Thomas E. Nelson

Dantrolene sodium can increase or attenuate activity of skeletal muscle ryanodine receptor calcium release channel : Clinical implications

Background Dantrolene sodium (DS) is a direct‐acting skeletal muscle relaxant whose only known action is to block calcium release from intracellular storage sites. The exact site of action for DS is unknown, but its efficacy in treating and preventing anesthetic‐induced malignant hyperthermia (MH) is well established. Methods Single ryanodine (Ry1) receptor calcium release channels were incorporated into a planar lipid bilayer for electrophysiologic recording and for subsequent analysis of the channels gating and conductance properties. The cellular effects of low DS concentrations were investigated by isometric contracture tension responses in biopsied MH human and dog muscle fascicles and in normal, single fibers from human vastus lateralis muscle. Results Two concentration‐dependent DS effects on the isolated Ry sub 1 receptor were discovered, suggesting at least two different binding sites. At nanomolar concentrations, DS activated the channel by causing three‐ to fivefold increases in open‐state probability and dwell times. At micromolar concentrations, DS first increased then reduced activity in the channels; with the dominant effect being reduced activity. A 20 nM concentration of DS produced significant contracture tension in human muscle from one MH subject and caused potentiation of twitch in muscle from another MH patient. Halothane contracture in MH dog muscle was followed by an additional increase in tension when treated with 20 nM DS. Other investigations on chemically skinned, human fibers showed that calcium loaded in the sarcoplasmic reticulum was partially released by nM DS. Conclusions The study results suggest that at least two binding sites for DS exist on the Ry1 receptor calcium channel. A low‐affinity (micro Meter) site is associated with reduced channel gating and open‐state dwell time and may relate to the established pharmacologic muscle relaxant effect of DS. The proposed high‐affinity (nM) DS binding site activates the channel, producing Calcium2+ release to the myoplasm, which, under environmentally adverse conditions, could damage genetically predisposed MH muscle. Such a phenomenon, if it occurs in DS‐treated MH patients, could generate a recrudescence of the syndrome.

Autosomal Dominant Canine Malignant Hyperthermia Is Caused by a Mutation in the Gene Encoding the Skeletal Muscle Calcium Release Channel (RYR1 )

BackgroundMalignant hyperthermia (MH) is an inherited disorder of skeletal muscle characterized by hypercarbia, rhabdomyolysis, generalized skeletal muscle contracture, cardiac dysrhythmia, and renal failure, that develops on exposure to succinylcholine or volatile anesthetic agents. All swine and up to 50% of human MH events are thought to be associated with mutations in the calcium release channel of the sarcoplasmic reticulum, also known as the ryanodine receptor (RYR1). Events resembling MH have been reported in other species, but none have undergone genetic investigation to date. MethodsTo determine the molecular basis of canine MH, a breeding colony was established with a male, mixed-breed, MH-susceptible (MHS) dog that survived an in vivo halothane–succinylcholine challenge. He was mated to three unaffected females to produce four litters and back-crossed to an affected daughter to produce one litter. One of his MHS sons was mated to an unaffected female to produce an additional litter. Forty-seven dogs were phenotyped with an in vitro contracture test and diagnosed as MHS or MH normal based on the North American in vitro contracture test protocol. Nine microsatellite markers in the vicinity of RYR1 on canine chromosome 1 (CFA01) were tested for linkage to the MHS phenotype. Mutational analysis in two MHS and two MH-normal dogs was performed with direct sequencing of polymerase chain reaction products and of cloned fragments that represent frequently mutated human RYR1 regions. A restriction fragment length polymorphism was chosen to detect the candidate mutation in the pedigree at large. ResultsPedigree inspection revealed that MHS in this colony is transmitted as an autosomal dominant trait. FH2294, the marker closest to RYR1, is linked to MHS at a &thgr; = 0.03 with a LOD score of 9.24. A T1640C mutation gives rise to an alanine for valine substitution of amino acid 547 in the RYR1 protein, generating a maximum LOD score of 12.29 at &thgr; = 0.00. All dogs diagnosed as MHS by in vitro contracture test were heterozygous for the mutation, and all MH-normal dogs were homozygous for the T1640 allele. ConclusionsThese results indicate that autosomal dominant canine MH is caused by a mutation in the gene encoding the skeletal muscle calcium release channel and that the MHS trait in this pedigree of mixed-breed dogs is in perfect cosegregation with the RYR1 V547A mutation.

