Changes in Ryanodine-Induced Contractures by Stimulus
Frequency in Malignant Hyperthermia Susceptible and
Malignant Hyperthermia Nonsusceptible Dog Skeletal Muscle
ROBERTO T. SUDO and THOMAS E. NELSON
Departments of Basic and Clinical Pharmacology, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (R.T.S.) and Anesthesia, Bowman Gray School of Medicine, Winston-Salem, North Carolina (T.E.N.)
Accepted for publication May 12, 1997
ABSTRACT
Elective diagnosis of malignant hyperthermia depends on hal-othane and caffeine contracture testing of biopsied skeletal muscle. Ryanodine-induced contractures may provide greater sensitivity and specificity for malignant hyperthermia (MH) di-agnosis. This study investigated the effects of ryanodine con-centration and stimulus frequency to distinguish between MH susceptible (MHS) and MH non-susceptible (MHN) dogs. In-creasing ryanodine concentrations (1, 2.5 and 5mM) increased peak isometric contracture tension, but similar responses in MHS and MHN muscle precluded use for diagnosis. Time to tension onset and to peak tension decreased with increasing
ryanodine concentration, and these times were shorter in MH skeletal muscle. Increasing stimulus frequency (0.1, 0.5 and 1 Hz) decreased the time to tension onset and to peak tension, but the effect was greater in MHN muscle which decreased the difference between MHN and MHS muscle responses. When ryanodine contracture tension onset time was selected to de-tect MHS muscle, combinations of either 0.1 Hz and 1 mM ryanodine or 0.5 Hz and 1mM ryanodine reduced the probabilty of a false diagnosis to less than 1%. Similar studies performed on human muscle might identify optimal stimulus frequency and ryanodine concentration for detecting MH in patients.
MH, a pharmacogenetic disease affecting skeletal muscle, is induced by general, volatile anesthetics and by depolariz-ing muscle relaxants. Before the clinical use of the skeletal muscle relaxant dantrolene sodium, the mortality from MH was.70%. The syndrome is expressed as a hypermetabolic state with as much as 5-fold increases in oxygen consump-tion, causing high CO2production, metabolic and respiratory acidosis, high plasma CK concentration and myoglobinuria due to rhabdomyolysis. The primary cause is an abnormal sustained increase in myoplasmic [Ca11] which produces the hypermetabolic state and in some cases, skeletal muscle ri-gidity (Britt and Kalow, 1970). The mechanism of MH is not completely understood but several electrophysiological, phar-macological and biochemical studies indicate abnormal re-lease of Ca11 from the ryanodine receptor calcium release channel (Ry1) (Otsuet al., 1994, Carrieret al., 1991, Mick-elsonet al., 1988; Lopezet al., 1988, Nelson, 1983). Several different single amino acid changes in the protein Ry1 have linked mutation in the Ry1 gene to MH susceptiblity in pigs
(Fujii, et al., 1991) and humans (MacLennan et al., 1990, McCarthyet al., 1990, Gillardet al., 1991, Otsuet al., 1994). These mutations may be responsible for altering the wild-type channel into a channel which has higher Ca11 perme-ability and is more susceptible to physiological and pharma-cological activators.
The Ry1 calcium channel is a large homotetrameric protein in which each subunit binds 1 molecule of ryanodine (Coro-nado et al., 1994, for review). High (nanomolar) and low (micromolar) affinity ryanodine binding sites have been de-scribed (Pessah and Zimanyi, 1991) and binding of ryanodine to these sites is very complex because of interaction with a negative cooperativity (Carrollet al., 1991; Laiet al., 1989). Binding to the high affinity site is associated with a sus-tained, open substate channel conductance whereas binding to the low affinity site produces inactivation of the channel (Laiet al., 1989; Zimanyiet al., 1992).
The CHCT is the method accepted by The North American and European MH Group to detect patients susceptible to MH (Larach, 1989; The European Malignant Hyperpyrexia Group, 1984). This method evaluates critical tensions in-duced by caffeine and/or halothane in biopsied, human skel-etal muscle. Although the CHCT continues to be important
Received for publication January 27, 1997.
