Malignant Hyperthermia Susceptible Pigs Research Articles

In vitro effects of levosimendan on muscle of malignant hyperthermia susceptible and non-susceptible swine

BackgroundThe calcium sensitizer levosimendan is increasingly used to improve hemodynamics in patients with acutely decompensated heart failure. By binding to cardiac troponin C the conformation of the calcium-troponin C complex is stabilized, which leads to acceleration of actin-myosin crossbrigde formation and increased force generating capacity of muscle fibers. Besides indications in cardiac failure, beneficial effects of levosimendan in skeletal muscle disorders are currently evaluated. The aim of this study was to investigate differential effects of levosimendan on skeletal muscle of pigs with and without susceptibility to malignant hyperthermia (MH) in order to identify possible risks of this emerging drug for patients with predisposition to MH.MethodsMuscle bundles of 17 pigs (9 MH susceptible (MHS); 8 MH non-susceptible (MHN)) were excised under general anesthesia and examined in the tissue bath with increasing concentrations of levosimendan (0.065; 0.125; 0.5; 1.0; 10 and 50 μg/ml). Baseline tension and twitch force were monitored continuously. Data are presented as median and interquartile range. Statistical evaluation was performed using D’Agostino & Pearson test for normal distribution and student’s t test and 2-way ANOVA for differences between the groups. P < 0.05 was considered significant.ResultsThere were no differences between the groups concerning length, weight, initial twitch force and pre-drug resting tension of the investigated muscle strips. After an initial decrease in both groups, twitch amplitude was significantly higher in MHN (− 3.0 [− 5.2–0.2] mN) compared to MHS (− 7.5 [− 10.8- -4.5] mN) (p = 0.0034) muscle at an applied levosimendan concentration of 50 μg/ml. A marked increase in resting tension was detected following levosimendan incubation with 50 μg/ml in MHS muscle bundles (3.3 [0.9–6.1] mN) compared to MHN (− 0.7 [− 1.3–0.0] mN) (p < 0.0001).ConclusionsThis in vitro investigation revealed the development of significant contractures in muscle bundles of MHS pigs after incubation with levosimendan. However, the effect appeared only at supra-therapeutic concentrations and further research is needed to determine the impact of levosimendan on MHS individuals in vivo.

Ultrasound Elastography for Rapid, Real-time Detection of Localized Muscular Reaction in Malignant Hyperthermia-susceptible Pigs.

WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Halothane and caffeine induce excessive sarcoplasmic calcium liberation and skeletal muscle contracture in patients susceptible to malignant hyperthermia (MH) and are utilized for diagnosis in the in vitro contracture test. Intramuscular injection previously caused a marked local lactate increase in MH-susceptible but not in MH-nonsusceptible individuals in vivo. Using shear-wave elastography, this study evaluated localized changes in muscle stiffness after intramuscular injection of halothane and caffeine. Microdialysis probes were placed into the gracilis muscle of 16 pigs (9 MH-susceptible and 7 MH-nonsusceptible). After local injection of either halothane or caffeine in different concentrations, changes of tissue elasticity surrounding the probe were examined by quantitative shear-wave elastography. Local lactate concentrations were analyzed spectrophotometrically. Ultrasound elastography detected a temporary increase in local muscle rigidity in MH-susceptible but not in MH-nonsusceptible pigs after 2.5 and 5 vol% halothane and after 10, 40, and 80 mM caffeine, whereas there were no differences in the control groups (median [interquartile range] for maximum effect after 5 vol% halothane: MH-susceptible: 97 [31 to 148] vs. MH-nonsusceptible: 5 [-6 to 18] kPa; P = 0.0006; maximum effect after 80 mM caffeine: 112 [64 to 174] vs. -3 [-6 to 35] kPa; P = 0.0002). These effects were seen rapidly within 5 min. Local lactate concentrations were higher in MH-susceptible versus nonsusceptible pigs after 1 and 2.5 vol% halothane and 10, 40, and 80 mM caffeine (2.5 vol% halothane: MH-susceptible: 2.8 [1.9 to 4.4] vs. MH-nonsusceptible: 0.6 [0.6 to 0.7] mmol/l; P < 0.0001; 80 mM caffeine: 5.2 [4.1 to 6.3] vs. 1.6 [1.2 to 2.4] mmol/l; P < 0.0001). After 10 vol% halothane, rigidity and lactate levels were increased in both MH-susceptible and MH-nonsusceptible animals. This pilot study revealed shear-wave elastography as a suitable technique for real-time detection of altered tissue elasticity in response to pharmacologic stimulation. By considering the variability of these results, further test protocol optimization is required before elastography could serve as a minimally invasive MH diagnostic test.

