MH-susceptible Pigs Research Articles

A REVIEW: MALIGNANT HYPERTHERMIA‐A GENETIC DISEASE OF SODIUM CHANNEL FUNCTION

Malignant Hyperthermia is inherited as a dominant gene in pigs and in humans. It is characterized by a rapidly increasing body temperature up to 118°F with a metabolic rate over 10× normal, an intense peripheral vasoconstriction with blood pressures over 400 mmHg, and a fatal outcome in most cases.The MH syndrome is triggered by exercise, hauling to market, breeding activity, hot weather, and other stress factors. In humans, the cases are triggered by depolarizing muscle relaxants, Halothane anesthesia, and other halogenated anesthesia compounds. Non‐depolarizing muscle relaxants such as Pancuronium and Vecuronium are safer to use. Organon 9426 (Rocuronium) is safe to use and provides carry over protection against the development of MH.The widespread use of Sevoflurane in humans has reduced the incidence of MH to 1:550,000. Only three cases of MH have been reported in the USA during the past 30 years. They developed in WI, FL and SC.The MH susceptible pig is an outstanding animal model for research purposes and has enabled the development of new muscle relaxants and anesthesia agents that are safer for human use.The sodium channels in MH susceptible animals are leaky and allow the influx of sodium into muscle cells which must be pumped out by sodium‐potassium ATPase, thereby using ATP and generating heat.Malignant Hyperthermia is inherited as a dominant gene in pigs and in humans. It is characterized by a rapidly increasing body temperature up to 118°F with a metabolic rate over 10× normal, an intense peripheral vasoconstriction with blood pressures over 400 mmHg, and a fatal outcome in most cases.The MH syndrome is triggered by exercise, hauling to market, breeding activity, hot weather, and other stress factors. In humans, the cases are triggered by depolarizing muscle relaxants, Halothane anesthesia, and other halogenated anesthesia compounds. Non‐depolarizing muscle relaxants such as Pancuronium and Vecuronium are safer to use. Organon 9426 (Rocuronium) is safe to use and provides carry over protection against the development of MH.The widespread use of Sevoflurane in humans has reduced the incidence of MH to 1:550,000. Only three cases of MH have been reported in the USA during the past 30 years. They developed in WI, FL and SC.The MH susceptible pig is an outstanding animal model for research purposes and has enabled the development of new muscle relaxants and anesthesia agents that are safer for human use. The sodium channels in MH susceptible animals are leaky and allow the influx of sodium into muscle cells which must be pumped out by sodium‐potassium ATPase, thereby using ATP and generating heat.Support or Funding Information

Malignant hyperthermia: A runaway thermogenic futile cycle at the sodium channel level

