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Effects of the phosphodiesterase-III inhibitor enoximone on skeletal muscle specimens from malignant hyperthermia susceptible patients
Original Contributions Effects of the Phosphodiesterase-III Inhibitor Enoximone on Skeletal Muscle Specimens From Malignant Hyperthermia Susceptible Patients Marko Fiege, MD,* Frank Wappler, MD,† Jens Scholz, MD,‡ Ralf Weisshorn, MD,* Verena von Richthofen, MD,* Jochen Schulte am Esch, MD§ Department of Anesthesiology, University-Hospital Eppendorf, Hamburg, Germany
*Staff Anesthesiologist †Associate Professor of Anesthesiology ‡Professor of Anesthesiology §Professor Chairman of Anesthesiology Address correspondence to Dr. Fiege at the Department of Anesthesiology, UniversityHospital Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail: fiege@ uke.uni-hamburg.de Presented in part at the Annual Meeting of the American Society of Anesthesiologists, Orlando, FL, October 17–21, 1998. Received for publication October 22, 1999; revised manuscript accepted for publication January 18, 2000.
Study Objectives: To study the in vitro effects of the phosphodiesterase-III inhibitor enoximone on skeletal muscle specimens from malignant hyperthermia susceptible (MHS) and normal (MHN) patients. Design: Prospective study. Setting: Malignant hyperthermia (MH) laboratory at a university hospital. Patients: 47 patients with clinical suspicion for MH undergoing in vitro contracture test (IVCT) for diagnosis of MH susceptibility. Interventions: Biopsies of M. quadriceps femoris were performed in adult patients with a 3-in-1 nerve block and in children with trigger-free general anesthesia. Measurements and Main Result: Patients were first classified as MHS or MHN by the IVCT according to the protocol of the European MH Group (EMHG). Patients with equivocal results (MHE) or with neuromuscular diseases were excluded from the study. Enoximone was added to the organ bath to surplus vital muscle specimens in single bolus concentrations of 0.4, 0.6, 0.8, or 1.6 mmol/L. The in vitro effects of enoximone on muscle contractures and twitch were measured. Seventeen patients were classified as MHS and 30 as MHN by the EMHG criteria. Enoximone induced contractures in skeletal muscles in a dose-dependent manner. Contractures of MHS compared to MHN muscle specimens were significantly larger at all concentrations used in this study. No overlap in maximum contractures was seen between MHS and MHN muscles at a bath concentration of 0.6 mmol/L enoximone only. Conclusions: Diagnosis of MH by an IVCT test with a single bolus administration of enoximone seems to be possible using a concentration of 0.6 mmol/L. The findings of this study may indicate an involvement of the phosphodiesterase-III and cAMP system in pathogenesis of MH. Further in vivo investigation should determine the trigger potency of enoximone in MH susceptible individuals. © 2000 by Elsevier Science Inc.
Journal of Clinical Anesthesia 12:123–128, 2000 © 2000 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
0952-8180/00/$–see front matter PII S0952-8180(00)00124-0
Original Contributions
Keywords: Cafffeine; enoximone; halothane; malignant hyperthermia; phosphodiesterase.
Introduction Malignant hyperthermia (MH) is a silent myopathy with autosomal-dominant heredity, which is usually triggered by volatile anesthetics and depolarizing muscle relaxants.1 It is widely accepted that susceptibility to MH is caused by abnormal calcium metabolism within the skeletal muscle fiber.1 A rapid and sustained calcium release of intracellular stores results in high myoplasmic calcium concentrations during anesthetic-induced MH crisis in skeletal muscle cells. This effect is accountable for muscle contractures as well as for hypermetabolism. The site of the defect in MH is supposed to be the skeletal muscle ryanodine receptor, which is the calcium release channel of skeletal muscle sarcoplasmic reticulum. Several pharmacologic and biochemical abnormalities of the ryanodine receptor have been found in MH susceptible (MHS) individuals.1,2 However, the activity of the ryanodine receptor is regulated by a variety of modulators, which have been shown to be altered in MH. An involvement of free fatty acids,3 inositolphosphates,4 and the serotonin-system5 in pathophysiology of MH has been described. The cAMP system also may play an important role in pathogenesis of MH. In skeletal muscle cells from MHS patients and MHS animals, higher cAMP levels could be measured compared to MH normal (MHN) individuals.6 – 8 Furthermore, during and after physical exercise, cAMP concentrations in blood serum increased to a higher extent and were more prolonged in MHS than in MHN patients.9 Phosphodiesterase (PDE)-III inhibitors are substances with receptor-independent, positive inotropic effects on the heart. PDE-III inhibitors act by decreasing the rate of cAMP degradation. The cAMP activates proteinkinase A, which results in changed transport rates of different intracellular calcium channels. In cardiac muscle cells, PDE-III inhibition increases calcium release from the sarcoplasmic reticulum via the ryanodine receptor.10,11 The effects of PDE-III inhibitors on human skeletal muscle are essentially unknown, but a similar mechanism of action may be possible. Previous studies have shown that the PDE-III inhibitor enoximone induced contractures in human skeletal muscles in vitro.12,13 Enoximone doses were increased cumulatively and muscle contractures started at lower concentrations in MHS than in MHN preparations. Furthermore, the contractures in all MHS specimens were greater than in MHN. Therefore, an in vitro diagnosis of MH by a contracture test with cumulative administration of enoximone seemed to be possible. The purpose of the current study was to investigate the in vitro effects of different bolus concentrations of enoximone on skeletal muscle specimens from MHS and MHN patients, and to investigate whether this bolus test enables a clear discrimination between both groups. 124
