Genotype and phenotype relationships for mutations in the ryanodine receptor in patients referred for diagnosis of malignant hyperthermia.

Anaesthesia-induced malignant hyperthermia (MH) may be caused by specific gene defects in the skeletal muscle ryanodine receptor. We have studied the frequency of occurrence of the C1840T mutation, analogous to the porcine mutation, and three mutations associated both with MH and central core disease (G7301A, C487T and C1209G). We investigated skeletal muscle specimens from up to 137 patients testing negative and 101 patients testing positive for MH susceptibility by the North American MH Group protocol. The presence or absence of the mutations was determined by polymerase chain reaction and restriction enzyme digestion. The frequencies of occurrence of the C1840T and C487T mutations were 2% and 1%, respectively, in MH-positive subjects and were the only two mutations identified. One subject with central core disease did not have any of the three mutations examined associated with this disorder. Therefore, the porcine and central core disease-associated mutations examined in the ryanodine receptor account for a small proportion (approximately 3%) of MH-positive diagnoses. The mutations examined did not occur in any of the MH-negative patients, supporting an association between defects in the ryanodine receptor and a positive diagnosis for MH.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of phenytoin on skeletal muscle from quarter horses with hyperkalaemic periodic paralysis.

The contractile activity, the threshold for calcium-induced calcium release in fractions of sarcoplasmic reticulum and the potassium concentration were determined in preparations of semimembranosus muscle from normal quarter horses and quarter horses with hyperkalaemic periodic paralysis before and after they were treated with phenytoin. Before the treatment there was no difference in caffeine contracture or electrically elicited twitch response between the two groups. For one week after the treatment, the time to peak tension of caffeine contractures was significantly (P < 0.005) reduced in the horses with hyperkalaemic periodic paralysis but unchanged in the normal horses. The variance but not the mean values for the threshold for Ca(2+)-induced Ca2+ release from the sarcoplasmic reticulum was greater for the horses with hyperkalaemic period paralysis before but not after the treatment with phenytoin.

Malignant hyperthermia: halothane- and calcium-induced calcium release in skeletal muscle.

The temperature dependence of oleic acid-enhanced halothane- induced Ca2+ release and the existence of a reduced threshold of Ca(2+)-induced Ca2+ release were examined to determine their roles in human malignant hyperthermia. Halothane (8-11 mM) induced Ca2+ release from terminal cisternae-containing preparations from skeletal muscle. Oleic acid (15 microM) markedly reduced the threshold of halothane-induced Ca2+ release to < 0.5 mM at 37 degrees C, but not at 25 degrees C, consistent with the temperature dependence of halothane action in intact muscle. Two states of the threshold of Ca(2+)-induced Ca2+ release were unrelated to malignant hyperthermia in humans, in contrast to pigs. Excess fatty acid production may be an important modulator of halothane action in malignant hyperthermia.

Species difference in modulation of calcium release by Naja naja kaouthia snake venom cardiotoxin in terminal cisternae from human and equine skeletal muscle.

The modulation of Ca2+ release by a cardiotoxin (CTX) from Naja naja kaouthia snake venom was examined in terminal cisternae-containing fractions from equine and human skeletal muscle. Pretreatment with CTX (10 microM) decreased by 27% (human muscle), or had no effect on (equine muscle), the threshold of Ca(2+)-induced Ca2+ release. If terminal cisternae fractions were first preloaded with Ca2+ to greater than 65% of the threshold of Ca(2+)-induced Ca2+ release and then CTX added, an immediate and sustained release of Ca2+ occurred in preparations from both species. Addition of CTX after a Ca2+ preload of less than 60% of the threshold of Ca(2+)-induced Ca2+ release did not elicit Ca2+ release in preparations from either species. Ruthenium red (10 microM) antagonized CTX-induced Ca2+ release, whereas dantrolene (10 microM) did not. These findings suggest that the effects of CTX on the Ca2+ release channel are dependent on Ca2+ preload and that CTX may be an important probe of the Ca(2+)-modulated Ca2+ release process and in understanding regulation of Ca2+ release in skeletal muscle from different species.

Caffeine contractures, twitch characteristics and the threshold for Ca(2+)-induced Ca2+ release in skeletal muscle from horses with chronic intermittent rhabdomyolysis.