Malignant Hyperthermia (“MH”)—the rapid onset of extremely high fever with muscle rigidity—is caused by a runaway heat production futile cycle mediated via the sodium channels at the myoneural receptor sites. MH is not triggered by non-depolarizing muscle relaxants; however, depolarizing muscle relaxants may trigger it [1]. Here we present a de novo hypothesis of how MH is triggered and develops. We believe that the acetylcholine receptor/sodium channels in the muscles of MH susceptible pigs initiate MH by allowing an increased flux of sodium ions when it is depolarized by acetylcholine or other depolarizing agents, such as succinylcholine and Halothane. Our theory is consistent with our observations of the effects of general anesthetics over twenty years. Succinylcholine is a depolarizing agent that is a potent MH trigger. Acetylcholine, the natural depolarizing muscle activator, may trigger MH if the susceptible patient or animal is exposed to sufficient stress, i.e., during strenuous activity, such as transport, fighting, breeding, etc. Halothane apparently destabilizes the myoneural sodium channels, which rapidly induces MH. The increased sodium channel activity releases heat with cascades that further releases of heat which results in the rapid onset of MH. MH susceptible pigs have increased action potential amplitudes at their myoneural junctions that are abnormally long in duration. This increased activity is thought to induce hypertrophy of muscle mass, increase metabolic rate, and cause other physical manifestations. When slaughtered, this increased metabolic activity causes the rapid post mortem release of heat in the muscles of MH susceptible pigs and, at the same time, the accumulation of low acidity, all of which denatures the muscle proteins to result in a pale, soft, exudative, pork meat considered to be of lesser quality for human consumption. The potency of inhalation anesthetics as a MH triggers varies widely. The inhalation anesthetic Halothane is a strong trigger of MH, causing MH within minutes of exposure. In contrast, the anesthetic Sevoflurane is a very weak trigger of MH, requiring several hours of inhalation exposure to trigger MH. Because of this, changing from Halothane to Sevoflurane as the general anesthetic of choice for surgeries in hospitals in the Greater Kansas City area during 1994 to 2006 led to an 11-fold decrease in the incidence of MH, from 1:50,000 to 1:550,000 [11]. One non-depolarizing muscle relaxant, Organon 9426 (“Rocuronium”) temporarily prevents MH in MH susceptible pigs when they are given sufficient dosages of it before being challenged with either Halothane or succinylcholine. Binding Rocuronium to the myoneural receptor sites apparently stabilizes them, thereby preventing increased sodium channel activity, and resulting MH. However, other non-depolarizing muscle relaxants do not have this protective effect— for examples Vecuronium, Arduan, and Organon 9616 do not. Uncoupling of mitochondria is not the source of accelerated heat production in MH susceptible pigs, as heart, liver, and skeletal muscle mitochondria isolated from MH susceptible pigs are all competent.

Reduced Threshold for Luminal Ca2+ Activation of RyR1 Underlies a Causal Mechanism of Porcine Malignant Hyperthermia

Naturally occurring mutations in the skeletal muscle Ca(2+) release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca(2+) release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca(2+) release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca(2+) release during store Ca(2+) overload, a process we refer to as store overload-induced Ca(2+) release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca(2+) activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca(2+) and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca(2+) activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca(2+) regulation of RyR1 represents a primary causal mechanism of MH.

Postulated role of interdomain interaction between regions 1 and 2 within type 1 ryanodine receptor in the pathogenesis of porcine malignant hyperthermia

We have demonstrated recently that CICR (Ca2+-induced Ca2+ release) activity of RyR1 (ryanodine receptor 1) is held to a low level in mammalian skeletal muscle ('suppression' of the channel) and that this is largely caused by the interdomain interaction within RyR1 [Murayama, Oba, Kobayashi, Ikemoto and Ogawa (2005) Am. J. Physiol. Cell Physiol. 288, C1222-C1230]. To test the hypothesis that aberration of this suppression mechanism is involved in the development of channel dysfunctions in MH (malignant hyperthermia), we investigated properties of the RyR1 channels from normal and MHS (MH-susceptible) pig skeletal muscles with an Arg615-->Cys mutation using [3H]ryanodine binding, single-channel recordings and SR (sarcoplasmic reticulum) Ca2+ release. The RyR1 channels from MHS muscle (RyR1MHS) showed enhanced CICR activity compared with those from the normal muscle (RyR1N), although there was little or no difference in the sensitivity to several ligands tested (Ca2+, Mg2+ and adenine nucleotide), nor in the FKBP12 (FK506-binding protein 12) regulation. DP4, a domain peptide matching the Leu2442-Pro2477 region of RyR1 which was reported to activate the Ca2+ channel by weakening the interdomain interaction, activated the RyR1N channel in a concentration-dependent manner, and the highest activity of the affected channel reached a level comparable with that of the RyR1MHS channel with no added peptide. The addition of DP4 to the RyR1MHS channel produced virtually no further effect on the channel activity. These results suggest that stimulation of the RyR1MHS channel caused by affected inter-domain interaction between regions 1 and 2 is an underlying mechanism for dysfunction of Ca2+ homoeostasis seen in the MH phenotype.

Dantrolene Stabilizes Domain Interactions within the Ryanodine Receptor

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.