The effect of succinylcholine on malignant hyperthermia events in susceptible swine

BackgroundWhile the impact of volatile anaesthetics to induce malignant hyperthermia (MH) is abundantly clear, the role of succinylcholine still remains controversial. To evaluate the influence of succinylcholine on porcine MH events, the authors investigated the hemodynamic and metabolic responses in MH susceptible (MHS) and non-susceptible (MHN) swine following either succinylcholine or halothane application alone or a combination of both substances.MethodsWith approval of the local animal care committee 27 MHS and 30 MHN pigs were anaesthetized and mechanically ventilated. Fiberoptic probes for continuous PCO2 measurement were inserted into the femoral vein and the triceps muscle. Group A received succinylcholine 4 mg/kg, group B incremental doses of halothane (0.5, 1.0 vol%) and group C succinylcholine and halothane simultaneously. Vital signs were recorded continuously.ResultsPrior to drug application measured values did not differ between MHS and MHN. While MHN pigs did not show relevant alterations, succinylcholine, halothane and the combination of both lead to significant hemodynamic and metabolic changes in MHS swine.ConclusionsHemodynamic and metabolic alterations following succinylcholine were similar to halothane in MHS pigs. The combination of both pharmacological agents potentiated the observed effects. According to these results succinylcholine acted as an independent and supportive factor during onset of an MH episode.

A Review of the Factors Affecting the Incidence of Malignant Hyperthermia in the Greater Kansas City Area

Malignant Hyperthermia (MH) is a rare genetic disease. However, it is devastating when it occurs in a patient. MH is usually triggered by inhalational anesthetics and/or depolarizing muscle relaxants. Public awareness of MH has increased with the presentation of an episode on the television program, “House”, and the availability of web-based information. For over 20 years, the MH susceptible pig has been used in experiments by our group as an animal model for MH in humans. The incidence of Malignant Hyperthermia in the Greater Kansas City Area has declined dramatically since the introduction of Sevoflurane in 1992 as the anesthetic of choice (over 60% usage rate) in most surgical procedures. Historically, Malignant Hyperthermia was reported to occur at a rate of 1:50,000 during surgical procedures [1]. In the Greater Kansas City Area, Malignant Hyperthermia (MH) occurred at a rate of 1:53,636 during the 1965-1985 time period, as there were 38 MH cases in 35 patients [2]. During the past ten years (1996-2006), there were only 2 cases of MH, representing an incidence rate of 1:597,240. That decrease is an 11.13 fold (or 89%) decrease which is very significant. Despite the reduced incidence of Malignant Hyperthermia, two recent cases of MH that result in deaths in Wisconsin and Florida make it imperative that MH is recognized early and appropriate treatment initiated without delay. We have expanded our analysis of the futile cycle mechanism that underlies the MH syndrome. MH is equivalent to the rapid discharge of a battery by a short circuit.

Succinylcholine in malignant hyperthermia: Evaluation of a novel in vivo model

Malignant hyperthermia (MH) is a potentially lethal anesthetic complication characterized by muscle hypermetabolism and generalized rigor. The exact mechanism of succinylcholine as an MH trigger cannot be examined in existing in vitro models. Therefore, a novel in vivo model was used to examine the metabolic response to succinylcholine. With institutional review board approval, 6 MH susceptible (MHS) and 6 MH non-susceptible (MHN) pigs were anesthetized with hemodynamic and systemic metabolic monitoring. Microdialysis catheters were placed intramuscularly. After equilibration, succinylcholine, halothane, and Ringer solution were injected. Lactate was measured in the dialysate and statistically analyzed by Mann-Whitney U-test (significance level P < 0.05). Hemodynamic and systemic metabolic parameters were not different between the groups throughout the experiment. In the MHS pigs, halothane induced a significant increase of lactate. In MHN pigs, no substance induced a reaction. Halothane, but not succinylcholine, induced a hypermetabolic reaction in this model. Therefore, the role of succinylcholine as an MH trigger remains questionable.