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Materials and Methods Following approval by the local University-Hospital Eppendorf Ethic Committee, written informed consent for the different investigations was obtained from the patients or their parents, as appropriate. Forty-seven patients, 6 to 65 years of age, with clinical suspicion of MH were included in the study. A complete personal and family history was performed for each patient and relevant laboratory parameters, including creatine kinase (CK) levels, were measured. Adult muscle bundles were excised in regional anesthesia (3-in-1 nerve block) with 40 mL prilocaine 1% supplemented by sedation with midazolam when required. Biopsies in children were obtained during trigger-free general anesthesia using propofol, alfentanil, and nitrous oxide in oxygen. Muscle bundles were excised carefully from the vastus lateralis and immediately placed in Krebs-Ringer solution (constituents [mM]: NaCl 118.1; KCl 3.4; CaCl2 2.5; MgSO4 0.8; KH2PO4 1.2; NaHCO3 25.0; glucose 11.1) equilibrated with carbogen (95% oxygen:5% carbon dioxide). Apart from these specimens, an additional small muscle sample (weight approximately 250 mg) was excised for evaluation of histomorphological changes. All in vitro investigations were performed within a time period of 5 hours maximum after muscle biopsy. Muscle bundles for in vitro contracture test (IVCT) were split into 6 to 10 specimens (length 12 to 23 mm; width 2 to 3 mm; weight 95 to 220 mg). Only viable muscle specimens with a twitch response ⱖ10 mN to supramaximal stimulation were used for IVCT. Each muscle sample was secured with silk sutures to a fixed point and connected with a force displacement transducer (Lectromed, Welwyn Garden City, UK). The specimens were suspended in a 20-ml tissue bath perfused with Krebs-Ringer solution bubbled with carbogen continuously. Temperature was constant at 37°C, and pH was 7.4. The muscles were stimulated electrically with square waves to achieve a supramaximal response by a HSE Stimulator Type 215/I (Hugo Sachs Elektronik, March, Germany) with a duration of 1 ms and a frequency of 0.2 Hz. Contracture curves were displayed on a Linseis L2200 II (Selb, Germany) and recorded with a computer-based data evaluation program (MusCo™, RS BioMedTech, Sinzing, Germany). The resting length of the specimens was measured before testing, and the initial baseline tension prior to testing was achieved by stretching the samples slowly (4 mm/s) to 150 ⫾ 10% of the resting length. The patients were first classified by the IVCT according to the procedure of the European MH Group (EMHG).14 Halothane was added to the carbogen with a Draeger vaporizer (Luebeck, Germany), the concentration of halothane was measured with an anesthetic gas monitor (Normac威, Datex, Helsinki, Finland) and confirmed by gas chromatography periodically. The caffeine (Sigma, Deisenhofen, Germany) was dissolved fresh before each investigation in carboxygenated Krebs-Ringer solution and administered directly to the tissue bath. In each patient, a minimum of two samples was tested with each drug. MH status was evaluated by halothane and caffeine
Enoximone and malignant hyperthermia: Fiege et al.
Table 1. Patient Demographics
Age (yrs) Gender (male/female) Weight (kg) Height (cm) CK (U/L)
MHS (n ⴝ 17)
MHN (n ⴝ 30)
31.5 ⫾ 15.2 10/7 78.6 ⫾ 14.1* 173.5 ⫾ 7.8* 108.5 ⫾ 74.7*
26.7 ⫾ 17.1 14/16 56.9 ⫾ 22.7 156.9 ⫾ 21.7 51.8 ⫾ 63.1
Note: Values are means ⫾ SD. MHS ⫽ malignant hyperthermia susceptible; MHN ⫽ malignant hyperthermia normal; CK ⫽ creatine kinase concentration at rest. *p ⬍ 0.05 vs. MHN.
thresholds as follows. MHS ⫽ muscle contractures ⱖ2 mN at a halothane concentration ⱕ0,44 mmol/l and a caffeine concentration ⱕ2 mmol/L. MHN ⫽ muscle contractures ⬍2 mN at a halothane concentration ⱕ0.44 mmol/L and a caffeine concentration ⱕ2 mmol/L. Patients with neuromuscular diseases were excluded from the study. After investigation of MH susceptibility, viable muscle specimens surplus to diagnostic requirements were used for the study. After achieving at least a 10-minute stable baseline tension, enoximone (Perfan威, Hoechst, Frankfurt, Germany) was added as a bolus directly to the tissue bath to obtain concentrations of 0.4, 0.6, 0.8, or 1.6 mmol/L. The in vitro effects on contracture development and muscle twitch were observed for at least 30 minutes. Statistical evaluation was performed using a computerbased program (StatView 4.57, Abacus Concepts, Berkeley, CA). Contracture data are presented as medians and ranges; demographic characteristics and twitch data are shown as means ⫾ SD. Statistical analysis of demographic and contracture data were calculated using the MannWhitney test. The effects of enoximone on muscle twitch variables were assessed with repeated measures analysis of variance. When appropriate, subsequent comparisons were performed using Scheffe´’s post hoc method. MannWhitney test was used for comparisons between the groups. Results were considered significant for p-values less than 0.05.
Results Seventeen patients were characterized as MHS and 30 as MHN by IVCT, according to EMHG criteria. Patients demographics are listed in Table 1. Patients did not differ in age; however, there was a shift to male patients in the MHS group, and weight, height, and CK values at rest (normal values ⫽ 30 to 80 U/L) were significantly greater in MHS than in MHN patients. IVCT with different concentrations of enoximone were performed with 30 MHS muscle and 65 MHN muscle specimens. Typical original IVCT tracings of an MHS muscle and an MHN muscle following administration of enoximone in a concentration of 0.6 mmol/L are shown in Figure 1. In the MHS muscle preparation, a contracture developed immediately after administration of the enoximone. Maximum contracture of 64.2 mN was reached
Figure 1. Effects of enoximone bolus administration of 0.6 mmol/l on contracture development in skeletal muscle specimens of a malignant hyperthermia susceptible (MHS) and a malignant hyperthermia normal (MHN) patient. Original tracings of representative specimens are shown.