Caffeine sensitivity of native RyR channels from normal and malignant hyperthermic pigs: effects of a DHPR II–III loop peptide

Enhanced sensitivity to caffeine is part of the standard tests for susceptibility to malignant hyperthermia (MH) in humans and pigs. The caffeine sensitivity of skeletal muscle contraction and Ca(2+) release from the sarcoplasmic reticulum is enhanced, but surprisingly, the caffeine sensitivity of purified porcine ryanodine receptor Ca(2+)-release channels (RyRs) is not affected by the MH mutation (Arg(615)Cys). In contrast, we show here that native malignant hyperthermic pig RyRs (incorporated into lipid bilayers with RyR-associated lipids and proteins) were activated by caffeine at 100- to 1000-fold lower concentrations than native normal pig RyRs. In addition, the results show that the mutant ryanodine receptor channels were less sensitive to high-affinity activation by a peptide (C(S)) that corresponds to a part of the II-III loop of the skeletal dihydropyridine receptor (DHPR). Furthermore, subactivating concentrations of peptide C(S) enhanced the response of normal pig and rabbit RyRs to caffeine. In contrast, the caffeine sensitivity of MH RyRs was not enhanced by the peptide. These novel results showed that in MH-susceptible pig muscles 1). the caffeine sensitivity of native RyRs was enhanced, 2). the sensitivity of RyRs to a skeletal II-III loop peptide was depressed, and 3). an interaction between the caffeine and peptide C(S) activation mechanisms seen in normal RyRs was lost.

Effects of a 5HT2 receptor agonist on anaesthetized pigs susceptible to malignant hyperthermia

The pathophysiology of the serotoninergic system in malignant hyperthermia (MH) is not completely understood. The serotonin-2 (5HT(2A)) receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) induces typical MH symptoms, including skeletal muscle rigidity, an increase in body temperature, hyperventilation and acidosis in conscious MH-susceptible (MHS) pigs. Whether these symptoms are directly generated in skeletal muscle, result from central serotonergic overstimulation or from a porcine stress syndrome remains unresolved. In this study the in vivo effects of DOI on anaesthetized (and thus stress-protected) MHS and MH-normal (MHN) pigs were investigated. and results. DOI 1 mg kg(-1) was administered three times at 40-min intervals to five MHS and five MHN anaesthetized pigs. Body temperature, heart rate, muscle tone, arterial carbon dioxide pressure (Pa(CO(2))), pH and creatine kinase concentrations were measured. The clinical occurrence of MH was defined by Pa(CO(2)) above 70 mm Hg and an increase in body temperature of more than 2 degrees C. Intragroup differences were analysed with the Friedman test as an overall non-parametric ANOVA and, in case of significance, with the Wilcoxon test. Intergroup comparisons were performed with the Mann-Whitney U-test (statistical significance P<0.05). MHS and MHN pigs developed muscle fasciculations, significant increases in body temperature and Pa(CO(2)) and a significant decrease in pH after the administration of DOI. These changes were comparable in both groups until the third dose of DOI, when in MHS pigs heart rate and Pa(CO(2)) rose significantly and pH fell significantly compared with MHN pigs. All MHS pigs fulfilled the MH criteria. Body temperature increased by more than 2 degrees C in all MHN pigs and Pa(CO(2)) exceeded 70 mm Hg in two. Thus, two MHN pigs fulfilled the criteria of MH. The comparability of the clinical presentation following DOI administration in MHS and MHN animals and the order of the development of MH-like symptoms favour the hypothesis of a central serotonergic overstimulation, leading to a serotonin syndrome.

Changes in thiol reactivity and extractability of myofibril bound cardiac troponin C in porcine malignant hyperthermia.