1
This work was supported by National Institutes of Health Grant GM23875 and by CAPES/MEC/CNPq/Brazil.
ABBREVIATIONS: MHS, malignant hyperthermia susceptible; MHN, malignant hyperthermia nonsusceptible; Ry1, ryanodine receptor calcium release channel; CHCT, caffeine-halotrane-contracture test; RCT, ryanodine-contracture test; Pt, maximal tension amplitude; CSA, cross-sectional area; MHE, malignant hyperthermia equivocal.
THEJOURNAL OFPHARMACOLOGY ANDEXPERIMENTALTHERAPEUTICS Vol. 282, No. 3 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282:1331–1336, 1997
for the diagnosis of MH susceptible individuals, its sensitiv-ity and specificsensitiv-ity have been questioned; especially after dis-covery of several different Ry1 MH mutations (Deufelet al., 1995; MacLennan, 1995). With the aim of improving thein vitrocontracture test, the European MH group suggested the use of an RCT in addition to the CHCT (Wappleret al., 1996). Data from these laboratories show that RCT is more specific for detecting MH equivocal patients; however, some overlaps between MHN and MHS patients occurred, especially at higher ryanodine concentrations (i.e.,.2mM) (Lenzenet al., 1993). Recently, Wappler (Wappler et al., 1996) demon-strated that the overlap can be reduced at lower ryanodine concentration (1mM).
Experiments using different methods show that the bind-ing of ryanodine to Ry1 is very slow (Pessah and Zimanyi, 1991). This time can be reduced by pre-activation of Ry1 with caffeine, ATP or Ca11and prolonged by Mg11(Chuet al., 1990; Rousseauet al., 1987). Increasing the open state prob-ability of the Ca11 channel increases accessibility of Ry to the binding site. In relation to MH diagnostic testing, the CHCT, involving two tests, takes less than 30 minutes while the RCT takes 2 to 3 hr if 1mM Ry is used. In our study we tested a hypothesis that ryanodine binding is use dependent and that by increasing the frequency of muscle stimulation the time to ryanodine-induced contracture tension would be shortened. If correct, this method could be used to reduce the time of RCT for MH diagnosis and possibly improve diagnos-tic sensitivity and specificity. The objective of our study was to investigate if the kinetics of ryanodine-induced contrac-ture in intact muscle fibers could be changed by use-depen-dence mechanisms and, if so, then to determine the stimulus frequency and ryanodine concentration optimal for distin-guishing MHN from MHS.
Methods
MH phenotyping. This study was perfomed in nine nonsuscep-tible (MHN) and seven MHS dogs. The experimental protocol was approved by our Institutional Animal Care and Use Committee. MH susceptibility of each dog was determined by anin vivotest with a halothane-succinylcholine challenge protocol (Nelson, 1991) and by the in vitro CHCT (Larach, 1989). During the in vivo test, body temperature, muscle rigidity, end tidal CO2level and arterial blood
gases were evaluated. Forin vitrotesting, specimens from gracilis muscle were biopsied, under pentobarbital anesthesia, on the same day and immediately before thein vivotest and submitted to the contracture protocol approved by the North American MH Group for human MH diagnosis (Larach, 1989).