after 4.5 minutes, afterwards muscle contracture decreased slowly. At first, the MHS muscle twitch increased from 85 mN to 110 mN but then showed a marked decrease. The MHN-muscle preparation showed no contracture response to administration of enoximone. However, the muscle twitch of 93 mN at the start increased to 240 mN within 20 minutes after bolus administration of enoximone. The MHS muscle specimens reached a higher contracture level compared to the MHN-muscles at all enoximone concentrations. The individual data for maximum contractures are summarized in Figure 2. At an enoximone concentration of 0.4 mmol/L, six of seven MHS-muscle fascicles developed a contracture of 3.1 mN, but only four of 17 MHN fascicles showed contractures of maximum 0.8 mN. Enoximone in a concentration of 0.6 mmol/L induced marked contractures of 19.7 mN in all 11 tested MHS muscles, but only slight contractures of maximum 1.1 mN in 12 of 23 MHN muscles. Thus, no overlap in maximum contracture was found between the MHS and MHN groups at this enoximone concentration. After bolus administration of 0.8 mmol/l enoximone to the tissue bath, all nine MHS muscle specimens developed contractures of 23.4 mN. The contractures from nine of 13 MHN muscles were somewhat more distinct with 1.5 mN at this enoximone concentration. An enoximone concentration of 1.6 mmol/L led to contracture development in all muscle specimens, in the MHS group to 69.6 mN and in the MHN group to 13.8 mN. The muscle twitch before administration of enoximone was comparable with 37.6 ⫾ 30.0 in the MHS group and 48.4 ⫾ 37.2 in the MHN group. The data for the change in muscle twitch after adminstration of different concentrations of enoximone are summarized in Figure 3. Concentrations of 0.4 and 0.6 mmol/L enoximone induced an J. Clin. Anesth., vol. 12, March 2000
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Figure 2. Maximum contracture following administration of enoximone in concentrations of 0.4, 0.6, 0.8, or 1.6 mmol/L in skeletal muscle specimens of malignant hyperthermia susceptible (MHS) and malignant hyperthermia normal (MHN) patients. Shown are the median (—) and individual maximum contractures (■/E). Numbers above the boxes indicate the number of muscle specimens used for the different enoximone concentrations. *p ⬍ 0.05 versus MHN.
increase of the twitch in both groups after 10 minutes of observation. The increase continued in the MHN group after 20 and 30 minutes, but it was not detectable in the MHS group. At concentrations of 0.8 and 1.6 mmol/L enoximone, twitch amplitude increased only in the MHN group; therefore, in the MHS group no change in muscle twitch was found. Marked differences between the groups were found 30 minutes after adminstration of 0.6 mmol/L enoximone and after 10 minutes of observation at a concentration of 0.8 mmol/L enoximone.
Discussion The IVCT with halothane and caffeine is at present the gold standard for diagnosis of MH disposition and is used according to the protocol of the EMHG14 and the North American Malignant Hyperthermia Group (NAMHG).15 The IVCT with halothane and caffeine is highly sensitive. An investigation, attended by all European MH laboratories, demonstrated a sensitivity of 99.0% and a specifity of 93.6% for the standard IVCT,14 which is comparable to the results of a study using the NAMHG protocol.15 Despite the high sensitivity, there are two reports of supposed false-negative results of the standard IVCT.16,17 Because of uncertainties in the present IVCT such as those described, additional developments are desirable for safe diagnosis of MH susceptibility. A valid and reproducable noninvasive test for susceptibility of MH has not been found until now, and to date none of the drugs used for IVCT yields unambiguous discrimination between patients with and without disposition to MH. The ryanodine receptor has a key position in intracellular calcium metabolism and has been found to be modified in MH.1 Therefore, an IVCT with the physiological ligand ryano126
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dine, acting directly at the receptor, has been established in the last few years.18 However, a recently published multicenter study showed19 that the results of the ryanodine IVCT were dependent on the testing center and that, as a result, no threshold data were yet available. Moreover, an overlap of IVCT results was found between MHS and MHN muscle specimens; thus, IVCT with ryanodine lacks a clear discrimination between the diagnostic groups. A widely used preservative in a large number of preparations, 4-chloro-m-cresol (4-CmC) was found to be a specific activator of ryanodine receptor mediated Ca2⫹ release in skeletal muscle cells. Several published studies examined the potential of IVCT with 4-CmC for diagnosis of MH susceptibility.20 IVCT with 4-CmC discriminated between MHS and MHN. However, the published data do not yet allow a final evaluation of specifity and sensitivity. Multicenter studies with this new 4-CmC-IVCT are still in progress, but its future value for MH diagnostic is still unknown. In the last few years, two IVCT studies with the PDE-III inhibitor enoximone were published.12,13 The IVCT was performed with cumulative administration of enoximone to human skeletal muscle specimens. Enoximone induced in vitro a contracture development in skeletal muscles. These contractures arose in MHS muscle fascicles at lower enoximone concentrations than in MHN, so that a discrimination seemed to be possible. In this study, IVCTs with bolus adminstration of different enoximone concentrations were performed. With the enoximone concentrations used in this investigation, the human skeletal muscle specimens developed contractures in a concentration-dependent manner. A bolus concentration of 0.6 mmol/L enoximone induced marked contractures in all MHS muscles but none or only slight contrac-
Enoximone and malignant hyperthermia: Fiege et al.