We recently showed that a shift ( approximately 44%) in cardiac myosin isozyme from V3 to V1 occurs in the hearts of malignant hyperthermia (MH)-susceptible pigs and may be important in the disease. To follow up this finding, we investigated whether this myosin isozyme shift results in conformational changes in cardiac troponin C (cTnC). The two cysteine residues (Cys-35 and Cys-84) in the regulatory domain of cTnC make it possible to attach conformational probes to this region. Incorporation of a fluorescent probe, 7-diethylamino-3-(4'maleimidylphenyl)-4-methylcoumarin (CPM), into myofibril-bound cTnC was measured by alkaline urea gel electrophoresis, followed by quantification of the protein and the fluorescent label on the gels. The structural stability of cTnC incorporated into cardiac myofibrils was compared between normal and MH-susceptible pigs by selective cTnC extraction and re-incorporation. Changes were detected in both the reactivity of cTnC with CPM in rigor myofibrils and cTnC incorporation into myofibrils from the hearts of MH-susceptible pigs. These changes are very likely to be a consequence of the cardiac myosin isozyme shift in the hearts of MH-susceptible pigs, which may contribute to the changes in the myofilament response to Ca(2+) binding and to the modulation of cardiac contractility seen in this disease.

Altered Expression of Cardiac Myosin Isozymes Associated with the Malignant Hyperthermia Genotype in Swine

Anesthetic-induced malignant hyperthermia (MH) in humans and pigs is associated with dramatic alterations in cardiac function. However, it remains controversial as to whether MH-associated cardiac symptoms represent a primary difference of myocardium or a secondary alteration consequent to increases in the hyperthermic stress. Here the authors describe changes in myosin isoform expression in the hearts of MH-susceptible pigs with and without prior exposure to halothane. One group of pigs was diagnosed as MH susceptible by halothane challenge and Hal-1843 nucleotide examination. To determine if there is an effect of halothane exposure, another group of pigs was diagnosed by simple MH genotyping without exposure to halothane. After diagnosis and genotyping, animals with and without exposure to halothane were killed to study cardiac myosin isozyme distributions, cardiac myofibrillar adenosine triphosphatase (ATPase) activity, and the steepness of the Ca2+-ATPase activity relation in the hearts of normal and susceptible pigs. The altered myosin isozyme expression was analyzed by pyrophosphate gel electrophoresis. Malignant hyperthermia-susceptible animals with the prior halothane challenge showed an increased V1 myosin (-44%) expression, increased myofibrillar ATPase activity (-25%) and increased steepness of the Ca2+-ATPase activity relation. Without exposure to halothane, no change of myofibrillar ATPase activity was found in the hearts of different genotyped pigs, but there was a small increase in expression of V1 myosin (-5%) in the mutant (TT). The potential modulation of V1 myosin expression occurs in the hearts of MH-susceptible pigs. The added stress by halothane challenge would further cause a V3 --> V1 shift, which may be attributed to the long-term effects of hyperthermic stress.

Room I, 10/16/2000 2: 00 PM - 4: 00 PM (PS) Local Intramuscular Application of Halothane in MH-Susceptible Pigs without Systemic Effects

<b>Room I, 10/16/2000 2: 00 PM - 4: 00 PM (PS) Local Intramuscular Application of Halothane in MH-Susceptible Pigs without Systemic Effects</b>

Hemodynamic and metabolic manifestations of acute endotoxin infusion in pigs with and without the malignant hyperthermia mutation.

The hypermetabolic state induced by acute endotoxemia and malignant hyperthermia (MH) may be indistinguishable. The aims of this study were (1) to investigate the differences between MH and sepsis, (2) to determine whether acute endotoxemia can trigger MH, and (3) to establish the effects of dantrolene in these two disorders. Three groups of swine were studied. All pigs were invasively monitored and initially anesthetized with nontriggering agents. A placebo MH-susceptible group (n = 5) received normal saline whereas the endotoxin groups (MH-susceptible, n = 6; MH-negative, n = 4) received intravenous endotoxin (250 microg/kg total) during 2.5 h. Halothane (1.5%) and succinylcholine (2-4 mg/kg) were then administered, followed by two doses of dantrolene (4 mg/kg total). Endotoxin infusion resulted in pulmonary hypertension and systemic hypotension in pigs with and without the MH mutation, but did not trigger MH. Halothane and succinylcholine triggered MH, evidenced by a markedly higher oxygen consumption in the MH-susceptible pigs that received endotoxin (325+/-196 ml/min) and those that did not (374+/-110 ml/min) compared to the MH-negative pigs (69+/-15 ml/min, P<0.0009), as well as muscular rigidity in the susceptible animals. Dantrolene reversed these changes. Three of the six MH-susceptible pigs that received endotoxin died; two died soon after triggering and one after dantrolene administration. In contrast, none of the MH-negative pigs or the MH-susceptible pigs that did not receive endotoxin died (0 of 9 vs. 3 of 6, P = 0.044). Endotoxemia does not trigger MH, but may worsen outcome if it occurs.

