Inhibition of sarcoplasmic Ca2+-ATPase increases caffeine- and halothane-induced contractures in muscle bundles of malignant hyperthermia susceptible and healthy individuals
© Schuster et al; licensee BioMed Central Ltd. 2005
Received: 24 November 2004
Accepted: 09 June 2005
Published: 09 June 2005
Malignant hyperthermia (MH) is triggered by halogenated anaesthetics and depolarising muscle relaxants, leading to an uncontrolled hypermetabolic state of skeletal muscle. An uncontrolled sarcoplasmic Ca2+ release is mediated via the ryanodine receptor. A compensatory mechanism of increased sarcoplasmic Ca2+-ATPase activity was described in pigs and in transfected cell lines. We hypothesized that inhibition of Ca2+ reuptake via the sarcoplasmic Ca2+-ATPase (SERCA) enhances halothane- and caffeine-induced muscle contractures in MH susceptible more than in non-susceptible skeletal muscle.
With informed consent, surplus muscle bundles of 7 MHS (susceptible), 7 MHE (equivocal) and 16 MHN (non-susceptible) classified patients were mounted to an isometric force transducer, electrically stimulated, preloaded and equilibrated. Following 15 min incubation with cyclopiazonic acid (CPA) 25 μM, the European MH standard in-vitro-contracture test protocol with caffeine (0.5; 1; 1.5; 2; 3; 4 mM) and halothane (0.11; 0.22; 0.44; 0.66 mM) was performed. Data as median and quartiles; Friedman- and Wilcoxon-test for differences with and without CPA; p < 0.05.
Initial length, weight, maximum twitch height, predrug resting tension and predrug twitch height of muscle bundles did not differ between groups. CPA increased halothane- and caffeine-induced contractures significantly. This increase was more pronounced in MHS and MHE than in MHN muscle bundles.
Inhibition of the SERCA activity by CPA enhances halothane- and caffeine-induced contractures especially in MHS and MHE skeletal muscle and may help for the diagnostic assignment of MH susceptibility. The status of SERCA activity may play a significant but so far unknown role in the genesis of malignant hyperthermia.
In skeletal muscle, the action potential passes along the surface membrane of the muscle fibre into the transverse tubular system. Depolarisation of the voltage sensitive dihydropyridine receptor leads to an opening of the ryanodine receptor in the nearby sarcoplasmic reticulum (SR). Sarcoplasmic calcium (Ca2+) release via the ryanodine receptor raises cytosolic Ca2+ and activates muscle contraction. Energy-dependent Ca2+ reuptake into the SR is caused by the SR Ca2+-ATPase (SERCA) and enables skeletal muscle relaxation . In individuals susceptible to the autosomal dominant skeletal muscle disorder malignant hyperthermia (MH), electro-mechanical coupling is disturbed. Due to MH-associated mutations in the ryanodine receptor, triggering agents such as halogenated anaesthetics cause an excessive Ca2+ release from the SR resulting in intracellular hypermetabolism, increased mitochondrial energy-turnover and metabolic failure with a deficiency of adenosine-triphosphate . This may also lead to energetic exhaustion of the SERCA, the main transporter for Ca2+ ions across the sarcoplasmic membrane. Cyctosolic Ca2+ concentration is determined by sarcoplasmic Ca2+ release and it's reuptake via the SERCA . The mycotoxin cyclopiazonic acid (CPA) is a selective inhibitor of SR Ca2+ reuptake  that has been used previously to study SERCA in different tissues [5, 6].
We hypothesized that in skeletal muscle, preincubation with CPA enhances halothane- and caffeine-induced contractures in MH susceptible (MHS) more than in non-susceptible (MHN) skeletal muscle.
Muscle bundles of 30 patients undergoing a diagnostic in-vitro contracture test (IVCT) were investigated to detect MH susceptibility. With informed consent, surplus muscle bundles were studied by the same IVCT protocol following SERCA inhibition by CPA.
