Hyperthermia Susceptible Pigs Research Articles

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.

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.

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.

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>

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.

Slow calcium current is not reduced in malignant hyperthermic porcine myotubes

Malignant hyperthermia-susceptible (MHS) pigs express a sarcoplasmic reticulum (SR) Ca(2)+-release channel mutation that results in lower than normal contractile thresholds in skeletal muscles. In adult MHS pig muscles the L-type calcium current (ls) is also reduced. We tested the hypothesis that there is a causal relationship between ls and the lower contractile threshold by recording ls from MHS and normal porcine myotubes using the whole cell patch-clamp technique. Current voltage relationships for both MHS and normal myotubes were similar, with peak ls between +20 and +30 mV. Maximum ls amplitudes were not different from (normal: 4976 +/- 566 pA; MHS:6516 +/- 1088 pA) nor was ls specific density (normal: 9.0 +/- 0.8; MHS: 8.8 +/- 1.1 pA/pF). In both MHS and normal myotubes, both the dihydropyridine antagonist PN200-110 (200 nmol/L) and holding the membrane potential at -10mV for 5 min decreased ls significantly (by more than 50%). There was no apparent direct relationship between the mutation in the SR Ca(2)+ -release channel mutation on muscle development.

Decreased phospholipid polyunsaturated fatty acid content and superoxide dismutase activity in cardiac muscle of malignant hyperthermia-susceptible swine

Homogenates of cardiac left ventricle from malignant hyperthermia-susceptible (MHS) pigs produced a circa 72% more pentane than those from malignant hyperthermia-resistant (MHR) animals, indicating enhanced peroxidation of n-6 fatty acids. This is consistent with the observed circa 70% decrease in total phospholipid polyunsaturated fatty acids (PUFA) in MHS compared with MHR tissue, a decrease mainly due to the quantitatively greater loss of n-6 PUFA. Although the percentage loss of n-3 PUFA was greater than that of n-6 PUFA (90% vs 60%), absolute amounts were insufficient to register as ethane production. Three-fold greater phospholipid content of MHS compared with MHR ventricles indicates reduced neutral lipid content probably due to increased catecholamine stimulation. These findings were associated with a small but significant decrease in superoxide dismutase activity in MHS tissues.

Tissue distribution of ryanodine receptor isoforms and alleles determined by reverse transcription polymerase chain reaction.

The tissue distribution of mRNA for ryanodine receptor (ryr) isoforms in various porcine tissues has been determined using the reverse transcription-polymerase chain reaction (RT-PCR). First strand cDNA was synthesized from total tissue RNA with reverse transcriptase and random hexamer primers. PCR primers were selected to amplify an approximately 500-base pair segment from homologous regions near the 5' end of the skeletal (ryr1), cardiac (ryr2), or brain (ryr3) ryr cDNA sequences. The specific amplification of each of the ryr isoforms was confirmed by restriction enzyme mapping and DNA sequencing. A ryr1 RT-PCR product was identified in skeletal muscle and esophagus, a ryr2 RT-PCR product was identified in cardiac muscle, aorta and esophagus, and a ryr3 RT-PCR product was identified in skeletal and cardiac muscle, aorta, esophagus, adrenal gland, small intestine, and lung. All three ryr isoforms were identified throughout the brain, including the parietal, frontal, and temporal lobes of the cerebrum, thalamus/hypothalamus, cerebellum, and brain stem. The normal (Arg615) and mutant (Cys615) ryr1 alleles were expressed in the brains of normal and malignant hyperthermia susceptible pigs, respectively. These results thus demonstrate expression of two ryr isoforms in each type of striated muscle, and all ryr isoforms in a number of regions of the nervous system. The wide distribution of ryr1 in the brain provides a possible neurogenic etiology of malignant hyperthermia.

Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation

The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel.