Reduced inhibitory effect of Mg2+ on ryanodine receptor-Ca2+ release channels in malignant hyperthermia

Malignant hyperthermia (MH) is a potentially fatal, inherited skeletal muscle disorder in humans and pigs that is caused by abnormal regulation of Ca2+ release from the sarcoplasmic reticulum (SR). MH in pigs is associated with a single mutation (Arg615Cys) in the SR ryanodine receptor (RyR) Ca2+ release channel. The way in which this mutation leads to excessive Ca2+ release is not known and is examined here. Single RyR channels from normal and MH-susceptible (MHS) pigs were examined in artificial lipid bilayers. High cytoplasmic (cis) concentrations of either Ca2+ or Mg2+ (>100 microM) inhibited channel opening less in MHS RyRs than in normal RyRs. This difference was more prominent at lower ionic strength (100 mM versus 250 mM). In 100 mM cis Cs+, half-maximum inhibition of activity occurred at approximately 100 microM Mg2+ in normal RyRs and at approximately 300 microM Mg2+ in MHS RyRs, with an average Hill coefficient of approximately 2 in both cases. The level of Mg2+ inhibition was not appreciably different in the presence of either 1 or 50 microM activating Ca2+, showing that it was not substantially influenced by competition between Mg2+ and Ca2+ for the Ca2+ activation site. Even though the absolute inhibitory levels varied widely between channels and conditions, the inhibitory effects of Ca2+ and Mg2+ were virtually identical for the same conditions in any given channel, indicating that the two cations act at the same low-affinity inhibitory site. It seems likely that at the cytoplasmic [Mg2+] in vivo (approximately 1 mM), this Ca2+/Mg2+-inhibitory site will be close to fully saturated with Mg2+ in normal RyRs, but less fully saturated in MHS RyRs. Therefore MHS RyRs should be more sensitive to any activating stimulus, which would readily account for the development of an MH episode.

Effects of the 5-HT2 receptor antagonist ritanserin on halothane-induced increase of inositol phosphates in porcine malignant hyperthermia.

Recent studies have shown a significant increase of inositol phosphates (IPs) in skeletal muscle during episodes of halothane-induced malignant hyperthermia (MH) in pigs. After treatment with dantrolene and disappearance of MH crisis the IP concentrations returned to basal levels. In order to examine if the increase of IPs during halothane-induced MH may be related to an enhanced IP synthesis in response to activation of 5-HT2 (5-hydroxytryptamine) receptors, the effects of ritanserin, a selective 5-HT2 receptor antagonist, on IP levels were investigated. Biopsies of skeletal muscle of the hindlimbs were obtained in random order and IPs were determined in homozygous MH-susceptible (MHS) and MH-non-susceptible (MHN) swine in the following order: (1) basal, (2) after treatment with ritanserin (2.0 mg/kg), (3) after halothane challenge (3 vol% for 20 min). Basal concentrations of all IPs were higher in MHS than in MHN swine. Ritanserin did not cause any significant changes of IP levels compared to the basal concentrations in MHS and MHN pigs. In MHS pigs, ritanserin did not prevent a halothane-induced MH-crisis. After halothane challenge, 1,3,4-IP3, 1,3,4,6-IP4 and 1,3,4,5-IP4 levels were increased in MHS (during MH crisis) vs. basal concentrations, whereas no changes were found in MHN pigs. Since the increases of IP levels in MHS pigs during MH crisis found in the present study were comparable to those without pretreatment with ritanserin, shown by recent studies, it may be concluded that ritanserin does not prevent the increase of IPs during a halothane-induced MH. Thus, the present data indicate that increases of IP levels during halothane-induced MH in swine are due to other mechanisms than 5-HT mediated enhancement of IP synthesis.