Comparison of the therapeutic effectiveness of a dantrolene sodium solution and a novel nanocrystalline suspension of dantrolene sodium in malignant hyperthermia normal and susceptible pigs

Stopping trigger agents and prompt administration of dantrolene are the cornerstones of treatment of malignant hyperthermia. However, significant time is lost in treatment of the condition because of the cumbersome preparation and administration of the commercially available dantrolene sodium for injection. A potential improvement has become available in the form of a novel nanocrystalline dantrolene sodium suspension (DSS), which is 150 times more concentrated (50 mg ml(-1)) than the standard dantrolene sodium solution (0.33 mg ml(-1)). The aims of this study were to measure the effects of DSS on clinical and laboratory variables in malignant hyperthermia normal pigs and to compare the therapeutic management and clinical effectiveness of DSS with standard dantrolene sodium in a fulminant malignant hyperthermia crisis in susceptible pigs. The pig model is a well accepted method of studying the malignant hyperthermia crisis and is an ideal way to evaluate the variables of interest in this study. Seven malignant hyperthermia normal and 10 malignant hyperthermia susceptible pigs were studied. Malignant hyperthermia susceptible pigs (body weight approximately 24 kg) were allocated to a dantrolene sodium group or a DSS group. After induction of anaesthesia, a 22-gauge catheter was placed in an ear vein and trigger-free anaesthesia was performed. After achieving stable conditions, administration of halothane was started with 0.1% and then 0.15%. Halothane was discontinued after the administration of 0.2% (malignant hyperthermia normal pigs) or when a fulminant malignant hyperthermia crisis was achieved (malignant hyperthermia susceptible pigs). After halothane was discontinued, FIO2 was set to 1.0, respiratory minute volume was doubled and sodium bicarbonate 2 mmol kg(-1) was administered. The time required to prepare and administer each formulation was measured. To simulate the administration of the substances under typical clinical conditions for a child weighing approximately 24 kg, dantrolene sodium (5 mg kg(-1)) or DSS (5 mg kg(-1)) was prepared and injected via the intravenous 22-gauge cannula. Bolus administrations of dantrolene sodium or DSS were repeated after 24 min. Arterial pH, arterial pCO2, mean arterial pressure and arterial lactate concentration remained stable during the experiment with DSS in malignant hyperthermia normal pigs. A significant decrease in cardiac index and increases in systemic vascular resistance and serum potassium concentration occurred after administration of DSS. In all malignant hyperthermia susceptible animals, the inhaled administration of halothane 0.15% led to a fulminant malignant hyperthermia crisis. The therapeutic regimens with administration of dantrolene sodium or DSS were successful in treating the malignant hyperthermia crisis in all animals. The course of the malignant hyperthermia crisis and the therapeutic effects of dantrolene sodium or DSS were comparable in the two groups. The time needed to prepare DSS for administration was significantly shorter (51 ± 9 s) compared to dantrolene sodium (860 ± 202 s). The time taken to inject DSS (4 ± 2 s) was significantly shorter than for dantrolene sodium (472 ± 51 s). The therapeutic action of DSS in a malignant hyperthermia crisis in pigs was effective and comparable to that of standard dantrolene sodium. However, preparation and administration of DSS were significantly faster, which may offer a clinically significant advantage in the treatment of a fulminant malignant hyperthermia crisis and may result in a reduction in stress for the anaesthesia team.

Statins Alter Intracellular Calcium Homeostasis in Malignant Hyperthermia Susceptible Individuals