Malignant hyperthermia in North America: genetic screening of the three hot spots in the type I ryanodine receptor gene.

Background:Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle, manifested as a life-threatening hypermetabolic crisis after exposure to anesthetics. Type I ryanodine receptor 1 is the primary gene responsible for susceptibility to MH as well as central core disease, a congenital myopathy that predisposes susceptibility to MH. More than 40 mutations in the RyR1 gene cluster in three coding regions: the N-terminus, central, and C-terminus regions. However, the frequency of mutations in each region has not been studied in the North American MH-susceptible population. Methods:The authors tested 124 unrelated patients with MH susceptibility for the presence of mutations in the N-terminus (exons 2, 6, 9, 11, 12, and 17), central (exons 39, 40, 44, 45, and 46), and C-terminus (exons 95, 100, 101, and 102) regions. Results:Fourteen mutations have been identified in 29 of 124 MH-susceptible patients (23%). Approximately 70% of the mutations, which include a novel mutation, Ala 2437Val, were in the central region. In 8 patients (28%), mutations were identified in the N-terminus region. Screening the C-terminus region yielded a novel mutation, Leu4824Pro, in a single patient with a diagnosis of central core disease. Conclusions:The detection rate for mutations is only 23% by screening mutations (or exons) listed in the 2002 North American consensus panel. The implications from this study suggest that testing the central region first is currently the most effective screening strategy for the North American population. Screening more exons in the three hot spots may be needed to find an accurate frequency of mutations in the RyR1 gene.

Identification and functional characterization of a novel ryanodine receptor mutation causing malignant hyperthermia in North American and South American families.

Malignant hyperthermia is a pharmacogenetic disorder associated with mutations in Ca(2+) regulatory proteins. It manifests as a hypermetabolic crisis triggered by commonly used anesthetics. Malignant hyperthermia susceptibility is a dominantly inherited predisposition to malignant hyperthermia that can be diagnosed by using caffeine/halothane contracture tests. In a multigenerational North American family with a severe form of malignant hyperthermia that has caused four deaths, a novel RYR1 A2350T missense mutation was identified in all individuals testing positive for malignant hyperthermia susceptibility. The same A2350T mutation was identified in an Argentinean family with two known fatal MH reactions. Functional analysis in HEK-293 cells revealed an altered Ca(2+) dependence and increased caffeine sensitivity of the expressed mutant protein thus confirming the pathogenic potential of the RYR1 A2350T mutation.

Is Comparative Cardiotoxicity of S(−) and R(+) Bupivacaine Related to Enantiomer-selective Inhibition of L-type Ca2+ Channels?

Cardiac toxicity can occur after accidental intravascular injection of bupivacaine. Racemic bupivacaine can inhibit both cardiac Na+ and Ca2+ channels, but the contribution of these actions to cardiac depression is not totally understood. We tested whether the effect of R(+) bupivacaine on cardiac electrical activity in isolated hearts and on L-type Ca2+ channels (ICa-L) in isolated cardiac myocytes could be responsible for its increased cardiotoxicity compared with S(−) bupivacaine. Cardiac electrical activity of spontaneously beating isolated hearts was recorded before and after exposure to increasing concentrations of R(+) and S(−) bupivacaine. An increase of the PR interval (80%) and the QRS duration (370%) by 10&mgr;M R(+) bupivacaine (80% and 370%) was significantly higher than for S(−) bupivacaine (25% and 200%, respectively). R(+) but not S(−) bupivacaine produced severe arrhythmias at concentrations larger than 2.5&mgr;M. The intensity of ICa-L inhibition did not differ between bupivacaine isomers. At 0 mV, ICa-L was irreversibly reduced by 40.2% ± 8.8% and 51.4% ± 3.8% in the presence of 10&mgr;M R(+) and S(−) bupivacaine, respectively. The arrhythmogenic effect of R(+) bupivacaine could not be explained by stereoselectivity on the ICa-L inhibition. Thus, other mechanisms could contribute to the cardiac electrical and contractile dysfunction induced by R(+) bupivacaine. Implications Accidental intravascular injection of bupivacaine can induce toxic effects on the heart. We investigated the sensitivity of different bupivacaine structures’ actions on the Ca2+ conducting channels in rat ventricular cells and concluded that the increased toxicity of R(+) bupivacaine is not explained by actions on the Ca2+ channels’ inhibition.