Malignant hyperthermia: excitation-contraction coupling, Ca2+ release channel, and cell Ca2+ regulation defects.

Malignant hyperthermia (MH) is a disorder of skeletal muscle in which certain anesthetic agents trigger a sustained elevation in myoplasmic Ca2+ concentration that activates metabolic and contractile activity. This review focuses on the biochemical and physiological alterations in the skeletal muscle of MH-susceptible (MHS) pigs and humans that appear responsible for this inherited disorder. In porcine MH, these studies identified the skeletal muscle sarcoplasmic reticulum Ca2+ release channel gene (RYR1) as the site of the defect. A mutation in this protein results in altered excitation-contraction coupling and secondary changes in porcine muscle structure and function. Although RYR1 mutations have been reported in many MHS human families, there is also significant genetic heterogeneity, and much less is known as to the underlying mechanism responsible for altered human myoplasmic Ca2+ regulation. The effects of caffeine and anesthetic agents on MHS and normal muscle are also discussed to better understand the basis for the in vitro clinical test for this disorder and mechanisms responsible for the initiation and maintenance of MH episodes in susceptible individuals. Finally, we examine the possiblity of a defect in Ca2+ regulation in tissues other than skeletal muscle. Current understanding of the molecular basis of MH elegantly illustrates the successful integration of knowledge obtained from all fields of biological and clinical science.

Cardiovascular and Metabolic Responses to Anesthetic-induced Malignant Hyperthermia in Swine

Several cardiovascular disturbances, such as tachycardia and hypotension, are observed during human and porcine malignant hyperthermic (MH) crises. However, the pathophysiologic mechanisms responsible for the deterioration of cardiovascular function during MH are not completely known. The purpose of this study was to elucidate the changes in left ventricular (LV) function and metabolism and the systemic and regional hemodynamics during anesthetic-induced MH in swine. The study was carried out in 12 open-chest MH-susceptible pigs and in 8 healthy control (non-MH-susceptible) pigs under the same conditions. The cardiovascular and metabolic responses to halothane (1% inspired) and succinylcholine (3 mg.kg-1 intravenously 15 min after the start of halothane administration) were studied. Global hemodynamic and LV variables (expressed as means +/- SEM) were determined over a period of 90 min after the beginning of halothane exposure. Simultaneous investigations were performed on hindleg and cardiac muscle to compare the regional functional and metabolic changes in these tissues. MH was triggered in all MH-susceptible pigs. Early (10-30 min) cardiovascular changes during the development of MH consisted of a rapid increase in heart rate (from 86 +/- 4 to 204 +/- 8 beats.min-1), cardiac index (+84%), and peak rate of change in LV pressure (+150%); stroke volume index (-24%) and mean aortic pressure (-13%) decreased progressively even in the early stage of MH. These alterations were accompanied by an early and persistent reduction in systemic vascular resistance (maximally -57%) with an increase in aortic pressure amplitude. Early changes in coronary and peripheral hemodynamics during the development of MH consisted of a three-fold increase in coronary blood flow in conjunction with a marked decrease (-77%) in coronary vascular resistance. The early circulatory changes were associated with a fourfold increase in myocardial and a 2.5-fold increase in peripheral O2 consumption. The ratio of the LV stroke work index (LVWI) to myocardial O2 consumption (MVO2) was significantly decreased, by a factor of 5. Increased catecholamine concentrations and myocardial lactate and H+ production could be demonstrated throughout the MH crisis. In the late stage of MH (> 30 min), pronounced hypotension and a subsequent decrease in cardiac index were noted. These changes were associated with a significant reduction in LV end-diastolic pressure, from 9 +/- 1 to 6 +/- 1 mmHg (P < 0.05), and in the rate of change in LV pressure, by a maximum of -25%. Coronary vascular resistance remained reduced while coronary blood flow decreased. Peripheral (hind-leg) blood flow initially increased by 48% while peripheral vascular resistance decreased by 42%, followed by a fivefold increase in peripheral vascular resistance with a marked decrease in peripheral blood flow (-88%) in the late phase of MH. The current findings indicate that metabolic status during MH is characterized by a demand ischemia of the heart and of the skeletal muscle. Insufficient coronary blood flow and increased metabolism as a result of tachycardia and increased concentrations of catecholamines are the dominant factors contributing to the dramatic alteration in cardiac performance during porcine MH. Acidosis, hypovolemia, and hyperkalemia, especially in the late phase of MH, are additional essential factors responsible for the progressive cardiovascular deterioration and cardiac death.