A muscle biopsy of the vastus lateralis muscle was performed following a femoral nerve block. Muscle bundles were immediately placed in carboxygenated (95% oxygen, 5% carbon dioxide) Krebs-Ringer's solution (NaCl 118.1 mM; KCl 3.4 mM; CaCl2 2.5 mM; MgSO4 0.8 mM; KH2PO4 1.2 mM; NaHCO3 25.0 mM; Glucose 11.1 mM) and transported to the laboratory.
In brief, after length and wet weight of each muscle bundle was measured, single muscle strips were mounted vertically in the experimental bath perfused with carboxygenated Krebs-Ringer's solution at 37°C, fixed to an isometric force transducer (Lectromed Type 4150, UK) and stimulated electrically with a supramaximal square wave stimulus at 1 ms duration and a frequency of 0.2 Hz (Hugo-Sachs-Elektronik, Type 215/I, Germany). Resting tension and twitch height of the muscle strips were recorded continuously by a digital recording system (MusCo, RS BioMed, Germany). After equilibration, caffeine (Sigma Chemicals, Germany) respectively halothane (Abott, Germany) were given at increasing concentrations of 0.5; 1; 1.5; 2; 3; 4; and 32 mM respectively 0.11; 0.22; 0.44 and 0.66 mM at 3 min intervals. A contracture < 2 mN at caffeine 2 mM and halothane 0.44 mM was classified MHN. A stronger contracture following only one of both drugs lead to the diagnosis MH equivocal (MHE). If both drugs developed a significant contracture the patient was assigned as MHS. Investigations were performed within 5 hours after muscle biopsy .
A modified contracture test was carried out studying the drug CPA (M = 336.38 g mol-1) that was prepared in a stock solution at 2.5 mM dissolved in dimethylsulphoxide 0,5% (DMSO) (all Sigma Chemicals, Germany). Following equilibration as described above, muscle bundles were incubated with CPA 25 μM for 15 min. The contracture test was then carried out as described above.
Data are shown as median and quartiles. IVCT results of skeletel muscle contractures with CPA were statistically evaluated in comparison to the results without CPA by using the Friedman- and Wilcoxon-test for differences with and without CPA. p < 0.05 was considered significant.
Biometric data of muscle bundles used for the In-vitro Contracture-Test without (IVCT) and with preincubation with cyclopiazonic acid (CPA-IVCT); median and quartiles.
18 (16 – 20)
18 (15 – 20)
220 (190 – 233)
205 (180 – 240)
Maximum twitch height (mN)
24 (22 – 27)
25 (23 – 27)
Predrug resting tension (mN)
11 (9 – 14)
11 (8 – 14)
Predrug twitch height (mN)
57 (37 – 75)
42 (16 – 82)
Caffeine-induced contractures with and without preincubation by cyclopiazonic acid 25 μM (CPA); median and quartile; * p < 0.05 for differences with CPA and without CPA.
1 (0 – 1)
1 (1 – 1)
3 (1 – 6)
8 (4 – 12)
20 (15 – 31)
21 (14 – 35)
171 (136 – 137)
MHS CPA [mN]
3 (2 – 8)*
14 (11 – 31)*
27 (17 – 47)*
32 (25 – 38)*
31 (27 – 37)
34 (29 – 38)
131 (108 – 191)
1 (1 – 2)
1 (1 – 1)
0 (0 – 0)
1 (0 – 1)
2 (1 – 3)
4 (3 – 7)
127 (108 – 190)
MHEh CPA [mN]
0 (0 – 1)
3 (0 – 5)
7 (3 – 19)*
12 (11 – 27)*
31 (29 – 37)*
46 (31 – 47)*
199 (156 – 227)
1 (0 – 1)
1 (0 – 1)
1 (1 – 1)
1 (0 – 1)
1 (0 – 1)
1 (0 – 2)
158 (108 – 176)
MHN CPA [mN]
2 (1 – 2)
2 (1 – 2)
1 (1 – 5)
1 (1 – 6)
4 (1 – 21)*
17 (4 – 27)*
167 (153 – 180)
Halothane-induced contractures with and without cyclopiazonic acid 25 μM (CPA) pre-incubation; median and quartile; * p < 0.05 for differences between IVCT and CPA-IVCT.