Figure 3. Change in muscle twitch (mN) after administration of A: 0.4, B: 0.6, C: 0.8, and D: 1.6 mmol/L enoximone in skeletal muscle specimens of malignant hyperthermia susceptible (MHS; ■) and malignant hyperthermia normal (MHN; E) patients. Data are given as means ⫾ SD. *p ⬍ 0.05 versus MHN. #p ⬍ 0.05 versus 0 min.
tures in MHN muscles. In this study, a clear discrimination between MHS and MHN was possible by an IVCT with this enoximone concentration. Further investigation of enoximone IVCT should include MHE patients and patients with neuromuscular diseases, and be performed with larger populations. Such studies could then possibly determine the value of the new enoximone IVCT for MH diagnostic purposes. Phosphodiesterases of cyclic nucleotides posses a key position in metabolism of cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP). Many cellular functions are regulated by activation of adenylcyclase and guanylcyclase, leading to an increase of cAMP and cGMP. Phosphodiesterases lower the effects of these intracellular messengers by enhancing the degradation of cyclic nucleotides.21 Different isoenzymes of phosphodiesterases exist, classificated into five subgroups22 and with varying distribution in the tissues. In human cardiac muscle, the PDE-isoenzymes I to IV are detectable. Selective inhibition of PDE-III in cardic muscle has a receptor-independent positive inotropic effect.10,11 Enoximone is an established substance in clinical use, with high PDE-III selectivity. In contrast to cardiac muscle, very little is known about the distribution of the PDE isoenzymes in human skeletal muscle. In this study, the PDE-III inhibitor
enoximone induced an increase in twitch response in human skeletal muscle specimens. This effect was marked at lower enoximone concentrations and could be a hint for PDE-III activity in human skeletal muscle. In some investigations, indicators for an altered cAMP regulation in skeletal muscle cells from MHS individuals were found. In 1979, a higher cytoplasmic cAMP content and adenylcyclase activity was reported in three children who suffered anesthetic-induced hyperthermia, and in two children with stress-induced hyperthermia compared to a control group.23 A study of 33 MHS patients and 29 MHN individuals demonstrated a higher cytoplasmic cAMP content and adenylcyclase activity in the MHS-patients.6 However, differences in cAMP-PDE activity could not be found in this study. Another investigation confirmed the higher cAMP content in human skeletal muscle from MHS patients, and a lower cytoplasmic ATP content was found.7 In addition, during and after physical exercise, cAMP concentrations in blood serum increased more and were prolonged in MHS than in MHN patients.9 Furthermore, the higher cAMP content in skeletal muscle was found in MHS compared to MHN swine.8 To date, the importance and cause of high cAMP content in skeletal muscle cells in pathogenesis of MH is unknown. The site of the defect in MH is thought to be J. Clin. Anesth., vol. 12, March 2000
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the skeletal muscle ryanodine receptor, but the triggering mechanism of MH and the variability of clinical presentation have yet been defined conclusively. It has not been elucidated that the cAMP system may be a cofactor in disturbed calcium homeostasis in MH. In vivo administration of PDE-III inhibitors might enhance intracellular release of Ca2⫹ and potentiate an MH crisis. PDE-III inhibitors possibly also have an MH trigger potency of their own. The marked in vitro contractures of MHS compared to MHN muscles after adminstration of a PDE-III inhibitor found in this study hint at this hypothesis. In this study, the MHS muscle specimens developed minor contractures at a concentration of 0.4 mmol/L (0.1 mg/mL) enoximone, in earlier investigations at 0.2 mmol/L (0.05 mg/mL) enoximone.13 These concentrations are markedly higher than therapeutic plasma concentrations (0.5 to 3 g/mL) in humans.24,25 Multiple proposals for therapeutic administration of enoximone were published, and short-term higher plasma concentrations of enoximone or the active metabolite enoximone sulfoxide were measured.26 However, no data exist to date concerning concentrations in skeletal muscle during enoximone therapy. In this study, a clear discrimination between MHS and MHN by an IVCT with enoximone at a concentration of 0.6 mmol/L was possible. Further investigation should determine the value of this test for MH diagnostic purposes. The present in vitro experiments provide evidence that the PDE-III and cAMP system might be involved in pathogenesis of MH in humans. Further in vivo investigation should determine the trigger potency of enoximone in MH-susceptible individuals.
9.
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11. 12.
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References 1. Mickelson JR, Louis CF: Malignant hyperthermia: excitationcontraction coupling, Ca2⫹ release channel, and cell Ca2⫹ regulation defects. Physiol Rev 1996;76:537–92. 2. Fill M, Stefani E, Nelson TE: Abnormal human sarcoplasmic reticulum Ca2⫹ release channels in malignant hyperthermic skeletal muscle. Biophys J 1991;59:1085–90. 3. Fletcher JE, Tripolitis L, Erwin K, et al: Fatty acids modulate calcium-induced calcium release from skeletal muscle heavy sarcoplasmic reticulum fractions: implications for malignant hyperthermia. Biochem Cell Biol 1990;68:1195–201. 4. Foster PS, Gesini E, Claudianos C, Hopkinson KC, Denborough MA: Inositol 1,4,5-trisphosphate phosphatase deficiency and malignant hyperpyrexia in swine. Lancet 1989;2:124 –7. 5. Wappler F, Roewer N, Ko¨chling A, et al: Effects of the serotonin2 receptor agonist DOI on skeletal muscle specimens from malignant hyperthermia-susceptible patients. Anesthesiology 1996;84: 1280 –7. 6. Willner JH, Cerri CG, Wood DS: High skeletal muscle adenylate cyclase in malignant hyperthermia. J Clin Invest 1981;68:1119 – 24. 7. Ellis FR, Halsall PJ, Allam P, Hay E: A biochemical abnormality found in muscle from unstressed malignant-hyperpyrexia-susceptible humans. Biochem Soc Trans 1984;12:357– 8. 8. Scholz J, Steinfath M, Roewer N, et al: Biochemical changes in malignant hyperthermia susceptible swine: cyclic AMP, inositol
