Jeffrey E. Fletcher

Modulation of Human Muscle Sodium Channels by Intracellular Fatty Acids Is Dependent on the Channel Isoform

Free fatty acids (FFAs), including arachidonic acid (AA), are implicated in the direct and indirect modulation of a spectrum of voltage-gated ion channels. Skeletal muscle sodium channels can be either activated or inhibited by FFA exposure; the response is dependent on both FFA structure and site of exposure. Recombinant human skeletal muscle sodium channels (hSkM1) were transfected into heterologous human renal epithelium HEK293t cells. Cytoplasmic delivery of 5 μM AA augmented the voltage-activated sodium current of hSkM1 channels by 190% (±54 S.E., n = 7) over a 20-min period. Similar results were seen with 5 μM oleic acid. Sodium currents in HEK293t cells transfected with human cardiac muscle sodium channels (hH1) were insensitive to AA treatment, and exposure to oleic acid inhibited the hH1 currents over a 20-min period by 29% (±13 S.E., n = 5). The increase in hSkM1 current was not accompanied by shifts in voltage dependence of activation, steady-state inactivation, or markedly altered kinetics of inactivation of the macroscopic current. The FFA-induced increase in sodium currents was not dependent on protein kinase C activity. In contrast, both isoforms were reversibly inhibited by external application of unsaturated FFA. Thus, the differential effects of FFA on skeletal muscle sodium channels first noted in cultured muscle cells can be reproduced by expressing recombinant sodium channels in epithelial cells. Although the responses to applied FFAs could be direct or indirect, we suggest that: 1) SkM1 has two classes of response to FFA, one which produces augmentation of macroscopic currents with intracellular FFA, and a second which produces inhibition with extracellular FFA; 2) H1 has only one class of response, which produces inhibition with extracellular FFA. A testable hypothesis is that the presence or absence of each response is due to a specific structure in SkM1 or H1. These specific structures may directly interact with FFA or may interact with intermediate components.

Evidence for genetic heterogeneity in malignant hyperthermia susceptibility

Malignant hyperthermia susceptibility (MHS) is a clinically heterogeneous pharmacogenetic disorder characterized by accelerated metabolism, hyperthermia, and frequently muscle rigidity. MHS is elicited by all commonly used potent inhalation anesthetics and depolarizing neuromuscular blockers and remains an important cause of death due to anesthesia. Recent linkage studies suggest a single genetic locus for this disorder on chromosome 19q13.1. The results of our linkage analyses exclude several loci on 19q13.1 as a site for the gene(s) that produces the MHS phenotype in three unrelated families and clearly establish genetic heterogeneity in this disorder. These results are consistent with the hypothesis that the genetic defect that alters thermoregulation may vary in MHS and that clinical variability in the expression of MHS may be explained by genetic heterogeneity.

Melittin and phospholipase A2 from bee (Apis mellifera) venom cause necrosis of murine skeletal muscle in vivo

Melittin and phospholipase A2 (PLA2) from bee (Apis mellifera) venom were rested for their ability to induce necrosis of skeletal muscle cells after intramuscular injection into mice. Light and electron microscopic examination of tissue indicated that both melittin (4 micrograms/g) and bee venom PLA2 (4 micrograms/g) caused necrosis of skeletal muscle cells within 30 min after i.m. injection. Early changes in the cells consisted of delta lesions, indicating a ruptured plasma membrane, and hypercontraction of myofibrils. By 24 hr the affected cells appeared as an amorphous mass of disorganized and disrupted myofibrils contained in an intact basal lamina. To ensure that the myotoxic activity of the melittin preparation was not due to contaminating. PLA2 activity, the preparation was treated with p-bromophenacyl bromide (p-BPB), a known inhibitor of PLA2 activity. The p-BPB-treated melittin was determined to have no detectable PLA2 activity using a sensitive muscle cell culture assay, and it still induced myonecrosis, although to a lesser extent and of a slower onset. Additionally, p-BPB treatment of purified bee venom PLA2 completely inhibited its myotoxic activity. These results indicate that both melittin and bee venom PLA2 are capable of inducing necrosis of skeletal muscle cells upon i.m. injection, and that the catalytic and myotoxic activities of bee venom PLA2 are inihibited by p-BPB. Also, melittin and contaminating PLA2 in the melittin fraction may be acting synergistically to induce a stronger and more rapid myotoxic effect than occurs with either alone.