Preparation of muscle specimens for ryanodine study. At least 1 mo after the MH phenotyping, each dog was anesthetized with pentobarbital, 25 mg/kg, i.v., the trachea intubated and the lungs mechanically ventilated with 70% O2and 30% N2O. During
this anesthesia, 14 to 16 muscle specimens (length 5 2–3 cm, width 51.5–3 mm, weight5 0.1–0.2 g) were dissected from the gracilis muscle of each animal. Four specimens were mounted in 50 ml vertical chambers, filled with Krebs-Ringer solution (in mmol/ liter, NaCl, 118.1; KCl, 3.4; MgSO4, 0.8; KH2PO4, 1.2; glucose, 11.1;
NaHCO3, 25; CaCl2.6 H2O, 2.5, pH 7.4), saturated with carbogen
(95% O2/5% CO2) and the temperature maintained at 37°C. Others
specimens for later testing were stored in Krebs-Ringer solution at room temperature and continuously oxygenated until used. One end of each fascicle was tied to a fixed clamp and the other end attached to a FT-03 force transducer (Grass Instruments, West Warwick, RI) mounted on a micromanipulator. The muscles were electrically
stim-ulated by a pair of platinum electrodes connected to a Grass model S88 stimulator. To maximize twitch tension each fiber received a different current via a Med-Lab Stimu-Splitter II connected between the Grass stimulator and platinum electrodes. The muscle length was adjusted to obtain twitches of Pt. The frequency and duration of stimulation were 0.1 Hz and 2 msec, respectively, during the period of muscle preparation. The total time of each experiment varied from 3 to 7 hr. Over the period of these experiments, the muscle twitch was not significantly affected. The muscle twitches were recorded by a Grass mod 7400 (Astro-Med, Inc, Instr, West Warwick, RI) instru-ment. The analog signal was conditioned and digitized by a Cyber-Amp 320 Programmable Signal Conditioner and Digidata 1200 In-terface, respectively (Axon Instruments, Foster City, CA). The data were further analyzed using pClamp6 software (Axon Instruments). Experimental protocol. To investigate the effect of use-depen-dence on the kinetics and intensity of ryanodine-induced contrac-tures, 12 viable fascicles were divided in 3 sets of 4 fascicles each. Each set was stimulated at a fixed frequency (0.1, 0.5 or 1 Hz) and each fascicle exposed to a fixed ryanodine concentration (0, 1, 2.5 or 5mM). The ryanodine concentration in the bath was randomly se-lected. Therefore, each specimen was stimulated at a fixed frequency and tested with a fixed concentration of Ry. The control (0 ryanodine) was necessary to measure the effect of time and stimulus frequency on the baseline tension of the muscle specimen. After adding ryan-odine into the Krebs-Ringer solution, we determined the time to tension onset, half-time to peak tension and time to peak tension. We also correlated the tension with the time of exposure to Ry. The peak tension was also correlated to Pt measured just before adding Ry into solution and to CSA calculated at the end of experiments using the equation: CSA (cm2)5weight (g)/length (cm)31.06 g/cm3.
The fascicles were exposed to Ry during the time necessary to reach the contracture plateau. Then, the ryanodine solution was replaced by a fresh Krebs-Ringer solution without ryanodine. We did not wait to complete recovery of tension to baseline, except for a few experiments where we desired to investigate the offset time of ryan-odine from the Ry1.
Drugs. High purity ryanodine was purchased from Calbiochem and the stock solution was prepared by dissolving the powder in distilled water and frozen until used.
Data collections and analysis. The time to onset tension was determined at the moment that the trace starts to rise from the baseline after addition of ryanodine into solution. The values are presented as mean6S.E. The data from MHN and MHS dogs were compared between groups by Student’sttest.
Results
Effect of ryanodine concentration. Exposure of normal and MHS dog gracilis muscle to increasing concentrations of ryanodine produced slowly developing isometric contractures that increased with increasing ryanodine concentration (fig. 1). In normal dog muscle 1mM ryanodine produced an aver-age peak contracture tension of 2.076 0.49 g, and the ten-sions at 5mM ryanodine averaged 4.1160.77 g. The average tensions produced by 1, 2.5 and 5mM ryanodine in MHS dog muscle were greater than those in normal muscle but these were not statistically significantly different (fig. 1). The time to onset of ryanodine contracture tension was inversely af-fected by increasing ryanodine concentrations (fig. 2). In MHN muscle the time to 1 mM ryanodine-induced tension onset was almost 1 hr although at 5mM the time to tension onset was reduced to 19 min. This effect of ryanodine con-centration on time to tension onset was much greater in MHN compared to MHS muscle (fig. 2). The tension onset time was 10 times shorter for 1mM ryanodine in MHS mus-cle;i.e.,5.560.9 min in MHSvs.556 10.4 min for MHN Vol. 282
muscle (fig. 2). Because of the shorter time to tension onset in MHS muscle, increasing the concentration of ryanodine had less effect in this muscle. The difference in time to tension
onset between MHS and MHN muscle is greater at 1 mM ryanodine and diminishes as the concentration of ryanodine is increased (fig. 2). Similar to the time to onset of ryanodine contractures, the time to peak contracture was inversely affected by ryanodine concentration and was shorter for MHS muscle (fig. 3). At 1mM ryanodine the average time to peak tension was 152.7611.7 min for MHN and 36.468.1 min for the MHS muscles. Increasing the ryanodine concen-tration from 1 to 5mM decreased the time to peak tension by approximately 50% in both MHN and MHS muscle (fig. 3).