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phosphates, ␣1, 1- and 2-adrenoceptors in skeletal and cardiac muscle. Acta Anaesthesiol Scand 1993;37:575– 83. Stanec A, Stefano G: Cyclic AMP in normal and malignant hyperpyrexia susceptible individuals following exercise. Br J Anaesth 1984;56:1243– 6. Schmitz W, von der Leyen H, Meyer W, Neumann J, Scholz H: Phosphodiesterase inhibition and positive inotropic effects. J Cardiovasc Pharmacol 1989;14:S11– 4. Scholz H: PDE inhibition as a therapeutic principal. Z Kardiol 1994;83:1–5. Hartung E, Rauch A, Ru¨bsam B, Preis I, Sold M, Engelhardt W: Cyclic AMP and phosphodiesterase inhibition in skeletal muscle: a potent trigger or a cofactor for malignant hyperthermia? Minerva Anestesiol 1994;60:65–71. Fiege M, Wappler F, Scholz J, von Richthofen V, Brinken B, Schulte am Esch J: Diagnosis of susceptibility to malignant hyperthermia with an in vitro contracture test with the phosphodiesterase-III-inhibitor enoximone. Ana¨sthesiol Intensivmed Notfallmed Schmerzther 1998;33:557– 63. Ørding H, Brancadoro V, Cozzolino S, et al: In vitro contracture test for diagnosis of malignant hyperthermia following the protocol of the European MH Group: results of testing patients surviving fulminant MH and unrelated low-risk subjects. The European Malignant Hyperthermia Group. Acta Anaesthesiol Scand 1997;41:955– 66. Allen GC, Larach MG, Kunselman AR, The North American Malignant Hyperthermia Registry of MHAUS: The sensitivity and specificity of the caffeine-halothane contracture test: a report from the North American Malignant Hyperthermia Registry. Anesthesiology 1998;88:579 – 88. Isaacs H, Badenhorst M: False-negative results with muscle caffeine halothane contracture testing for malignant hyperthermia. Anesthesiology 1993;79:5–9. Wedel DJ, Nelson TE: Malignant hyperthermia— diagnostic dilemma: false-negative contracture responses with halothane and caffeine alone. Anesth Analg 1994;78:787–92. Hopkins PM, Ellis FR, Halsall PJ: Ryanodine contracture: a potentially specific in vitro diagnostic test for malignant hyperthermia. Br J Anaesth 1991;66:611–3. Hopkins PM, Hartung E, Wappler F: Multicentre evaluation of ryanodine contracture testing in malignant hyperthermia. Br J Anaesth 1998;80:389 –94. Tegazzin V, Scutari E, Treves S, Zorzato F: Chlorocresol, an additive to commercial succinylcholine, induces contracture of human malignant hyperthermia-susceptible muscles via activation of the ryanodine receptor Ca2⫹-channel. Anesthesiology 1996;84:1380 –5. Nicholson CD, Challiss RA, Shahid M: Differential modulation of tissue function and therapeutic potential of selective inhibitors of cyclic nucleotide phosphodiesterase isoenzymes. Trends Pharmacol Sci 1991;12:19 –27. Thompson WJ: Cyclic nucleotide phosphodiesterases: pharmacology, biochemistry and function. Pharmacol Ther 1991;51:13– 33. Willner JH, Cerri CJ, Wood DS: Malignant hyperthermia: abnormal cyclic AMP metabolism in skeletal muscle. Neurology 1979; 29:557. Ja¨hnchen E, Trenk D: Pharmacology and pharmacokinetics of enoximone. Z Kardiol 1991;80:21– 6. Trenk D, Ja¨hnchen E: Clinico-pharmacologic aspects of enoximone therapy. Z Kardiol 1994;83:7–14. Dage RC, Okerholm RA: Pharmacology and pharmacokinetics of enoximone. Cardiology 1990;77:2–13.
*Staff Anesthesiologist †Associate Professor of Anesthesiology ‡Professor of Anesthesiology §Professor Chairman of Anesthesiology Address correspondence to Dr. Fiege at the Department of Anesthesiology, UniversityHospital Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail: fiege@ uke.uni-hamburg.de Presented in part at the Annual Meeting of the American Society of Anesthesiologists, Orlando, FL, October 17–21, 1998. Received for publication October 22, 1999; revised manuscript accepted for publication January 18, 2000.
Study Objectives: To study the in vitro effects of the phosphodiesterase-III inhibitor enoximone on skeletal muscle specimens from malignant hyperthermia susceptible (MHS) and normal (MHN) patients. Design: Prospective study. Setting: Malignant hyperthermia (MH) laboratory at a university hospital. Patients: 47 patients with clinical suspicion for MH undergoing in vitro contracture test (IVCT) for diagnosis of MH susceptibility. Interventions: Biopsies of M. quadriceps femoris were performed in adult patients with a 3-in-1 nerve block and in children with trigger-free general anesthesia. Measurements and Main Result: Patients were first classified as MHS or MHN by the IVCT according to the protocol of the European MH Group (EMHG). Patients with equivocal results (MHE) or with neuromuscular diseases were excluded from the study. Enoximone was added to the organ bath to surplus vital muscle specimens in single bolus concentrations of 0.4, 0.6, 0.8, or 1.6 mmol/L. The in vitro effects of enoximone on muscle contractures and twitch were measured. Seventeen patients were classified as MHS and 30 as MHN by the EMHG criteria. Enoximone induced contractures in skeletal muscles in a dose-dependent manner. Contractures of MHS compared to MHN muscle specimens were significantly larger at all concentrations used in this study. No overlap in maximum contractures was seen between MHS and MHN muscles at a bath concentration of 0.6 mmol/L enoximone only. Conclusions: Diagnosis of MH by an IVCT test with a single bolus administration of enoximone seems to be possible using a concentration of 0.6 mmol/L. The findings of this study may indicate an involvement of the phosphodiesterase-III and cAMP system in pathogenesis of MH. Further in vivo investigation should determine the trigger potency of enoximone in MH susceptible individuals. © 2000 by Elsevier Science Inc.
Journal of Clinical Anesthesia 12:123–128, 2000 © 2000 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
0952-8180/00/$–see front matter PII S0952-8180(00)00124-0
Original Contributions
Keywords: Cafffeine; enoximone; halothane; malignant hyperthermia; phosphodiesterase.