Muscle from horses with intermittent exercise associated rhabdomyolysis was examined to determine if calcium regulation was abnormal. In vitro studies on semimembranosus muscle fibre bundles showed the time to 50 per cent relaxation of caffeine-induced contractures was shorter and the electrically elicited twitch longer in horses with exercise associated rhabdomyolysis. Substitution of strontium for calcium eliminated the difference in caffeine contracture between the normal and rhabdomyolysis horses. The threshold of calcium-induced calcium release was lower than normal in terminal cisternae-containing fractions of muscle from horses with rhabdomyolysis. Thoroughbreds with rhabdomyolysis had a shorter time to peak twitch tension than standardbreds, and normal thoroughbreds had a shorter caffeine contracture than normal standardbreds. There was no difference in fibre typing between breeds or groups. Either no histological changes or low grade to moderate degenerative myopathy was seen in muscle from horses with rhabdomyolysis. These results suggest horses with intermittent exercise associated rhabdomyolysis have abnormal calcium regulation.

Effects of phenytoin in two myotonic horses with hyperkalemic periodic paralysis.

The effects of phenytoin treatment were evaluated in 2 myotonic horses with hyperkalemic periodic paralysis (HPP). Phenytoin treatment abolished the clinical signs of muscle fasciculations following oral potassium challenge and decreased or abolished repetitive firing and myotonic discharges found on electromyographic examination. In both horses, an abnormally low threshold for calcium-induced calcium release was measured in heavy sarcoplasmic reticulum fractions from skeletal muscle, and this threshold increased with phenytoin treatment. Results suggest phenytoin is useful in modifying disordered ion regulation in the sarcolemma and sarcoplasmic reticulum of skeletal muscle in equine hyperkalemic periodic paralysis.

Bee venom melittin is a potent toxin for reducing the threshold for calcium-induced calcium release in human and equine skeletal muscle.

The modulation of Ca2+ release by synthetic bee venom melittin was examined in equine and human terminal cisternae-containing fractions. Melittin (0.1 microM) decreased the threshold of Ca(2+)-induced Ca2+ release by 20% in equine muscle and by 36% in human muscle. If terminal cisternae fractions were first preloaded with Ca2+ to greater than about 75% of the threshold of Ca(2+)-induced Ca2+ release and then melittin added, an immediate and sustained release of Ca2+ occurred in preparations from both species. Addition of melittin after a Ca2+ preload of < 50% of the threshold of Ca(2+)-induced Ca2+ release did not elicit sustained Ca2+ release. Ruthenium red (10 microM) antagonized all effects of melittin on Ca2+ release. Melittin (0.1-10 microM) did not affect [3H]ryanodine binding. Melittin (0.1 microM) slightly (10%) inhibited the Ca2+ pump and this action was not antagonized by ruthenium red. These findings suggest that melittin may be an important new probe of the Ca(2+)-modulated Ca2+ release process that does not act at the ryanodine binding site.

Altered [3H]ryanodine binding is not associated with malignant hyperthermia susceptibility in terminal cisternae preparations from swine.

Based on comparisons between Pietrain malignant hyperthermia susceptible swine and Yorkshire control swine, other investigators have reported a 3-fold lower Kd for [3H]ryanodine binding to terminal cisternae in the malignant hyperthermia swine. However, the Kd of [3H]ryanodine binding did not correlate with malignant hyperthermia susceptibility when examined within the same strain (a Yorkshire/Duroc cross) in the present study. The values of Kd for the malignant hyperthermia susceptible and control swine in the present study were similar to those previously reported for the Pietrain strain, suggesting that the control strain chosen, not malignant hyperthermia susceptibility, accounts for what appeared to be a low Kd in Pietrain muscle.

Fatty acids markedly lower the threshold for halothane-induced calcium release from the terminal cisternae in human and porcine normal and malignant hyperthermia susceptible skeletal muscle.

Malignant hyperthermia is caused by an abnormal increase in Ca2+ levels in skeletal muscle in response to anesthetics, including halothane. Since fatty acid production is elevated in skeletal muscle from individuals with malignant hyperthermia, the effects of fatty acids on the threshold of halothane-induced Ca2+ release were examined. In the absence of fatty acids halothane caused Ca2+ release from porcine and human heavy sarcoplasmic reticulum fractions, but only at concentrations above the clinically relevant range. Oleic acid (20 microM), an unsaturated fatty acid, reduced the threshold at which halothane induced Ca2+ release to concentrations used for anesthesia. Stearic acid, a saturated fatty acid had considerably less effect on the threshold of halothane action. The greater sensitivity of malignant hyperthermia muscle to halothane can be explained by elevated fatty acid production.