Effect of stimulus frequency on ryanodine contrac-tures. Increasing the frequency of electrical stimulation dra-matically decreased the time to onset of ryanodine-induced contracture in MHN but had little effect in the MHS dog muscles (fig. 4). This effect of increasing stimulus frequency was greater at the lower concentrations of ryanodine where increasing from 0.1 to 1 Hz caused decreases of 74, 71 and 68% in the time to tension onset at 1, 2.5 and 5mM ryanodine respectively. In comparison, this same increase in frequency produced decreases in time to tension onset of only 10.8, 22.2 and 3% at 1, 2.5 and 5mM ryanodine-induced contractures of MHS muscle. The time to peak tension was also decreased by increasing the rate of stimulation (fig. 5). In MHN muscles, a 10-fold increase in stimulus frequency decreased the time to peak contracture by 65, 65.3 and 60.9% at 1, 2.5 and 5mM ryanodine. A similar, but smaller effect of increasing stimu-lus frequency was observed in the MHS muscle where a 10-fold increase in frequency produced 52, 45.7 and 42.4% decreases in the time to peak tension at 1, 2.5, and 5 mM ryanodine. The peak tension produced at each ryanodine concentration was not significantly affected by the rate of stimulation of either MHN or MHS muscle (fig. 5). Correcting the tension for grams per cross-sectional area or for Pt of each fascicle did not alter these responses (data not shown). Fig. 1. Relationship between ryanodine concentration and the peak
isometric contracture tension in gracilis muscle fascicles biopsied from malignant hyperthermia susceptible (MHS) and non-susceptible (MHN) dogs. Each data point represents the mean 6 S.E. for seven MHS (closed symbols) and nine MHS (open symbols) muscle fascicles. In this protocol each fascicle was stimulated at 0.1 Hz. See figure 3 for the time to obtain peak tension in these fascicles.
Fig. 2. Relationship between ryanodine concentration and time to on-set of isometric contracture tension in gracilis muscle fascicles biop-sied from malignant hyperthermia susceptible (MHS) and non-suscep-tible (MHN) dogs. Each data point represents the mean6S.E. for seven MHS (closed symbols) and nine MHN (open symbols) muscle fascicles.
Fig. 3. Relationship between ryanodine concentration and time to peak isometric contracture tension in gracilis muscle fascicles biopsied from malignant hyperthermia susceptible (MHS) and nonsusceptible (MHN) dogs. Each data point represents the mean6S.E. for seven MHS (closed symbols) and nine MHN (open symbols) muscle fascicles.
Discussion
Caffeine and halothane cause Ca11 release from the SR membrane by activating the Ry1 calcium release channel.