5 (2 – 6)
14 (13 – 20)
20 (16 – 26)
19 (11 – 24)
MHS CPA [mN]
40 (26 – 58)*
52 (29 – 76)*
59 (33 – 73)*
48 (30 – 57)*
0 (0 – 0)
(0 – 1)
4 (2 – 4)
3 (2 – 4)
MHEh CPA [mN]
7 (1 – 11)*
25 (13 – 39)*
45 (24 – 55)*
45 (25 – 48)*
1 (0 – 2)
1 (0 – 1)
1 (1 – 1)
1 (0 – 1)
MHN CPA [mN]
1 (0 – 3)
2 (0 – 25)
16 (4 – 34)*
In MH uncontrolled SR Ca2+ release, caused by MH associated mutations mainly in the ryanodine receptor gene, is widely accepted as the underlying pathophysiological mechanism of hypermetabolism . However, the detection of a mutation in the alpha 1-subunit of the voltage sensitive dihydropyridine receptor in a French MH family suggests a more complex pathogenesis of MH . According to the unique mechanism of intracellular Ca2+ cycling that induces contraction and relaxation in vertebrate skeletal muscle, sarcoplasmic Ca2+ release and sarcoplasmic Ca2+ reuptake determine the mainstays of Ca2+ regulation. Undoubtly, an altered SR Ca2+ release plays a crucial role in the development of MH. However, it is completely unclear why many MHS individuals may suffer from MH only after several uneventful exposures to trigger agents during anaesthesia. Several modulating factors have been postulated to modulate cytosolic Ca2+ concentrations, e.g. magnesium , sympathetic activity , temperature , volatile anesthetics  or channel's redox state . While SR Ca2+ release was extensively studied in MH , the impact of an altered SR Ca2+ reuptake on the pathogenesis of MH by intrinsic or extrinsic factors is poorly understood. Theoretically, a reduced activity of the skeletal muscular SERCA type 1 may result in an elevated cytosolic Ca2+ level due to a persistent slow Ca2+ efflux out of the SR that is otherwise balanced by reuptake . A critical threshold of cytosolic Ca2+ may then be exceeded and may lead to contracture development in vitro and to the MH syndrome in susceptible patients. Interestingly, CPA alone did not induce skeletal muscle contractures at 25 μM . We assume that in our study SERCA was inhibited almost completely, since CPA 10 μM reduced the SERCA activity approximately by 70% in frog skinned fibres  and nearly by 100% in rat skinned fibres .
In the presented study, CPA preincubation lead to a high variability of halothane- respectively caffeine-induced contractures especially in the MHS and MHEh group, despite SERCA distribution does not differ between MHS and MHN muscle . Interestingly, the response of MHEh muscle bundles to caffeine was enhanced by CPA preincubation. However, at this stage, our results do not suggest CPA as an alternative approach to improve differentiation of MHE from MHN respectively MHS individuals.
Ca2+ uptake capacity and SERCA activity was found to be significantly increased in MHS pigs  and in HEK-293 cells transfected by MH mutants  but was described to be lower in MHS muscle compared to normal human skeletal muscle . Since a leaky ryanodine receptor in MHS individuals may lead to increased cytosolic calcium, it looks feasible that SR-Ca-ATPase may be upregulated by a compensatory mechanism.
Another option is that CPA itself modulates directly the effect of the trigger agent. This is less likely since halothane and caffeine do have different binding sites at the sarcoplasmic membrane .
The role of a reduced SERCA activity in the pathogenesis of Brody's disease, a skeletal muscular myopathy, is well known and characterized by painless muscle cramping and exercise-induced muscle stiffness linked to a mutation in the gene encoding SERCA [24, 25]. The left-shift of the dose-response curve for halothane- and caffeine-induced contractures following inhibition of the sarcoplasmic Ca2+ reuptake by CPA points out the essential part of SERCAs in the regulation of cytoplasmic Ca2+. We believe this may be an explanation why some MH susceptible patients develop a MH crisis while others never or only after several trigger exposures suffer from MH despite a proven in vitro susceptibility. In this context, an altered activity of SERCA due to intrinsic or extrinsic factors may play a crucial role in the evolution of MH.
