other gene(s) could be responsible for some MHS cases in the remaining fraction. However, although our results indicate that CASQ1 ablation in mice results in a phenotype that resembles MHS/EHS,19,20,40 loss-of-function mutations in the CASQ1 gene have not been linked to MHS/EHS in humans. Indeed, Kraeva et al.25 failed to identify any mutations within the CASQ1 coding region in 75 unrelated MHS patients diagnosed by caffeine–halothane contracture test, thus concluding CASQ1 is an unlikely genetic locus for MHS within the North American population. A similar conclusion was reached in another study of European MHS patients (Vincenzo Sorrentino, M.D., Professor, Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy, March 2014, personal communication). To date only one disease mutation in the CASQ1 gene (N244G) has been detected in a group of patients that exhibit a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions that result from the aggregation of SR proteins.45 However, an increase in susceptibility to MH has not been demonstrated in these families.
In vitro contracture tests used to diagnose MHS only work when extracellular Ca2+ is included in the solution bathing the muscle biopsy,46–48 suggesting that Ca2+ influx may play a critical role in the MH crises. Interestingly, store-operated Ca2+ entry (SOCE), a Ca2+ influx mechanism triggered by store depletion in which STIM1 Ca2+ sensors in the SR activate Ca2+ influx through Orai1 Ca2+ channels in the transverse tubule membrane,49,50 is significantly enhanced in the human skeletal fibers from MHS patients.51
Figure 8 depicts a proposed cascade of events linking SR Ca2+ leak (step 1) to rhabdomyolysis of skeletal muscle fibers (step 7). The model is based on both results presented here and in related previous studies. CASQ1 deficiency increases susceptibility of mice to heat- and halothane-induced contractures: indeed, CASQ1-deficient SR decreases Ca2+ buffering and increases RYR1 Ca2+ leak,52 which together serve to increase SR evacuability53 and promote SR Ca2+ depletion.22,54 Even under resting conditions (follow orange labeling), SR Ca2+ leak (step 1) would lead to an increased energy demand (required for continual SERCA-mediated SR Ca2+ reuptake) and enhanced oxidative stress (step 2) because of increased mitochondrial content21 and activity, a scenario that predisposes CASQ1-null mice to lethal reactions under stress.19 During exposure to heat or halothane, the sequence of events leading to lethal crises involve (follow purple labeling) (a) excessive RYR1 Ca2+ leak and increased oxidative and nitrosative stress (steps 1 and 219,31 ); (b) RYR1 S- nitrosylation, resulting in a feed-forward mechanism to further increase RYR1 leak (step 326 ); (c) SR Ca2+ store depletion (step 422 ); (d) activation of SOCE resulting in massive Ca2+ entry (step 524 ); and (e) uncontrolled contractures and rhabdomyolysis (steps 6 and 719 ).
Possible Molecular Mechanisms by Which Antioxidants Prevent Lethal Hyperthermic Episodes
The full understanding of the molecular mechanism by which antioxidants are capable of preventing lethal episodes in CASQ1-null mice may require additional investigation. Several publications have demonstrated that an increased production of ROS and RNS results in RYR1 S- nitrosylation/glutathionylation, which increases its opening probability.26,55–58 At physiologic conditions, the normal levels of oxidative stress could be responsible for a basal RYR1 low-opening probability, which would cause a small SR Ca2+ leak and in turn influence the total SR Ca2+ content. This hypothesis seems to be confirmed by data in figure 5, B and D showing how NAC has an effect also on Ca2+ transient of WT fibers: indeed in these experiments, the SR of WT fibers treated with NAC releases more Ca2+ during prolonged stimulation. Furthermore, data presented in figure 3, B and D show that WT muscles treated with NAC sustain twitch-tension better than untreated WT muscles, because their SR is likely more depletion resistant when exposed to increasing caffeine concentrations. Our results are well in agreement with those reported in Durham et al.26 : indeed, in those experiments, NAC treatment of WT mice resulted in a modest increase in frequency-dependent contractile force in soleus and also maximal caffeine-induced contractile force. Together, these findings suggest that basal levels of oxidative stress have a modest inhibitory effect on maximal Ca2+ release and contraction also in WT, possibly via an effect on basal SR Ca2+ leak and store content. According to this model, the protective effect of NAC and Trolox results from a reduction in oxidative stress in muscle (supported by data in figs. 6 and 7), thus reducing Ca2+ leak and blocking the feed-forward mechanism that promotes SR Ca2+ depletion22 (supported by data in figs. 3, B and D and 5, A–E) and SOCE activation.24 Whether RYR1 S-nitrosylation26 occurs in CASQ1-null mice and the degree to which NAC and Trolox treatment prevents RYR1 S-nitrosylation and/or uncontrolled Ca2+ entry through SOCE channels require further investigation.
The model proposed in figure 8 provides a framework for future investigations aiming to identify and develop drugs to prevent hyperthermic crises by blocking either SR Ca2+ leak or SOCE, two key steps that represent potentially viable therapeutic targets. Indeed, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside, acadesine, N1-(β-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide, an activator of the energy-sensing AMP kinase59 and a partial RYR1 agonist, reduces SR Ca2+ leak and prevents heat-induced sudden death in RYR1Y522S/WT knockin mice.60 In addition, dantrolene (or its analog azumolene), the drug of choice for acute treatment of MH crises,61 reduces RYR1 Ca2+ release62 and limits excessive Ca2+ entry because of activation of SOCE.63,64
Significance of the Study
Because MH reactions during anesthesia are rare, patients are not routinely tested/screened for MHS and preventive anesthetic measures are not typically taken to avoid hyperthermic crises in individuals who are not known to be at an increased risk for MHS. Indeed, in most cases, diagnosis of MH performed using standard protocols6,7 follows a suspected MH episode (i.e., proband and/or blood-related family members). Our results demonstrating that pretreatment with antioxidant (NAC and Trolox) protects CASQ1-null mice from lethal hyperthermic events induced by exposure to either halothane or heat stress may suggest a possible preventive (and inexpensive) therapy that could be used to reduce the risk of MH reactions in patients undergoing anesthesia who have not prescreened for MHS. However, further investigation is needed to determine (a) the minimal effective antioxidant dose and (b) the efficacy of acute antioxidant treatment in preventing hyperthermic crises in vivo. Indeed, in our experiments, the NAC dose used here (1 to 1.5 mg/g body weight), which was similar to that used in RYR1Y522S/WT knockin mice,26 is approximately 10X higher than that tested in a recent human clinical trial (acute oral administration) evaluating the effect of NAC on muscle fatigue during exercise (0.15 mg/g of body weight).65 In addition, because CASQ1 mutations have not been identified in MHS patients,25 it will be important for future studies to validate whether chronic antioxidant treatment also provides protection from heat- and halothane-induced episodes in RYR1 mouse and pig models of MHS and EHS.15–18
This study was supported by the grants GGP13213 from Italian Telethon ONLUS Foundation, Rome, Italy (to Drs. Protasi and Reggiani); AR059646 from National Institutes of Health, Bethesda, Maryland (to Dr. Dirksen and subcontract to Dr. Protasi); MDA275574 from Muscular Dystrophy Association, Chicago, Illinois (to Dr. Dirksen and subcontract to Dr. Protasi); AR053349 from National Institutes of Health (to Dr. Dirksen and subcontract to Dr. Protasi); and the Academia Dei Lincei Fund, Rochester, New York (to Dr. Wei-Lapierre).
The authors declare no competing interests.