Introduction Malignant hyperthermia (MH) is a silent myopathy with autosomal-dominant heredity, which is usually triggered by volatile anesthetics and depolarizing muscle relaxants.1 It is widely accepted that susceptibility to MH is caused by abnormal calcium metabolism within the skeletal muscle fiber.1 A rapid and sustained calcium release of intracellular stores results in high myoplasmic calcium concentrations during anesthetic-induced MH crisis in skeletal muscle cells. This effect is accountable for muscle contractures as well as for hypermetabolism. The site of the defect in MH is supposed to be the skeletal muscle ryanodine receptor, which is the calcium release channel of skeletal muscle sarcoplasmic reticulum. Several pharmacologic and biochemical abnormalities of the ryanodine receptor have been found in MH susceptible (MHS) individuals.1,2 However, the activity of the ryanodine receptor is regulated by a variety of modulators, which have been shown to be altered in MH. An involvement of free fatty acids,3 inositolphosphates,4 and the serotonin-system5 in pathophysiology of MH has been described. The cAMP system also may play an important role in pathogenesis of MH. In skeletal muscle cells from MHS patients and MHS animals, higher cAMP levels could be measured compared to MH normal (MHN) individuals.6 – 8 Furthermore, during and after physical exercise, cAMP concentrations in blood serum increased to a higher extent and were more prolonged in MHS than in MHN patients.9 Phosphodiesterase (PDE)-III inhibitors are substances with receptor-independent, positive inotropic effects on the heart. PDE-III inhibitors act by decreasing the rate of cAMP degradation. The cAMP activates proteinkinase A, which results in changed transport rates of different intracellular calcium channels. In cardiac muscle cells, PDE-III inhibition increases calcium release from the sarcoplasmic reticulum via the ryanodine receptor.10,11 The effects of PDE-III inhibitors on human skeletal muscle are essentially unknown, but a similar mechanism of action may be possible. Previous studies have shown that the PDE-III inhibitor enoximone induced contractures in human skeletal muscles in vitro.12,13 Enoximone doses were increased cumulatively and muscle contractures started at lower concentrations in MHS than in MHN preparations. Furthermore, the contractures in all MHS specimens were greater than in MHN. Therefore, an in vitro diagnosis of MH by a contracture test with cumulative administration of enoximone seemed to be possible. The purpose of the current study was to investigate the in vitro effects of different bolus concentrations of enoximone on skeletal muscle specimens from MHS and MHN patients, and to investigate whether this bolus test enables a clear discrimination between both groups. 124
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Materials and Methods Following approval by the local University-Hospital Eppendorf Ethic Committee, written informed consent for the different investigations was obtained from the patients or their parents, as appropriate. Forty-seven patients, 6 to 65 years of age, with clinical suspicion of MH were included in the study. A complete personal and family history was performed for each patient and relevant laboratory parameters, including creatine kinase (CK) levels, were measured. Adult muscle bundles were excised in regional anesthesia (3-in-1 nerve block) with 40 mL prilocaine 1% supplemented by sedation with midazolam when required. Biopsies in children were obtained during trigger-free general anesthesia using propofol, alfentanil, and nitrous oxide in oxygen. Muscle bundles were excised carefully from the vastus lateralis and immediately placed in Krebs-Ringer solution (constituents [mM]: NaCl 118.1; KCl 3.4; CaCl2 2.5; MgSO4 0.8; KH2PO4 1.2; NaHCO3 25.0; glucose 11.1) equilibrated with carbogen (95% oxygen:5% carbon dioxide). Apart from these specimens, an additional small muscle sample (weight approximately 250 mg) was excised for evaluation of histomorphological changes. All in vitro investigations were performed within a time period of 5 hours maximum after muscle biopsy. Muscle bundles for in vitro contracture test (IVCT) were split into 6 to 10 specimens (length 12 to 23 mm; width 2 to 3 mm; weight 95 to 220 mg). Only viable muscle specimens with a twitch response ⱖ10 mN to supramaximal stimulation were used for IVCT. Each muscle sample was secured with silk sutures to a fixed point and connected with a force displacement transducer (Lectromed, Welwyn Garden City, UK). The specimens were suspended in a 20-ml tissue bath perfused with Krebs-Ringer solution bubbled with carbogen continuously. Temperature was constant at 37°C, and pH was 7.4. The muscles were stimulated electrically with square waves to achieve a supramaximal response by a HSE Stimulator Type 215/I (Hugo Sachs Elektronik, March, Germany) with a duration of 1 ms and a frequency of 0.2 Hz. Contracture curves were displayed on a Linseis L2200 II (Selb, Germany) and recorded with a computer-based data evaluation program (MusCo™, RS BioMedTech, Sinzing, Germany). The resting length of the specimens was measured before testing, and the initial baseline tension prior to testing was achieved by stretching the samples slowly (4 mm/s) to 150 ⫾ 10% of the resting length. The patients were first classified by the IVCT according to the procedure of the European MH Group (EMHG).14 Halothane was added to the carbogen with a Draeger vaporizer (Luebeck, Germany), the concentration of halothane was measured with an anesthetic gas monitor (Normac威, Datex, Helsinki, Finland) and confirmed by gas chromatography periodically. The caffeine (Sigma, Deisenhofen, Germany) was dissolved fresh before each investigation in carboxygenated Krebs-Ringer solution and administered directly to the tissue bath. In each patient, a minimum of two samples was tested with each drug. MH status was evaluated by halothane and caffeine
Enoximone and malignant hyperthermia: Fiege et al.