Statines, HMG-CoA reductase inhibitors, are widely used to treat hypercholesterinemia. These substances are well tolerated, but myotoxic effects have been reported. The exact mechanisms of the induced myotoxicity are unknown but an involvement of intracellular calcium handling is suspected. Individuals susceptible to malignant hyperthermia (MH) have an impaired calcium homeostasis. An in vitro test measuring contracture responses of isolated muscle bundles is used to investigate cellular processes of MH. Aim of this study was to investigate if statins modify the contracture response of isolated muscle bundles from MH susceptible (MHS) and nonsusceptible (MHN) pigs. With approval of the local ethics committee muscle biopsies of 18 MH susceptible and 12 nonsusceptible pigs were performed. Muscle bundles were mounted on an isometric force transducer, preloaded, and electrically stimulated. After establishment of a stable baseline, muscle bundles were exposed to simvastatin, atorvastatin, gemfibrocil, and the pure solvent. Baseline tension was measured and analyzed for changes with P < 0.05 considered to be significant. There were no differences in weight, length, and predrug baseline tension between the groups. Both simvastatin and atorvastatin induced significant contractures in muscle bundles from MHS pigs. Gemfibrocil and the solvent methanol showed no effect. In MHN muscle bundles, none of the tested substances induced a contracture. Statines induce contractures only in MHS muscle bundles. We therefore conclude that the underlying mechanism may be a pathologic influence on intracellular calcium handling that is absent in MHN. A preexisting impairment of the calcium homeostasis seems to be necessary for this behavior because muscle bundles of MHN pigs showed no pathologic reaction. A higher muscle cell vulnerability toward statins is assumed in MHS patients. Statins ought to be used with caution in these individuals. Analogous a diagnostic workup for MH should be considered for patients with statin-induced rhabdomyolyis.

Isometric contraction of skeletal muscle of malignant hyperthermia (MH) susceptible and resistant Belgian landrace pigs under the influence of several drugs.

Summary In vitro measurement of isometric contraction of skeletal muscle enables classification of pigs into resistant, sensitive and very sensitive groups according to their response to treatment with halothane and/or caffeine at different concentrations. The m. gracilis is more appropriate for this measurement than the white m. semimembranosus. Previous exercise enhances the in vitro responses of the muscle. All abnormal contractions found in sensitive and very sensitive pigs ceased when calcium was omitted from the buffer medium. The role of external calcium in determining the degree of sensitivity is discussed. The classification of BL and LW pigs by this in vitro technique showed LW pigs to be mostly resistant and BL pigs to be mostly sensitive or very sensitive. The correlation between the in vitro technique and the in vivo halothane inhalation test is discussed. Zusammenfassung Untersuchungen uber die Halothan- und Koffein-Empfindlichkeit von Schweinen der Belgischen Landrasse hinsichtlich der malignen Hyperthermic und isometrischen Skelettmuskelkontraktion Aufgrund isometrischer Skelettmuskelkontraktionen in vitro, ausgelost durch Halothan und/oder Koffein in verschiedenen Konzentrationen, konnen Schweine entsprechend ihrer Ansprechbarkeit auf Halothan bzw. Koffein in drei Gruppen eingeteilt werden: 1. resistente Tiere, 2. sensible Tiere, 3. hochsensible Tiere. Der M. gracilis ist fur diesen Test besser geeignet als der M. semimembranosus. Muskelarbeit vor dem Test verstarkt die Muskelkontraktion. In Calcium-freier Krebs-Ringerlosung zeigten die Muskelstreifen von sensiblen und hochsensiblen Tieren keine Kontraktionen mehr. Der Einfluss des Calciums in der Nahrlosung auf die Ansprechbarkeit der Muskulatur wird diskutiert. Die meisten Tiere der Belgischen Landrasse (Belgian Landrace) erwiesen sich resistent, Tiere der weissen Rasse (Large White pigs) waren hingegen meistens sensibel bis hochsensibel. Die Beziehungen zwischen der in vitro-Methodik und der in vivo-Halothaninhalation werden besprochen. Resume Recherches sur la receptivite a l'halothane et a la cafeine des porcs de race blancs belges en fonction de l'hyperthermie maligne et de la contraction isometrique de la musculature du squelette Sur la base des contractions isometriques de la musculature du squelette declenchees in vitro par de l'halothane et/ou de la cafeine a differentes concentrations, on a pu repartir les porcs en trois groupes en fonction de leur receptivite a l'halothane et a la cafeine: 1) animaux resistants, 2) animaux sensibles, 3) animaux tres sensibles. M. gracilis convient mieux pour ce test que M. semimembranosus. Un travail musculaire precedant le test renforce la contraction musculaire. Les stries musculaires des animaux sensibles et tres sensibles n'ont presente aucune contraction dans une solution de Krebs-Ringer sans calcium. L'influence du calcium dans la solution nutritive sur le comportement de la musculature est discutee. La plupart des animaux de race porc blanc belge (Belgian Landrace) se sont montres resistants et les animaux de la race porc ameliore (Large White pigs) furent par contre le plus souvent sensibles et tres sensibles. Les rapports entre la methode in vitro et l'inhalation in vivo par l'halothane sont discutes. Resumen Estudios sobre la susceptibilidad al halothane y a la cafeina de cerdos de la raza blanca belga en funcion de la hipertermia maligna (HM) y de la contraccion isometrica de la musculatura del esqueleto Sobre la base de contracciones isometricas de los musculos del esqueleto in vitro, originadas por halothane y/o cafeina en concentraciones diversas, se pueden clasificar los cerdos en tres grupos en funcion de su receptividad a halothane y a la cafeina: 1) animales resistentes, 2) animales sensibles, 3) animales muy sensibles. El m. gracilis es mas adecuado para esta prueba que el m. semimembranoso. El trabajo muscular realizado antes de la prueba refuerza la contraccion muscular. Las estrias musculares de los animales sensibles y muy sensibles no presentaban ninguna contraccion mas en la solucion de Krebs-Ringer sin calcio. Se discute el influjo del calcio en la solucion nutritiva sobre la reaccion de la musculatura. La mayor parte de los cerdos de la raza blanca belga (rural) se muestran resistentes, mientras que los de la raza mejorada (Large White) solian ser sensibles o muy sensibles. Se discuten las correlaciones entre la tecnica in vitro y la prueba in vivo de inhalacion de halothane.