Similarities and differences in mechanisms of cardiotoxins, melittin and other myotoxins

Myonecrosis induced in vivo by cardiotoxin, melittin, and Asp49 and Lys49 phospholipase A2 (PLA2) myotoxins involves rapid lysis of the sarcolemma, myofibril clumping, and hypercontraction of sarcomeres. In contrast, skeletal muscle necrosis induced by crotamine and myotoxin a is much slower, consisting of mitochondrial and sarcoplasmic reticulum swelling, myofibril degeneration, and lack of sarcolemma or transverse tubule damage. The mechanisms contributing to the myonecrosis induced by these peptides were evaluated. Two cardiotoxins and two Lys49 PLA2 myotoxins lysed primary cultures of human skeletal muscle within 24 hr at a concentration of 0.25 microM, while melittin, crotamine, and myotoxin a, and an Asp49 PLA2 myotoxin were non-cytolytic at concentrations up to 5.0 microM, suggesting that cytolysis is not a good measure of myotoxicity. Crotamine and the Lys49 PLA2 myotoxin altered Ca2+ ion flux in human heavy sarcoplasmic reticulum by opening the ryanocine receptor. Whole-cell patch-clamp studies demonstrated that administrating crotamine intracellularly increased Na+ currents. Free fatty acids, liberated by activation of tissue phospholipase C or by the PLA2 activity of the myotoxins, were monitored for crotamine, myotoxin a and a Lys49 PLA2 myotoxin in cell cultures in which the lipids had been radiolabeled. Only the Lys49 myotoxin produced significant amounts of fatty acid in cell cultures, supporting a potential role for fatty acid production only in the mechanism of sarcolemma-destroying myotoxins. These findings, coupled with those in the literature, support a hypothesis in which the myotoxins and/or products of lipase activity (e.g. fatty acids) are acting at a site existing on both the Na+ channel and a protein involved in Ca2+ release and probably serving a modulatory function for ion regulation. Based on the similarities in mechanisms between the toxins and fatty acids, the most likely site would be a fatty acid binding site on the protein (either similar to that on fatty acid binding proteins, or an acylated cysteine residue) or in the membrane.

Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachatus haemachatus snake venom—II: Pharmacological properties in relationship to enzymatic activity☆

Abstract Despite a remarkable degree of homology in amino acid sequence, the neutral phospholipase A 2 from Hemachatus haemachatus venom is much less toxic than the basic phospholipase A 2 from Naja nigricollis venom, the i.v. ld 50 in mice for the two being, respectively, 8.6 and 0.63 mg/kg. Similarly following intraventricular injection into rats, the neutral phospholipase showed convulsant and lethal dose 50 values of about 7.5 and 15 μg per rat, respectively, whereas corresponding values for the basic phospholipase were 0.5 and 0.5 μg per rat. Death appears to be due to congestion, hemorrhage and edema in the lungs. Consideration of dosages required and times until onset of action suggests that, dependent upon the route of administration, the effect is either mediated via a central action or is due to a direct effect on the cardiac and/or respiratory system in the periphery. The pattern and extent of phospholipid hydrolysis in various brain regions was similar following intraventricular injection of the two phospholipases so that no relationship between phospholipid hydrolysis and lethal potency could be established. Concentrations of 5 and 10 μmg/ml of the N. nigricollis and H. haemachatus phospholipases, respectively, were required to block electrical activity of the isolated single electroplax. The ultrastructural changes produced by both phospholipases were also similar. Parallel to the somewhat greater potency on the electroplax, N. nigricollis phospholipase produced slightly greater overall hydrolysis in the innervated and non-innervated membranes of the electroplax than did H. haemachatus phospholipase. The results suggest that these two phospholipases do not have a specific junctional effect and that the small difference in potency on the junction cannot be responsible for the large difference in lethality observed in mammalian species.