Fluidity state of lymphocyte plasma membrane in malignant hyperthermia susceptible pigs and humans

Recent studies suggest that abnormalities occur at the lipid level in malignant hyperthermia susceptible humans and pigs. To test this hypothesis, we first investigated the physical state of plasma membranes of lymphocytes isolated from normal and malignant hyperthermia susceptible swine. In halothane-challenged pigs, malignant hyperthermia susceptibility was also assessed by ryanodine binding assay on purified sarcoplasmic reticulum membranes. The results clearly show that plasma membrane of lymphocytes from malignant hyperthermic pigs are significantly more fluid than controls. We then attempted to apply the same methodology to lymphocytes prepared from human patients previously diagnosed by the halothane and caffeine contracture test. In that case, there was no clear relationship between malignant hyperthermia susceptibility and the fluidity state of lymphocyte plasma membranes.

Analysis of lipid composition of sarcoplasmic reticulum membranes from normal and malignant hyperthermic pig skeletal muscle.

In search of a general membrane defect hypothesis for malignant hyperthermia syndrome, we analysed the lipid profiles of heavy sarcoplasmic reticulum membranes isolated from normal and malignant hyperthermia longissimus dorsi pig muscle. Malignant hyperthermia susceptibility was assessed by halothane challenge of pigs. Sarcoplasmic reticulum membranes from malignant hyperthermia susceptible pigs differed significantly from control ones in the cholesterol content and phosphatidylethanolamine/phosphatidylcholine ratio; both were higher in former membranes. These latter lipid modifications were in agreement with the significant increase of their bulk lipid viscosity, as evidenced by an increase of diphenyl hexatriene fluorescence anisotropy. The increased level of phosphatidylethanolamine associated with the decreased content of phosphatidylcholine in malignant hyperthermic membranes was shown to be a potential consequence of depressed activities of both phospholipid N-methyltransferase I and II activities. Finally, the distribution of fatty acids in these particular phospholipids showed no change in phosphatidylcholine molecules, whereas the percentage of arachidonate and stearate in the phosphatidylethanolamine species were respectively higher and lower in malignant hyperthermic membranes. These differences in major phospholipids content and the enrichment of a metabolically important fatty acyl chains in malignant hyperthermia sarcoplasmic reticulum membranes strongly suggest that the lipid metabolism may contribute to the molecular mechanism of malignant hyperthermia syndrome.

Reconstitution of abnormalities in the malignant hyperthermia-susceptible pig ryanodine receptor

Malignant hyperthermia-susceptible (MHS) pigs homozygous for the Cys615 ryanodine receptor allele demonstrate altered sarcoplasmic reticulum (SR) ryanodine binding and Ca2+ release channel regulatory properties when compared with normal pigs homozygous for the Arg615 allele. While solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified MHS and normal ryanodine receptors had a similar dissociation constant (Kd) for ryanodine, maximum binding, and Ca2+ concentration for half-maximal stimulation and inhibition of ryanodine binding (Ca2+(0.5)); however, after reconstitution into proteoliposomes, the purified MHS and normal receptors had Kd values for ryanodine of 75 and 150 nM, respectively, which were significantly different. The purified MHS and normal porcine ryanodine receptors also had similar single-channel Cs+ conductance, optimal cis-Ca2+ for channel opening, and cis-Ca2+(0.5) for channel activation. Significantly, at inactivating levels of cis-Ca2+ (> 0.1 mM), MHS channels had a greater open probability, a higher cis-Ca2+(0.5) for inhibition of channel opening (250 vs. 75 microM for MHS and normal, respectively), longer mean open times, and shorter mean closed times than did normal channels. We conclude that the mutation at residue 615 causes a detectable alteration in ryanodine receptor/Ca2+ channel activity and thus may represent the primary defect responsible for the altered SR Ca2+ regulation characteristic of MHS porcine muscle.