Intramuscular Injection of Sevoflurane Detects Malignant Hyperthermia Predisposition in Susceptible Pigs

The authors hypothesized that intramuscular sevoflurane injection allows diagnostic differentiation between malignant hyperthermia-susceptible (MHS) and -nonsusceptible (MHN) pigs by measurement of intramuscular lactate and carbon dioxide partial pressure (PCO2), and that dantrolene reduces the sevoflurane-induced PCO2 increase. With approval of the local animal care committee, microdialysis probes with attached microtubing for sevoflurane injection were placed in the adductor muscles of nine MHS and six MHN pigs, and PCO2 probes with microtubing were positioned in the triceps muscle of eight MHS and six MHN pigs. After equilibration, sevoflurane boluses at different concentrations and a sevoflurane-dantrolene bolus were injected synchronously. Lactate, pyruvate, and glucose as well as PCO2 were measured spectrophotometrically, and the rate of PCO2 increase was calculated. Intramuscular sevoflurane injection increased local lactate and PCO2 dose dependently, and significantly higher in MHS than in MHN pigs. Measurement of the rate of PCO2 increase allowed a distinct differentiation between single MHS and MHN pigs. No significant increase in PCO2 was found with sevoflurane and dantrolene. Local sevoflurane induces a hypermetabolic reaction measured by PCO2 and lactate increases. The reduced PCO2 increase in MHS after sevoflurane and dantrolene injection is likely to be a result of the sevoflurane-mediated calcium release and its antagonism by dantrolene. Sevoflurane may be useful for a less invasive diagnostic test for malignant hyperthermia in humans.

Effects of ryanodine on skeletal muscle lactate and pyruvate in malignant hyperthermia-susceptible and normal swine as assessed by microdialysis

The caffeine/halothane contracture test in North America and the in vitro contracture test in Europe are currently the only validated bioassays for diagnosing malignant hyperthermia susceptibility and phenotyping families. Both tests are invasive requiring surgical muscle biopsy. Here, we report first use of the selective ryanodine receptor type I agonist ryanodine in a percutaneous microdialysis protocol designed to test whether microdialysis-induced local metabolic responses of skeletal muscle due to ryanodine receptor activation can differentiate between malignant hyperthermia-sensitive and normal pigs. Six microdialysis catheters were implanted percutaneously into the adductor muscles of the right and left thighs of malignant hyperthermia-susceptible (n = 9) and normal (n = 8) anaesthetized (ketamine/propofol) and mechanically ventilated swine. Systemic blood gases, haemodynamic parameters and creatine kinase levels were measured before, during and after microdialysis perfusion of ryanodine. After a post-implantation equilibration period of 30 min, one catheter perfused (2 micro min-1) with 0.9% NaCl (control) and was compared with the remaining five catheters perfused with increasing concentrations of ryanodine (0.2-100 micromol). Lactate and pyruvate levels were measured enzymatically. Continuous perfusion with ryanodine revealed dose-dependent sigmoidal increases in the dialysate lactate and lactate-pyruvate ratio parameters; these effects were greatly augmented in malignant hyperthermia-susceptible pigs compared to normal pigs (two- to threefold): estimated EC50 greatly decreased (>19-fold) while the maximum effect increased (>twofold) in the malignant hyperthermia-susceptible group. The in vivo percutaneous microdialysis protocol for skeletal muscle, using ryanodine as the ryanodine receptor type I agonist and dialysed lactate-pyruvate parameters as metabolic index, can reproducibly differentiate between malignant hyperthermia-susceptible and normal swine.