Table 1. Patient Demographics
Age (yrs) Gender (male/female) Weight (kg) Height (cm) CK (U/L)
MHS (n ⴝ 17)
MHN (n ⴝ 30)
31.5 ⫾ 15.2 10/7 78.6 ⫾ 14.1* 173.5 ⫾ 7.8* 108.5 ⫾ 74.7*
26.7 ⫾ 17.1 14/16 56.9 ⫾ 22.7 156.9 ⫾ 21.7 51.8 ⫾ 63.1
Note: Values are means ⫾ SD. MHS ⫽ malignant hyperthermia susceptible; MHN ⫽ malignant hyperthermia normal; CK ⫽ creatine kinase concentration at rest. *p ⬍ 0.05 vs. MHN.
thresholds as follows. MHS ⫽ muscle contractures ⱖ2 mN at a halothane concentration ⱕ0,44 mmol/l and a caffeine concentration ⱕ2 mmol/L. MHN ⫽ muscle contractures ⬍2 mN at a halothane concentration ⱕ0.44 mmol/L and a caffeine concentration ⱕ2 mmol/L. Patients with neuromuscular diseases were excluded from the study. After investigation of MH susceptibility, viable muscle specimens surplus to diagnostic requirements were used for the study. After achieving at least a 10-minute stable baseline tension, enoximone (Perfan威, Hoechst, Frankfurt, Germany) was added as a bolus directly to the tissue bath to obtain concentrations of 0.4, 0.6, 0.8, or 1.6 mmol/L. The in vitro effects on contracture development and muscle twitch were observed for at least 30 minutes. Statistical evaluation was performed using a computerbased program (StatView 4.57, Abacus Concepts, Berkeley, CA). Contracture data are presented as medians and ranges; demographic characteristics and twitch data are shown as means ⫾ SD. Statistical analysis of demographic and contracture data were calculated using the MannWhitney test. The effects of enoximone on muscle twitch variables were assessed with repeated measures analysis of variance. When appropriate, subsequent comparisons were performed using Scheffe´’s post hoc method. MannWhitney test was used for comparisons between the groups. Results were considered significant for p-values less than 0.05.
Results Seventeen patients were characterized as MHS and 30 as MHN by IVCT, according to EMHG criteria. Patients demographics are listed in Table 1. Patients did not differ in age; however, there was a shift to male patients in the MHS group, and weight, height, and CK values at rest (normal values ⫽ 30 to 80 U/L) were significantly greater in MHS than in MHN patients. IVCT with different concentrations of enoximone were performed with 30 MHS muscle and 65 MHN muscle specimens. Typical original IVCT tracings of an MHS muscle and an MHN muscle following administration of enoximone in a concentration of 0.6 mmol/L are shown in Figure 1. In the MHS muscle preparation, a contracture developed immediately after administration of the enoximone. Maximum contracture of 64.2 mN was reached
Figure 1. Effects of enoximone bolus administration of 0.6 mmol/l on contracture development in skeletal muscle specimens of a malignant hyperthermia susceptible (MHS) and a malignant hyperthermia normal (MHN) patient. Original tracings of representative specimens are shown.
after 4.5 minutes, afterwards muscle contracture decreased slowly. At first, the MHS muscle twitch increased from 85 mN to 110 mN but then showed a marked decrease. The MHN-muscle preparation showed no contracture response to administration of enoximone. However, the muscle twitch of 93 mN at the start increased to 240 mN within 20 minutes after bolus administration of enoximone. The MHS muscle specimens reached a higher contracture level compared to the MHN-muscles at all enoximone concentrations. The individual data for maximum contractures are summarized in Figure 2. At an enoximone concentration of 0.4 mmol/L, six of seven MHS-muscle fascicles developed a contracture of 3.1 mN, but only four of 17 MHN fascicles showed contractures of maximum 0.8 mN. Enoximone in a concentration of 0.6 mmol/L induced marked contractures of 19.7 mN in all 11 tested MHS muscles, but only slight contractures of maximum 1.1 mN in 12 of 23 MHN muscles. Thus, no overlap in maximum contracture was found between the MHS and MHN groups at this enoximone concentration. After bolus administration of 0.8 mmol/l enoximone to the tissue bath, all nine MHS muscle specimens developed contractures of 23.4 mN. The contractures from nine of 13 MHN muscles were somewhat more distinct with 1.5 mN at this enoximone concentration. An enoximone concentration of 1.6 mmol/L led to contracture development in all muscle specimens, in the MHS group to 69.6 mN and in the MHN group to 13.8 mN. The muscle twitch before administration of enoximone was comparable with 37.6 ⫾ 30.0 in the MHS group and 48.4 ⫾ 37.2 in the MHN group. The data for the change in muscle twitch after adminstration of different concentrations of enoximone are summarized in Figure 3. Concentrations of 0.4 and 0.6 mmol/L enoximone induced an J. Clin. Anesth., vol. 12, March 2000
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Figure 2. Maximum contracture following administration of enoximone in concentrations of 0.4, 0.6, 0.8, or 1.6 mmol/L in skeletal muscle specimens of malignant hyperthermia susceptible (MHS) and malignant hyperthermia normal (MHN) patients. Shown are the median (—) and individual maximum contractures (■/E). Numbers above the boxes indicate the number of muscle specimens used for the different enoximone concentrations. *p ⬍ 0.05 versus MHN.
increase of the twitch in both groups after 10 minutes of observation. The increase continued in the MHN group after 20 and 30 minutes, but it was not detectable in the MHS group. At concentrations of 0.8 and 1.6 mmol/L enoximone, twitch amplitude increased only in the MHN group; therefore, in the MHS group no change in muscle twitch was found. Marked differences between the groups were found 30 minutes after adminstration of 0.6 mmol/L enoximone and after 10 minutes of observation at a concentration of 0.8 mmol/L enoximone.