Single-amino-acid deletion in the RYR1 gene, associated with malignant hyperthermia susceptibility and unusual contraction phenotype

Malignant hyperthermia (MH) is an anesthetic-drug-induced, life-threatening hypermetabolic syndrome caused by abnormal calcium regulation in skeletal muscle. Often inherited as an autosomal dominant trait, MH has linkage to 30 different mutations in the RYR1 gene, which encodes a calcium-release-channel protein found in the sarcoplasmic reticulum membrane in skeletal muscle. All published RYR1 mutations exclusively represent single-nucleotide changes. The present report documents, in exon 44 of RYR1 in two unrelated, MH-susceptible families, a 3-bp deletion that results in deletion of a conserved glutamic acid at position 2347. This is the first deletion, in RYR1, found to be associated with MH susceptibility. MH susceptibility was confirmed among some family members by in vitro diagnostic pharmacological contracture testing of biopsied skeletal muscle. Although a single-amino-acid deletion appears to be a subtle change in the protein, the deletion of Glu2347 from RYR1 produces an unusually large electrically evoked contraction tension in MH-positive individuals, suggesting that this deletion produces an alteration in skeletal-muscle calcium regulation, even in the absence of pharmacological agents.

Malignant Hyperthermia in Dogs

Malignant hyperthermia (MH) is an anesthetic agent-induced hypermetabolic state. Human beings and several other animal species, including dogs, have been described to be genetically predisposed to development of MH. The halothane-triggered MH syndrome was characterized in genetically predisposed dogs, and in vitro contracture sensitivity of biopsied gracilis muscle exposed to halothane and caffeine was quantitated. Within 1 hour of halothane administration, each MH-susceptible dog developed rapid increases in CO2 production and rectal temperature. Reversal of the hypermetabolic state was achieved when halothane was discontinued and dantrolene sodium was given i.v. Biopsied gracilis muscle from MH-susceptible dogs had abnormal in vitro contracture responses to halothane and caffeine. These findings were consistent with those observed for MH-susceptible human beings and pigs in which a loss in regulation of muscle cell Ca(+)+ is believed to be the primary etiologic event for induction of MH.

Novel skeletal muscle ryanodine receptor mutation in a large Brazilian family with malignant hyperthermia.

Malignant hyperthermia (MH) is an autosomal dominant disorder that predisposes susceptible individuals to a potentially life‐threatening crisis when exposed to commonly used anesthetics. Mutations in the skeletal muscle calcium release channel, ryanodine receptor (RYR1) are associated with MH in over 50% of affected families. Linkage analysis of the RYR1 gene region at 19q13 was performed in a large Brazilian family and a distinct disease co‐segregating haplotype was revealed in the majority of members with diagnosis of MH. Subsequent sequencing of RYR1 mutational hot spots revealed a nucleotide substitution of C to T at position 7062, causing a novel amino acid change from Arg2355 to Cys associated with MH in the family. Haplotype analysis of the RYR1 gene area at 19q13 in the family with multiple MH members is an important tool in identification of genetic cause underlying this disease.

Heat production during anesthetic-induced malignant hyperthermia.

Malignant hyperthermia (MH) is a pharmacogenetic disease which predisposes to the trigger of a life-threatening, hypermetabolic syndrome by potent inhaled anesthetics and by depolarizing skeletal muscle relaxants. Heat production in the anesthetized MH can be profound with 5-fold increases in oxygen consumption. The trigger anesthetics cause an abnormal, sustained rise in myoplasmic calcium levels. Possible mechanisms by which continuous release of calcium from skeletal muscle sarcoplasmic reticulum stores can produce the profound hyperthermia are discussed. Mutations in the gene coding the ryanodine receptor calcium release channel have been found in MH families and these mutant channels may be the functionsl basis for MH.

Abnormal Function of Porcine Malignant Hyperthermia Calcium Release Channel in the Absence and Presence of Halothane

Malignant hyperthermia (MH) is an anesthetic-induced, life-threatening hypermetabolic state occurring in genetically predisposed pigs, dogs and humans. It is widely believed that MH is caused by an ab