Characterization of Ryanodine Receptor–mediated Calcium Release in Human B Cells

Mutations in the ryanodine type 1 receptor (RyR1) are causative for malignant hyperthermia. Studies in human B lymphocytes have shown that measurement of RyR1-mediated intracellular Ca(2+) (Ca(2+)(i)) release can differentiate between normal and malignant hyperthermia-susceptible individuals. The authors have further developed the B-cell assay by pharmacologically characterizing RyR1-mediated Ca release in two normal human B-cell lines and demonstrating increased sensitivity of lymphocytes to the RyR1 agonist 4-chloro-m-cresol (4-CmC) in the porcine model of MH. Ca(2+)(i) was measured fluorometrically using fura-2 in populations of cells in suspension or with fluo-4 in single cells using confocal microscopy. The Dakiki and PP normal human B cell lines were used, as well as lymphocytes obtained from normal and malignant hyperthermia-susceptible pigs. 4-CmC was used to elicit RyR1-mediated Ca release; all experiments were performed in the absence of external Ca(2+). EC(50) values for 4-CmC were 0.98 and 1.04 mm for Dakiki and PP cells, respectively, demonstrating reproducibility. The 4-CmC-induced increase in Ca(2+)(i) was eliminated by thapsigargin and was unaffected by xestospongin C. The Ca(2+)(i) increase was separable from mitochondrial stores and was inhibited by azumolene. Caffeine did not induce Ca(2+)(i) release, but ryanodine depleted intracellular stores by 50%. Lymphocytes from pigs carrying the Arg614Cys mutation in RyR1 showed increased sensitivity to 4-CmC (EC(50) = 0.47 vs. 0.81 mm for cells derived from normal animals). RyR1-mediated Ca(2+) signals can be pharmacologically distinguished from other intracellular sources in human B cells, and alterations of RyR1 function can be successfully detected using Ca(2+) release from intracellular stores as an end point.

The Dose-Response Relationship and Regional Distribution of Lactate After Intramuscular Injection of Halothane and Caffeine in Malignant Hyperthermia-Susceptible Pigs

We hypothesized that IM halothane and caffeine injection increases local lactate concentration dose-dependently in malignant hyperthermia-susceptible (MHS) and nonsusceptible (MHN) pigs and that the hypermetabolic reaction measured by regional distribution of lactate and carbon dioxide is limited to a small muscle volume. Microdialysis probes were placed in the hindlimbs of 7 MHS and 7 MHN pigs and perfused with Ringer's solution. After equilibration, boluses of increasing halothane and caffeine concentrations were injected. For the second hypothesis regarding regional distribution, microdialysis probes were positioned in 7 MHS and 6 MHN pigs at the injection site for halothane and caffeine and at a distance of 10 mm and 25 mm. Lactate was measured in the dialysate by spectrophotometry. In addition, PCO2 was measured in the halothane experiments. Halothane and caffeine increased IM lactate dose-dependently in MHS pigs significantly more than in MHN pigs. Lactate and PCO2 were increased only at the injection site but not at 10 mm and 25 mm distance. MH susceptibility leads to a leftward shift of the dose-response curve for IM lactate after local injection of halothane and caffeine. The increase of lactate and carbon dioxide levels after local MH trigger injection is limited to a small area around the probe.