Alteration of intracellular Ca2+ transients in COS-7 cells transfected with the cDNA encoding skeletal-muscle ryanodine receptor carrying a mutation associated with malignant hyperthermia

Malignant hyperthermia (MH), an inherited neuromuscular disease triggered by halogenated inhalational anaesthetics and skeletal-muscle relaxants, appears to be due to an alteration of intracellular Ca2+ homoeostasis. MH occurs in 1 out of 20,000 anaesthetized adults and is characterized by hypermetabolism, skeletal-muscle rigidity and elevation in body temperature, which is frequently fatal [MacLennan and Phillips (1992) Science 256, 789-794]. The defect responsible for the disease may lie within the mechanism controlling the release of Ca2+ from sarcoplasmic reticulum via the ryanodine-receptor (RYR) Ca2+ channel; in fact a point mutation in the RYR has been associated with MH in some human families, as well as in the MH-susceptible pig. To date, however, no direct evidence has been obtained demonstrating that the point mutation is both necessary and sufficient to cause functional alterations in RYR-mediated Ca2+ release. In the present report we show that the presence of the Arg-to-Cys point mutation in the recombinant RYR expressed in COS-7 transfected cells causes abnormal cytosolic Ca2+ transients in response to 4-chloro-m-cresol, an agent capable of eliciting in vitro contracture of MH-susceptible muscles.

Increase of serotonin in plasma during onset of halothane-induced malignant hyperthermia in pigs

The levels of serotonin (5-HT) were determined in platelet-free and platelet-rich plasma before and during the onset of halothane-induced malignant hyperthermia (MH) in genetically MH-susceptible pigs. During MH onset, the free (i.e. physiologically active) levels of 5-HT in plasma rose concomitantly with the increases in muscle tone, body temperature, venous pCO 2 and plasma lactate. Since pharmacological stimulation of 5-HT 2 receptors has recently been shown to trigger MH in susceptible pigs, the finding of increased 5-HT plasma levels during onset of halothane-induced MH may indicate that 5-HT is involved in the mechanisms by which volatile anesthetics trigger this myopathic syndrome.

Calcium handling by platelets from normal and malignant hyperthermia-susceptible pigs

Platelets from normal and malignant hyperthermia (MH)-susceptible pigs were evaluated for differences in 45calcium uptake in the absence or presence of caffeine (2–16 mM), halothane (0.05–0.5%), or halothane and caffeine together. There were no statistically significant differences in basal or halothane-inhibited calcium uptake by platelets from either source. There was a small statistically significant difference in calcium uptake between platelets from normal and MH-susceptible pigs in the presence of 16 mM caffeine and 0.5% halothane. Calcium uptake by platelets from one pedigree of MH-susceptible pigs were stimulated in a concentration-dependent manner by caffeine. These data suggest that exposure of platelets to caffeine may have potential for identifying MH-susceptiblity.