Fatty acids modulate calcium-induced calcium release from skeletal muscle heavy sarcoplasmic reticulum fractions: implications for malignant hyperthermia.

Based on studies in swine, the malignant hyperthermia syndrome has been postulated to result from an enhanced sensitivity (low threshold) of the Ca2(+)-induced Ca2(+)-release process. However, fatty acid production is elevated in homogenates of skeletal muscle from pigs and humans susceptible to malignant hyperthermia. In the present study, we demonstrate that the threshold of Ca2(+)-induced Ca2+ release is normal in susceptible humans and in susceptible swine depleted of triglycerides. Exogenously added unsaturated fatty acids decreased the threshold of Ca2(+)-induced Ca2+ release to a much greater extent in porcine and equine muscle than in human muscle. When triglyceride and free fatty acid values were reduced to about 40 and 60%, respectively, of control values, malignant hyperthermia-susceptible swine did not exhibit muscle rigidity when challenged in vivo with halothane and succinylcholine and the threshold of the Ca2(+)-induced Ca2(+)-release process in heavy sarcoplasmic reticulum fractions was normal. Despite the reduced triglyceride and fatty acid levels, these swine had a positive in vitro contracture test for malignant hyperthermia. A low Ca2(+)-induced Ca2(+)-release threshold is not essential for malignant hyperthermia susceptibility, but appears to be the result of excessive free fatty acids produced during organelle isolation.

Interactions in red blood cells between fatty acids and either snake venom cardiotoxin or halothane.

Phospholipase A2 (PLA2) activity enhances snake venom cardiotoxin (CTX)-induced and general anesthetic (halothane)-induced hemolysis of red blood cells. In the case of halothane-induced hemolysis, this effect appears to be related primarily to free fatty acids. In the present study, the interaction between CTXs and halothane and the effects of different free fatty acids on cardiotoxin and halothane-induced hemolysis were examined. The hemolytic actions of halothane and a CTX from Naja naja kaouthia venom were examined in erythrocytes with different phospholipid and free fatty acid composition from five species. The extent of hemolysis by CTX or halothane was dependent upon the species examined and appeared to be inversely related to the amount of saturated free fatty acid in the membrane. The order of susceptibility of red blood cells from five species to hemolysis was similar for halothane- and N. n. kaouthia CTX-induced hemolysis, but very different for osmotic fragility. The slope of the hemolysis dose-response curve was considerably steeper for halothane than for CTX. Hemolysis due to N. n. kaouthia CTX was greatly increased by halothane in erythrocytes from humans and horses and to a lesser extent in erythrocytes from sheep, goats and cows. Hemolysis induced by halothane and the N. n. kaouthia or Naja naja atra CTXs was enhanced by unsaturated fatty acids. In contrast, hemolysis induced by halothane was decreased and that caused by the two CTXs was relatively unaffected by saturated fatty acids. Halothane and CTXs differ in their exact mechanisms, but appear to act upon similar fatty acid-sensitive processes.

Triglycerides, not phospholipids, are the source of elevated free fatty acids in muscle from patients susceptible to malignant hyperthermia.

In the absence of halothane challenge, incubates of skeletal whole-muscle homogenates from malignant-hyperthermia-susceptible humans and pigs exhibit greater free fatty acid production than controls, which has been attributed to elevated phospholipase A2 activity. The present study examines lipid profiles of human muscle (vastus lateralis) prior to halothane challenge, relating the lipid profiles to the magnitude of halothane contracture response in muscle from the same biopsy. No differences in cholesterol, phospholipids or free fatty acids were observed among preparations exhibiting low, moderate or strong responses to halothane. Two fatty acids associated with triglycerides (palmitoleic and oleic) were significantly (P less than 0.05) lower in muscle demonstrating a strong response to halothane. These results do not support altered phospholipase A2 activity as a defect in malignant hyperthermia, but rather support an enhanced turnover of triglycerides in biopsied skeletal muscle from MH-susceptible humans.