Exposure of biopsied skeletal muscle to these compounds and measurement of the critical contracture tension provides the basis for the North American and European MH diagnostic centers’ identification of MH susceptible patients (Larach, 1989; The European Malignant Hyperpyrexia Group, 1984). A third drug, ryanodine, has been introduced by the Euro-pean MH group to improve the separation between MHN, MHE and MHS responses (Wappleret al., 1996). Ryanodine binds specifically to the Ry1 calcium release channel and probably causes contracture in skeletal muscle by induction of a sustained open, subconducting state of the Ry1 channel, allowing Ca11 to slowly leak from the SR (Rousseauet al., 1987). Because mutations in Ry1 link to MH in some fami-lies, it might be expected that ryanodine-induced contrac-tures might provide greater sensitivity and specificity for MH diagnostic contracture testing. Preliminary results from the European group show that the RCT is effective for distin-guishing MHN from MHS patients and for reducing, but not abolishing the number of equivocal tests detected by the CHCT (Hopkins et al., 1991; Wappler et al., 1996). In con-trast, others studies showed that improvement of the RCT protocol would be necessary to reduce overlap between MHN and MHS responses (Wappler et al., 1996, Hartung et al., 1996). In our study we demonstrate that the amplitude and kinetics of ryanodine-induced contracture can be changed by altering ryanodine concentration and/or frequency of electri-cal stimulation in MHN but not in MHS specimens. Ry Contracture: MHNvs.MHN Dogs
Ry concentrationvs.tension. Using the MH dog model we found that measurement of peak tension in response to 1, 2.5 and 5mM ryanodine was not a reliable method for detect-ing MHS in the dog. There was no significant difference between the two groups across the range of Ry concentrations tested (fig. 1). We considered the possibility that variability of the data could be related to specimen size or viability. However, correction of the peak tension values for CSA or for the Pt did not improve the difference between MHN and MHS responses. We conclude that the ryanodine-induced peak tension values obtained in this study are not useful for separating MHN from MHS dog muscle. We observed a bet-ter and more reliable differentiation when time variables, instead of absolute tension, were considered. At 1mM Ry the time to onset of contracture tension in MHS dog muscle was 10 times shorter than for MHN muscle, and at 1 and 2.5mM ryanodine no overlap occurred between the groups (fig. 2). The time to peak tension in response to all tested ryanodine concentrations was also a very reliable indicator (fig. 3). However, measuring the time to ryanodine-induced peak ten-sion for MH diagnostic purposes may add several hours to the total testing time. This could have adverse effects on muscle viability when the additional time required for the CHCT is also considered. Thus, using time to peak ryanodine-induced tension as an adjunct diagnostic test may not be advanta-geous.
Stimulation frequency versus Ry contracture ten-sion and kinetics. The use-dependence for Ry-induced con-tracture was tested by changing the frequency of stimulation. Significant decreases in the tension onset time and time to peak tension were observed when the frequency was in-creased from 0.1 to 0.5 Hz. However, the higher the fre-quency, the worse the separation between MHN and MHS Fig. 4. Effect of stimulus frequency on the time to onset of
ryanodine-induced isometric contracture tension in gracilis muscle biopsied from malignant hyperthermia susceptible (MHS) and nonsusceptible (MHN) dogs. Each data point represents the mean 6 S.E. for seven MHS (closed symbols) and nine MHN (open symbols) muscle fascicles. Each line represents a different concentration of ryanodine as indicated by different symbols.
Fig. 5. Effect of stimulus frequency on the time to peak of the ryano-dine-induced isometric contracture tension in gracilis muscle biopsied from malignant hyperthermia susceptible (MHS) and nonsusceptible (MHN) dogs. Each data point represents the mean6S.E. for seven MHS (closed symbols) and nine MHN (open symbols) muscle fascicles. Each line represents a different concentration of ryanodine as indicated by different symbols.
dog muscle responses. This is primarily due to MHS muscle responses not being affected by stimulus frequency and to the movement of MHN response values closer to the MHS re-sponse values. The advantage of using higher frequency for MH diagnosis is the reduced time required to complete the test, but if too high a frequency is used, the power of diag-nostic discrimination is reduced. If onset time was chosen as the variable to detect MH patients, the probability of having a false negative with the combinations of 0.1 Hz and 1mM Ry or with 0.5 Hz and 1mM Ry was lower than 1%. In the MHS group the onset time for all fascicles used in this study was less than 10 minutes with very small variability (fig. 4). Therefore, if we select 10 min as the time to contracture tension onset for a positive MH diagnosis, the probability of obtaining a false negative would be , 1%. This time of muscle exposure to Ry is close to the exposure time for the halothane test and is shorter than that for the caffeine test in the CHCT.
The frequency-dependence for ryanodine contractures in human muscle should be investigated because it may be different from that in dog muscle. Studies from the European group have only changed the Ry concentration (Wappleret al., 1996; Hartunget al., 1996) and have fixed the frequency at 0.2 Hz. It could be that in human muscle the overlap between MHN and MHE could be decreased by lowering frequency of stimulation from 0.2 to 0.1 Hz.