Pharmacodynamic effects of serotonin (5-HT) receptor ligands in pigs: stimulation of 5-HT2 receptors induces malignant hyperthermia

In pigs, the serotonin-2 (5-HT2) receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), 0.8 mg/kg, induced "psychotic" behaviour (e.g., grimacing, backward locomotion, blank stare) and a muscular syndrome, which is known as malignant hyperthermia (MH) in pigs and humans. This syndrome is characterized by generalized skeletal muscle rigidity, leading to an increase in body temperature, marked acidosis, hyperkaliaemia, cyanosis and elevation of lactate, carbon dioxide and the muscle enzyme creatine kinase (CK) in plasma. In pigs which were selectively bred for susceptibility to MH induction by known triggering agents, such as halothane, the administration of DOI was fatal in 3 out of 5 animals. In genetically susceptible pigs, MH was also induced by 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), 0.5-1.8 mg/kg, and D-lysergic acid diethylamide (LSD), 60-110 micrograms/kg. Furthermore, 5-MeO-DMT and LSD induced head shakes in the animals, which had not been observed after DOI and could not be blocked by 5-HT2-antagonists, ketanserin (0.5-5 mg/kg) and ritanserin (1-2.5 mg/kg). The psychotomimetic effects of 5-MeO-DMT could be blocked by ketanserin or ritanserin, which, depending on the dose, also reduced or totally prevented the hyperthermia and metabolic changes induced by 5-MeO-DMT in pigs. Administration of 5-MeO-DMT, 1.8 mg/kg, was fatal in 4 of 5 MH-susceptible pigs, whereas pigs injected with this dosage after pretreatment with ketanserin (0.5-5 mg/kg) or ritanserin (1-2.5 mg/kg) did not die. In pigs from MH-resistant littermates, administration of 5-MeO-DMT was not fatal. Comparison of metabolic changes in susceptible and non-susceptible pigs suggested that the marked increase in plasma potassium, which arises principally from damaged muscle cells, is primarily responsible for the fatal effect of DOI and 5-MeO-DMT in genetically susceptible individuals. In MH-susceptible pigs, which were anesthetized, relaxed and artificially ventilated, 5-MeO-DMT did not induce hyperthermia, thus substantiating that the marked hyperthermia observed in conscious pigs was a result of muscle activation and not due to effects on thermoregulation or blood pressure. The results indicate that hallucinogenic drugs with 5-HT2 agonistic effects trigger a life-threatening syndrome, MH, in genetically susceptible pigs. 5-HT2 antagonists, such as ketanserin or ritanserin, are capable of counteracting the fatality of this syndrome.

Transverse tubule calcium regulation in malignant hyperthermia

Transverse tubule (TT) calcium transport and permeability were examined in the inherited skeletal muscle disorder malignant hyperthermia (MH). ATP-dependent calcium uptake by TT vesicles isolated from normal and MH-susceptible (MHS) pig muscle had a similar dependence on ionized Ca 2+ concentration ( K 1 2 for Ca 2+ of 0.21 ± 0.04 and 0.25 ± 0.05 μ m for MHS and normal TT, respectively), as well as a similar V max (20.9 ± 2.0 and 23.7 ± 4.5 nmol Ca/mg protein/min for MHS and normal TT, respectively). Furthermore, the stimulation of calcium uptake by either calmodulin or cAMP-dependent protein kinase was similar in normal and MHS TT. Halothane concentrations greater than 2 m m inhibited calcium uptake by either normal or MHS TT to a similar extent (IC 50 = 8 m m). Dantrolene (10 μ m), nitrendipine (1 μ m), and Bay K 8644 (1 μ m) had no significant effect on either the initial rates of calcium uptake or maximal calcium accumulation of either MHS or normal TT vesicles. However, in the absence of any added agents, maximum calcium accumulation by MHS TT was significantly less than by normal TT (90 ± 10 versus 130 ± 9 nmol Ca/mg protein after 15 min of uptake). This difference was not due to an increased permeability of MHS TT to calcium, nor was it due to a difference in the sarcoplasmic reticulum contamination (less than 5%) of the MHS and normal preparations. Although our results indicate there is no significant defect in MHS TT calcium regulation, the diminished maximum calcium accumulation by MHS TT may contribute to the abnormal sarcoplasmic calcium homeostasis in skeletal muscle during an MH Crisis.