Compositional Changes in Muscle of Malignant Hyperthermia–Susceptible Pigs Due to Postmortem Alterations in Lipid Metabolism, Lipid Peroxidation and Protein Oxidation

Pigs with mutated calcium release channels are stress-susceptible and are known for increased postmortem metabolism which results in inferior meat quality. The lipid composition, lipid peroxidation, and oxidation of proteins of skeletal muscle homogenates from the longissimus muscle of pigs were investigated. Muscle tissue samples were obtained from malignant hyperthermia-resistant (MHR) and hyperthermia-susceptible (MHS) pigs in a postmortem time course. Differences in the n-3 polyunsaturated fatty acid composition with higher values in the susceptible pigs were detected (MHR versus MHS: 141±5 versus 116±4 μg/g wet wt.; P<0.05). This was accompanied by a dramatic 40% postmortem vitamin E loss in MHS muscles (22 h MHR versus 22 h MHS: 1.66±0.11 versus 1.06±0.16 μg/g wet wt.; P<0.05). Muscle tissue from MHS pigs oxidized at a significantly higher rate in comparison with tissue from MHR animals. This difference was also observed for spontaneous malondialdehyde formation as well as for oxidant-induced peroxidation rate (0.75 h MHR versus 0.75 h MHS: 0.81±0.09 versus 1.33±0.09 nmol/g wet wt.; P<0.05). No difference was observed in protein oxidation level in MHS and MHR animals. It is concluded that the enhanced lipid oxidation rate could contribute to the dramatic changes in meat quality from MHS pigs.

Room H, 10/16/2000 9: 00 AM - 11: 00 AM (PS) Modulation of 4-Chloro-m-Cresol-Induced Contractures in Skeletal Muscle Specimen from Malignant Hyperthermia Susceptible Pigs by Dantrolene

<b>Room H, 10/16/2000 9: 00 AM - 11: 00 AM (PS) Modulation of 4-Chloro-m-Cresol-Induced Contractures in Skeletal Muscle Specimen from Malignant Hyperthermia Susceptible Pigs by Dantrolene</b>

Room H, 10/16/2000 9: 00 AM - 11: 00 AM (PS) Comparison of the Effects of Ryanodine on Skeletal Muscle Preparations from Malignant Hyperthermia Susceptible Humans and Pigs

Room H, 10/16/2000 9: 00 AM - 11: 00 AM (PS) Comparison of the Effects of Ryanodine on Skeletal Muscle Preparations from Malignant Hyperthermia Susceptible Humans and Pigs

Malignant hyperthermia: fatigue characteristics of skeletal muscle.

Although the defects in cellular Ca(2+) homeostasis associated with malignant hyperthermia (MH) have been extensively studied, the functional consequences of the MH mutation are not clear. We used continuous and intermittent high-frequency stimulation to determine whether this mutation might alter the fatigue resistance of muscle from MH susceptible (MHS) pigs. Force decline with 10 s continuous stimulation (150 Hz) was significantly less in MHS muscle (58.4 +/- 1.0%) than in normal muscle (50.5 +/- 3.0%). With intermittent stimulation, there was no significant difference in tension decline between muscle types. Post-stimulation twitch and tetanus responses were similar in MHS and normal muscles except: 1) twitch potentiation was significantly greater in normal muscle after continuous stimulation, and 2) recovery of tetanic tension was slowed in MHS muscle. Although the MH defect does not cause major functional abnormalities, subtle differences in MHS muscle response to fatiguing stimulation are apparent. Therefore, it is unlikely the work capacity of MH patients would be limited by any MH associated defect within the muscle.

Cell injury and meat quality of pig in the time period post mortem from two genotypes susceptible or resistant to malignant hyperthermia

Muscles of pigs with mutated calcium release channels show increased metabolic activities, and ultimately inferior meat quality results. Different cellular changes in the time period post mortem (PM) are reported by investigating longissimus samples obtained immediately after death (0 h), 45 min PM, 4 h PM, and 22 h PM. The meat quality of such malignant hyperthermia susceptible (MHS) pigs was inferior to that of malignant hyperthermia resistant (MHR) pigs. Histological analysis of muscle fibres showed an enhanced rate and extent of the development of extracellular spaces in MHS muscle compared to MHR muscle in the time period PM. The permeability of crude sarcoplasmic reticulum (SR) to Ca2+ increased at a higher rate in MHS than in MHR muscle. Total muscle phospholipids contained a higher concentration of long chain n-3 fatty acids in MHS compared with MHR animals immediately after slaughter. The MHS homogenate lipids were more susceptible to stimulated peroxidation by Fe2+/ascorbate in samples obtained 45 min PM and 4 h PM, but there were no differences between the two genotypes for 0 h PM and 22 h PM samples, suggesting a retarded consumption of antioxidants in MHR compared to MHS muscle. The results suggest that in addition to the well-known denaturation of proteins, altered biochemical processes in muscle cells of stress-susceptible pigs cause injury to cell membranes as well as to SR membranes early in the time period PM.