Malignant Hyperthermia Susceptibility in Neuroleptic Malignant Syndrome

The relationship between neuroleptic malignant syndrome (NMS) and malignant hyperthermia (MH) was investigated using the in vitro skeletal muscle contracture test to screen for MH-susceptibility in NMS patients. The maximum contracture tension which developed following exposure to halothane (1–3%), and incremental doses of fluphenazine (0.2–25.6 mM) was measured in muscle obtained from seven NMS, six MH, and six control patients. Comparison of the cumulative responses to fluphenazine revealed no significant differences among the groups. However, the response (mean ± SEM) to halothane in the NMS group (1.7 ± 0.7 g), which was similar to the response in the MH group (1.5 ± 0.2 g), was significantly greater than the response found in controls (0.2 ± 0.1 g). In addition, live of seven NMS patients could be diagnosed as MH-susceptible, based on the development of muscle contractures greater than 0.7 g in response to 1–3% halothane. In contrast, none of the controls were MH-susceptible. These findings appear to correlate with clinical evidence suggesting an association between NMS and MH.

Masseter Muscle Rigidity and Malignant Hyperthermia Susceptibility in Pediatric Patients An Update on Management and Diagnosis

BackgroundControversy exists regarding the definition of masseter muscle rigidity (MMR) and anesthetic management after MMR. This study reports current anesthetic management after MMR, estimates the incidence of clinical malignant hyperthermia (MH) in patients with MMR, and is the first to evaluate the coincidence of MMR with malignant hyperthermla susceptibility (MHS) according to the 1987 North American Malignant Hyperthermia Group protocol. MethodsPracticing anesthesiologists referred pediatric patients for biopsy between 1986 and 1991 based on evidence of MMR after succinylcholine (1975–1991). The clinical scenario was described as MMR alone or MMR followed by signs of MH, including arterial CO2 tension > 50 mmHg, arterial pH ± 7.25, and base deficit > 8. Patients had caffelne-halothane muscle contracture testing to determine MHS. ResultsSeventy patients (50 boys and 20 girls) were evaluated. Eighty-three percent (58 of 70) of anesthetics were halothane-succinylcholine. In 68% (48 of 70) of cases, the anesthetic was discontinued, whereas anesthesia was continued with nontriggering agents in 11% (8 of 70) and with triggering agents in 13% (9 of 70). Fifty-nine percent (41 of 70) of patients were diagnosed as MHS by muscle biopsy. In 7% (5 of 70) of patients, clinical MH developed within 10 min of MMR. ConclusionsThis study, by using the current North American Malignant Hyperthermla Group protocol, reaffirms the high incidence (59%, 41 of 70) of MHS associated with MMR as confirmed by muscle biopsy. Of the MHS patients, 5 developed signs of clinical MH. Most anesthesiologists in this study, when confronted with MMR, discontinued anesthesia. Because of the potential lethality of MH and the > 50% concordance between MMR and MHS, the most conservative course of action after MMR is to discontinue the anesthetic and observe the patient for clinical evidence of MH. An acceptable alternative, depending on the urgency of the surgery, would be to continue anesthesia with nontriggering agents for MH, with appropriate monitoring.

North American Malignant Hyperthermia Population: Screening of the Ryanodine Receptor Gene and Identification of Novel Mutations

BackgroundMalignant hyperthermia (MH) is a disorder of skeletal muscle manifested as a life-threatening hypermetabolic crisis in susceptible individuals after exposure to inhalational anesthetics and depolarizing muscle relaxants. Mutations in the gene encoding the skeletal muscle ryanodine receptor (RYR1) are considered a common cause of the disorder, and, to date, more than 20 RYR1 mutations have been reported in European and Canadian families. Some studies suggest that differences may exist in the frequencies and distribution of mutations in the RYR1 gene between European and North American MH families the frequency and distribution of mutations in the RYR1 gene. MethodsSkeletal muscle samples from 73 unrelated individuals diagnosed as MH susceptible according to the North American MH caffeine–halothane contracture test were studied. Genomic DNA of MH-susceptible patients was investigated by polymerase chain reaction–based restriction fragment length polymorphism, single-strand conformation polymorphism, and sequencing analysis. The majority of known RYR1 mutations were analyzed using the restriction fragment length polymorphism method, whereas new mutations were searched by single-strand conformation polymorphism in exons 12, 15, 39, 40, 44, 45, and 46 of the gene. ResultsSeven known RYR1 mutations (Arg163Cys, Gly248Arg, Arg614Cys, Val2168Met, Thr2206Met, Gly2434Arg, and Arg2454His) were detected at frequencies of 2.7, 1.4, 1.4, 1.4, 1.4, 5.5, and 4.1%, respectively. In addition, three novel amino acid substitutions (Val2214Ile, Ala2367Thr, and Asp2431Asn) were detected at frequency of 1.4% each. These 10 mutations account for 21.9% of the North American MH-susceptible population. ConclusionThree novel candidate mutations in the RYR1 gene were identified in these MH patients. The frequency and distribution of RYR1 mutations observed in this North American MH population was markedly different from that previously identified in Europe. Larger-scale studies are necessary to clarify the type and frequency of mutations in RYR1 associated with MH in North American families.