Variability in Ry responses. Regarding sources of vari-ability in the data, it is interesting to note that varivari-ability was smaller for time to peak tension than it was for time to tension onset. Variability was also smaller in MHS than MHN responses; a discrepancy that has also been observed in human muscle (Hartunget al., 1996). Hartunget al.(1996) described higher variability in muscle fascicle responses among control and MHN groups than in MHS fascicles. They also found the smallest variability in the MH fulminant group of patients clinically classified as grade 6 with raw score range$50 (Larachet al., 1994). The exact onset time is usually more difficult to determine than is time to peak tension because the development of Ry-induced tension is very slow. Consequently it is difficult to determine exactly when the trace starts to rise from the baseline because there is not a clear deflection. Another explanation for variability could be related to different sensitivities of the normal Ry1 population. This sensitivity could be modulated by activation level or by some conformational change of the Ry1 caused by mutations other than those predisposing to MH.
Mechanisms of Ry-induced tension and factors af-fecting them. Different mechanisms could be involved to explain the acceleration of Ry-induced contracture caused by Ry concentration and by frequency of stimulation. Binding analysis using3[H]Ry has shown low association and disso-ciation rates that require long incubation periods for analysis (Hawkeset al., 1992). Electrophysiological studies measuring Ry1 activity of incorporated channels in a lipid bilayer show that Ry increases the open state probability and maintains the Ca11 channel in sustained open substate (Lai et al., 1989; Zimanyiet al., 1992). In intact muscle exposed to low concentrations of Ry, the slow binding of Ry and partial activation of the channels results in a slow rate of Ca11 release into the sarcoplasm allowing most of it to be pumped back into the SR by Ca-ATPase. The dissociation of Ry is also slow, and over time there is a cumulative effect as more Ry1
molecules are bound. As more and more of the four Ry1 binding sites become occupied per molecule, more Ca11 is released, requiring more Ca11-ATPase activation. With time, the rate of Ca11release exceeds that for Ca11uptake, resulting in a critical level of Ca11 that activates the con-tractile proteins and leads to tension onset.
The rate of Ry-induced tension was significantly increased by increasing the frequency of stimulation. In our interpre-tation, Ry binds to the receptor when the Ca11 channel is activated to an open state. For this reason, tension is devel-oped slower at low stimulus frequency or in the absence of stimulation. Several others studies have demonstrated that the affinity of Ry1 can be changed by experimental condi-tions. Binding of [3H]Ry and Ca11 channel activation is optimal at 10 to 100mM Ca11concentration and inhibited in mM range (Chuet al., 1990). Preactivation of Ry1 by caffeine increased the rate of [3H]Ry association without changing the rate of dissociation (Chuet al., 1990; Zimanyiet al., 1992). In skinned skeletal and cardiac fibers, Su (1992a; 1992b) re-ported that the combination of caffeine with Ry decreased the Ca11sequestration in the SR. This effect was caffeine dose dependent.
In MHS specimens the onset time and the time to peak were not affected by either Ry concentration or by frequency of stimulation. Assuming that the greater sensitivity of MHS dog muscle to ryanodine-induced contracture is a conse-quence of higher affinity for Ry binding and not number of binding sites, we can question why neither Ry concentration nor stimulus frequency affected the onset time of MHS mus-cle. We conclude that the Ry1 channels in MHS muscle are in an optimal configuration for binding even when stimulus frequency is low. The most likely explanation for this is that the MHS mutation favors the binding of Ry. Therefore, in-creasing frequency of stimulation minimizes the separation of MHN from MHS dog muscle. In human muscle it may be that decreasing stimulus frequency from 0.2 Hz to 0.1 Hz or less will improve the ability to distinguish between MHN from MHS muscle. Further studies comparing ryanodine con-tractures in MHS and MHN human skeletal muscle will be necessary in order to determine the optimal concentration, stimulus frequency, and so on for the use of ryanodine in the diagnosis of MH in humans.
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