Malignant hyperthermia: effects of sarcoplasmic reticulum Ca2+ pump inhibition.

In porcine malignant hyperthermia-susceptible (MHS) skeletal muscles, calcium release is abnormal and resting calcium may be elevated. Thus MHS muscles may have prolonged twitch relaxation and lower fusion frequencies, which would be augmented by inhibition of sarcoplasmic reticulum (SR) Ca2+ adenosine triphosphatase (ATPase) activity; bundles of intact muscle cells from MHS and normal pigs were used to investigate this possibility. Cooling and low-frequency stimulation, in combination, enhanced twitch fusion and prolonged twitch relaxation significantly more in MHS than in normal muscles (e.g., 34 +/- 4% versus 16 +/- 4% fusion, and 82.4 +/- 9.4 ms versus 43.2 +/- 7.8 ms half-relaxation time, for MHS and normal muscles, respectively). Similarly, inhibition of the SR Ca2+ ATPase by cyclopiazonic acid resulted in significantly greater twitch fusion in MHS muscles. These results were consistent with predicted effects of enhanced SR Ca2+ release and/or elevated resting calcium in MHS muscles and indicate that cooling during a malignant hyperthermia crisis could actually increase the force of muscle contractures.

Metabolic events with spontaneous malignant hyperthermia crisis in an anaesthetized pig.

To analyze metabolic changes associated with a fulminant malignant hyperthermia (MH) crisis developed spontaneously in an MH susceptible pig which was part of 12 pigs undergoing metabolic investigation (six MH susceptible and six controls) and had been anaesthetized with a non-triggering agent (pentobarbitone). The pig was placed in a cradle and then inserted into a 4.7 T magnet bore. The semi-membranous muscle was submitted to three repetitive stimulation-recovery sessions. 31-P magnetic resonance spectra and mechanical data were recorded. The pig developed a non-rigid MH crisis during recovery from the second set of experiments. Although no mechanical work was performed, dramatic metabolic changes were noted. Twitch tension decreased progressively reaching zero while mouth temperature continuously increased to 44.5 degrees C. Phosphocreatine (PCr) consumption was coupled to Pi accumulation. Also, a marked intracellular acidosis and a large accumulation of phosphomonoesters (PME) were observed, probably as a result of massive glycolysis activation. Interestingly, ATP level remained constant. These irreversible mechanisms may constitute a metabolic dead-end coupling calcium pumping ATP-consuming processes and ATP synthesis through PCr breakdown and anaerobic glycolysis. They do not differ from metabolic changes previously reported in rigid forms of MH crisis.

Calmodulin sensitivity of the sarcoplasmic reticulum ryanodine receptor from normal and malignant-hyperthermia-susceptible muscle.

Ca2+ release from sarcoplasmic reticulum (SR) of malignant-hyperthermia-susceptible (MHS) muscle is hypersensitive to Ca2+ and caffeine. To determine if an abnormal calmodulin (CaM) regulation of the SR Ca(2+)-release-channel-ryanodine-receptor complex (RYR1) contributes to this hypersensitivity, we investigated the effect of CaM on high-affinity [3H]ryanodine binding to isolated SR vesicles from normal and MHS pig skeletal muscle. CaM modulated [3H]ryanodine binding in a Ca(2+)-dependent manner. In the presence of maximally activating Ca2+ concentrations, CaM inhibited [3H]ryanodine binding with no differences between normal and MHS vesicles. In the absence of Ca2+, however, CaM activated [3H]ryanodine binding with a 2-fold-higher potency in MHS vesicles. Significant differences between normal and MHS tissue were observed for CaM concentrations between 50 nM and 10 microM. A polyclonal antibody raised against the central region of RYR1 specifically inhibited this activating effect of CaM without affecting the inhibition by CaM. This indicates that the central region of RYR1 is a potential binding domain for CaM in the absence of Ca2+. It is suggested that in vivo an enhanced CaM sensitivity of RYR1 might contribute to the abnormal high release of Ca2+ from the SR of MHS muscle.