Structural and functional correlates of a mutation in the malignant hyperthermia-susceptible pig ryanodine receptor

The skeletal muscle ryanodine receptor of malignant hyperthermia-susceptible (MHS) pigs contains a mutation at residue 615 that is highly correlated with various abnormalities in the regulation of sarcoplasmic reticulum (SR) Ca 2+ channel activity. In isolated SR membranes the Arg 615 to Cys 615 ryanodine receptor mutation is now shown to be directly responsible for an altered tryptic peptide map, due to the elimination of the Arg 615 cleavage site. Furthermore, trypsin treatment released 86–99 kDa ryanodine receptor fragments encompassing residue 615 from the SR membranes. We conclude that the 86–99 kDa domain containing residue 615 is near the cytoplasmic surface of the ryanodine receptor and likely near important Ca 2+ channel regulatory sites.

Genomic organization and analysis of the 5′ end of the porcine ryanodine receptor gene ( ryr1)

In this study we describe the isolation of genomic clones of the 5′ region of the porcine ryanodine receptor gene, a candidate for malignant hyperthermia in pigs and humans. The recombinants were isolated from a porcine liver, genomic DNA library in phage EMBL3A after screening with PCR amplified DNA fragments. The exon/intron structure of the ryanodine receptor gene was determined by DNA sequencing. Based on the sequence data it was possible to develop a simple test for the detection of malignant hyperthermia susceptible and normal pigs.

Excitation-contraction coupling in pigs heterozygous for malignant hyperthermia.

A defect in the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel of malignant hyperthermia-susceptible (MHS) pigs greatly enhances SR calcium release in pigs homozygous for the malignant hyperthermia (MH) gene. In pigs heterozygous at this locus, rates of calcium release from isolated SR stimulated by Ca2+, ATP, or caffeine are intermediate to those of both MHS and normal SR [Mickelson et al. Am. J. Physiol. 257 (Cell Physiol. 26): C787-C794, 1989]. In this study bundles of intact muscle cells dissected from pigs of various genotypes were used to examine the effects of the MH gene on contractile responses to caffeine (direct stimulation of the SR) or to surface membrane (sarcolemma) depolarization (i.e., stimulation by way of the steps in excitation-contraction coupling). The caffeine threshold for contractures in the heterozygous muscles (5 mM) was intermediate to both types of homozygous muscles (2 mM for MHS and 10 mM for normal) as is the case with direct stimulation of calcium release from SR vesicles [Mickelson et al. Am. J. Physiol. 257 (Cell Physiol. 26): C787-C794, 1989]. Sarcolemmal depolarization was elicited by electrical stimuli or elevated extracellular potassium. Control twitch tension for MHS and heterozygous muscles did not differ and was significantly greater in both than in homozygous normal muscles. Potassium-induced contractures were significantly larger in MHS and heterozygous than in normal muscles. Thus, in heterozygous muscles, force production via sarcolemmal depolarization (twitches and potassium contractures) was enhanced as much as in homozygous MHS muscles. This could be the result of feedback from abnormal SR calcium channels producing altered (enhanced) transverse tubule to SR signal transduction.

Calcium ATPase in the sarcoplasmic reticulum of muscle from normal and malignant hyperthermia susceptible pigs

The density of Mg 2+-dependent Ca 2+ ATPase in the terminal cisternae of pig skeletal muscle fibres was investigated to discover whether a reduction in Ca 2+ ATPase content impairs Ca 2+ sequestration and contributes to the elevated myoplasmic Ca 2+ concentration in malignant hyperthermia. Unexpectedly, immunogold electron microscopy showed an increase in Ca 2+ ATPase, while densitometry of SDS-polyacrylamide gels suggested that the Ca 2+ ATPase content of terminal cisternae vesicles did not change. The affinity of Ca 2+ ATPase in vesicles for our monoclonal antibody was not altered. We suggest that the availability of antigenic sites in malignant hyperthermia increases after processing for electron microscopy, perhaps as a consequence of altered sarcoplasmic reticulum membrane properties.