No role for enzymatic activity or dantrolene-sensitive Ca2+ stores in the muscular effects of bothropstoxin, a Lys49 phospholipase A2 myotoxin

The role of low levels of phospholipase A2 (PLA2) activity and intracellular Ca2+ stores in the pharmacological action of bothropstoxin (BthTX), a myotoxic Lys49 PLA2 homologue isolated from the venom of Bothrops jararacussu, was investigated. We examined the muscular effects of BthTX in the mouse diaphragm and its PLA2 activity in radiolabeled human and rat primary cultures of skeletal muscle. Although it is a Lys49 PLA2 homologue, BthTX had a low, but easily detectable, level of enzymatic activity relative to two Asp49 PLA2 enzymes from Naja naja kaouthia and Naja naja atra venoms, and this activity was reduced by about 85% in the presence of Sr2+ (4.0 mM). However, the replacement of 1.8 mM Ca2+ by 4 mM Sr2+ did not alter the BthTX-induced contracture and blockade of the muscle twitch tension. In addition, Sr2+ decreased by 50% the time required to cause 50% paralysis, and evoked approximately a four-fold increase in the number of spontaneous spikes. In isolated sarcoplasmic reticulum preparations, BthTX opened the intracellular Ca2+ release channel (ryanodine receptor) and lowered the threshold of Ca(2+)-induced Ca2+ release by a second, as yet unidentified, mechanism. However, in intact muscle, dantrolene, an antagonist of some forms of intracellular Ca2+ release, had no effect on the actions of BthTX. These findings do not support any role for the low levels of PLA2 activity, or dantrolene-sensitive intracellular Ca2+ stores, in the action of BthTX. The mechanism whereby Sr2+ stimulates the pharmacological activity of BthTX remains to be clarified.

Effects of a cardiotoxin from Naja naja kaouthia venom on skeletal muscle: Involvement of calcium-induced calcium release, sodium ion currents and phospholipases A2 and C

Snake venom cardiotoxin (CTX) fractions induce contractures of skeletal muscle and hemolysis of red blood cells. The fractions also contain trace amounts of venom-derived phospholipase A2 (PLA2) contamination and activate tissue phospholipase C (PLC) activity. The present study examines the mechanisms of action of a CTX fraction from Naja naja kaouthia venom in skeletal muscle. Sphingosine competitively antagonized CTX-induced red blood cell hemolysis, but not skeletal muscle contractures. CTX rapidly lowered the threshold for Ca(2+)-induced Ca2+ release in heavy sarcoplasmic reticulum fractions, as monitored with arsenazo III. There was also a slower time-dependent reduction of Na+ currents, as assessed by whole cell patch-clamp techniques. The CTX fractions elevated levels of free fatty acids and diacylglycerol for 2 hr in primary cultures of human skeletal muscle by a combined action of venom-derived PLA2 contamination in the fraction and activation of endogenous PLC activity. The activation of tissue PLC activity could be readily distinguished from the contribution of the venom PLA2 by p-bromophenacyl bromide treatment of CTX fractions. The mechanism of action involved in contractures of skeletal muscle appears to be related to the immediate and specific effect of CTX (Ca2+ release by the sarcoplasmic reticulum), while the mechanisms involved in hemolysis of red blood cells and decreased Na+ currents in skeletal muscle most likely relate to long-term effects on lipid metabolism.