Discussion The IVCT with halothane and caffeine is at present the gold standard for diagnosis of MH disposition and is used according to the protocol of the EMHG14 and the North American Malignant Hyperthermia Group (NAMHG).15 The IVCT with halothane and caffeine is highly sensitive. An investigation, attended by all European MH laboratories, demonstrated a sensitivity of 99.0% and a specifity of 93.6% for the standard IVCT,14 which is comparable to the results of a study using the NAMHG protocol.15 Despite the high sensitivity, there are two reports of supposed false-negative results of the standard IVCT.16,17 Because of uncertainties in the present IVCT such as those described, additional developments are desirable for safe diagnosis of MH susceptibility. A valid and reproducable noninvasive test for susceptibility of MH has not been found until now, and to date none of the drugs used for IVCT yields unambiguous discrimination between patients with and without disposition to MH. The ryanodine receptor has a key position in intracellular calcium metabolism and has been found to be modified in MH.1 Therefore, an IVCT with the physiological ligand ryano126
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dine, acting directly at the receptor, has been established in the last few years.18 However, a recently published multicenter study showed19 that the results of the ryanodine IVCT were dependent on the testing center and that, as a result, no threshold data were yet available. Moreover, an overlap of IVCT results was found between MHS and MHN muscle specimens; thus, IVCT with ryanodine lacks a clear discrimination between the diagnostic groups. A widely used preservative in a large number of preparations, 4-chloro-m-cresol (4-CmC) was found to be a specific activator of ryanodine receptor mediated Ca2⫹ release in skeletal muscle cells. Several published studies examined the potential of IVCT with 4-CmC for diagnosis of MH susceptibility.20 IVCT with 4-CmC discriminated between MHS and MHN. However, the published data do not yet allow a final evaluation of specifity and sensitivity. Multicenter studies with this new 4-CmC-IVCT are still in progress, but its future value for MH diagnostic is still unknown. In the last few years, two IVCT studies with the PDE-III inhibitor enoximone were published.12,13 The IVCT was performed with cumulative administration of enoximone to human skeletal muscle specimens. Enoximone induced in vitro a contracture development in skeletal muscles. These contractures arose in MHS muscle fascicles at lower enoximone concentrations than in MHN, so that a discrimination seemed to be possible. In this study, IVCTs with bolus adminstration of different enoximone concentrations were performed. With the enoximone concentrations used in this investigation, the human skeletal muscle specimens developed contractures in a concentration-dependent manner. A bolus concentration of 0.6 mmol/L enoximone induced marked contractures in all MHS muscles but none or only slight contrac-
Enoximone and malignant hyperthermia: Fiege et al.
Figure 3. Change in muscle twitch (mN) after administration of A: 0.4, B: 0.6, C: 0.8, and D: 1.6 mmol/L enoximone in skeletal muscle specimens of malignant hyperthermia susceptible (MHS; ■) and malignant hyperthermia normal (MHN; E) patients. Data are given as means ⫾ SD. *p ⬍ 0.05 versus MHN. #p ⬍ 0.05 versus 0 min.
tures in MHN muscles. In this study, a clear discrimination between MHS and MHN was possible by an IVCT with this enoximone concentration. Further investigation of enoximone IVCT should include MHE patients and patients with neuromuscular diseases, and be performed with larger populations. Such studies could then possibly determine the value of the new enoximone IVCT for MH diagnostic purposes. Phosphodiesterases of cyclic nucleotides posses a key position in metabolism of cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP). Many cellular functions are regulated by activation of adenylcyclase and guanylcyclase, leading to an increase of cAMP and cGMP. Phosphodiesterases lower the effects of these intracellular messengers by enhancing the degradation of cyclic nucleotides.21 Different isoenzymes of phosphodiesterases exist, classificated into five subgroups22 and with varying distribution in the tissues. In human cardiac muscle, the PDE-isoenzymes I to IV are detectable. Selective inhibition of PDE-III in cardic muscle has a receptor-independent positive inotropic effect.10,11 Enoximone is an established substance in clinical use, with high PDE-III selectivity. In contrast to cardiac muscle, very little is known about the distribution of the PDE isoenzymes in human skeletal muscle. In this study, the PDE-III inhibitor
enoximone induced an increase in twitch response in human skeletal muscle specimens. This effect was marked at lower enoximone concentrations and could be a hint for PDE-III activity in human skeletal muscle. In some investigations, indicators for an altered cAMP regulation in skeletal muscle cells from MHS individuals were found. In 1979, a higher cytoplasmic cAMP content and adenylcyclase activity was reported in three children who suffered anesthetic-induced hyperthermia, and in two children with stress-induced hyperthermia compared to a control group.23 A study of 33 MHS patients and 29 MHN individuals demonstrated a higher cytoplasmic cAMP content and adenylcyclase activity in the MHS-patients.6 However, differences in cAMP-PDE activity could not be found in this study. Another investigation confirmed the higher cAMP content in human skeletal muscle from MHS patients, and a lower cytoplasmic ATP content was found.7 In addition, during and after physical exercise, cAMP concentrations in blood serum increased more and were prolonged in MHS than in MHN patients.9 Furthermore, the higher cAMP content in skeletal muscle was found in MHS compared to MHN swine.8 To date, the importance and cause of high cAMP content in skeletal muscle cells in pathogenesis of MH is unknown. The site of the defect in MH is thought to be J. Clin. Anesth., vol. 12, March 2000
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the skeletal muscle ryanodine receptor, but the triggering mechanism of MH and the variability of clinical presentation have yet been defined conclusively. It has not been elucidated that the cAMP system may be a cofactor in disturbed calcium homeostasis in MH. In vivo administration of PDE-III inhibitors might enhance intracellular release of Ca2⫹ and potentiate an MH crisis. PDE-III inhibitors possibly also have an MH trigger potency of their own. The marked in vitro contractures of MHS compared to MHN muscles after adminstration of a PDE-III inhibitor found in this study hint at this hypothesis. In this study, the MHS muscle specimens developed minor contractures at a concentration of 0.4 mmol/L (0.1 mg/mL) enoximone, in earlier investigations at 0.2 mmol/L (0.05 mg/mL) enoximone.13 These concentrations are markedly higher than therapeutic plasma concentrations (0.5 to 3 g/mL) in humans.24,25 Multiple proposals for therapeutic administration of enoximone were published, and short-term higher plasma concentrations of enoximone or the active metabolite enoximone sulfoxide were measured.26 However, no data exist to date concerning concentrations in skeletal muscle during enoximone therapy. In this study, a clear discrimination between MHS and MHN by an IVCT with enoximone at a concentration of 0.6 mmol/L was possible. Further investigation should determine the value of this test for MH diagnostic purposes. The present in vitro experiments provide evidence that the PDE-III and cAMP system might be involved in pathogenesis of MH in humans. Further in vivo investigation should determine the trigger potency of enoximone in MH-susceptible individuals.
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11. 12.
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