The present study demonstrates that CPA preincubation enhances halothane- and caffeine-induced muscle contractures in the IVCT of MHS, MHEh more than in MHN patients.
Modulation of SERCA may play a significant role in the development of malignant hyperthermia. Patients with a high activity may compensate an increased Ca2+ release or leakage from the SR while patients with a low activity of the SERCA do not. Further investigations with focus on extrinsic and intrinsic factors that modulate SERCA activity may be helpful to understand why MH patients may have had several anaesthesias including trigger agents without a significant reaction while developing a fulminate MH crisis at another occasion.
- Ca2+ :
In-Vitro Contracture Test
Malignant hyperthermia equivocal; susceptible only for halothane
Malignant hyperthermia non-susceptible
Malignant hyperthermia susceptible
Sarcoplasmic calcium adenosine triphosphatase
The study was performed at the Department of Anesthesiology at the University of Wuerzburg, Germany.
- Berchtold MW, Brinkmeier H, Müntener M: Calcium ion in skeletal muscle: Its crucial role for muscle function, plasticity, and disease. Physiol Rev. 2000, 80: 1215-1265.PubMedGoogle Scholar
- Gronert GA, Antognini JF, Pessah IN: Malignant Hyperthermia. Anesthesia. Edited by: Miller RD. 2000, Philadelphia: Churchill Livingstone, 1033-1052. 5Google Scholar
- Gommans IMP, Vlak MHM, De Haan A, Van Engelen BGM: Calcium regulation and muscle disease. J Muscle Res Cell Motil. 2002, 23: 59-63. 10.1023/A:1019984714528.View ArticlePubMedGoogle Scholar
- Seidler NW, Joan I, Vegh M, Martonosi A: Cyclopiazonic acid is a specific inhibitor of the Ca2+ ATPase of the sarcoplasmic reticulum. J Biol Chem. 1989, 264: 17816-17823.PubMedGoogle Scholar
- Duke AM, Steele DS: Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres. Eur J Physiol. 1998, 436: 104-111. 10.1007/s004240050610.View ArticleGoogle Scholar
- Enzmann NR, Balog EM, Gallant EM: Malignant Hyperthermia: Effects of sarcoplasmic reticulum Ca2+ pump inhibition. Muscle Nerve. 1998, 21: 361-366. 10.1002/(SICI)1097-4598(199803)21:3<361::AID-MUS10>3.0.CO;2-2.View ArticlePubMedGoogle Scholar
- The European Malignant Hyperpyrexia Group: A protocol for the investigation of malignant hyperpyrexia (MH) susceptibility. Br J Anaesth. 1984, 56: 1267-1269.View ArticleGoogle Scholar
- Urwyler A, Deufel T, McCarthy T, West S, for the European Malignant Hyperthermia Group: Guidelines for the molecular genetic detection of susceptibility to malignant hyperthermia. Br J Anaesth. 2001, 86: 283-287. 10.1093/bja/86.2.283.View ArticlePubMedGoogle Scholar
- Monnier N., Procaccio V., Stieglitz P., Lunardi J.: Malignant-hyperthermia susceptibility is associated with a mutation of the alpha 1-subunit of the human dihydropyridine-sensitive L-type voltage-dependent calcium-channel receptor in skeletal muscle. Am J Hum Genet. 1997, 60: 1316-1325.View ArticlePubMedPubMed CentralGoogle Scholar
- Meissner G, Henderson JS: Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin. J Biol Chem. 1987, 262: 3065-PubMedGoogle Scholar
- Gronert GA, Milde JH, Theye RA: Role of sympathetic activity in porcine malignant hyperthermia. Anesthesiology. 1997, 47: 411-415.View ArticleGoogle Scholar
- Nelson TE: Porcine malignant hyperthermia: critical temperatures for in vivo and in vitro responses. Anesthesiology. 1990, 73: 411-5.Google Scholar
- Kunst G, Graf BM, Schreiner R, Martin E, Fink RH: Differential effects of sevoflurane, isoflurane and halothane on Ca2+ release from the sarcoplasmic reticulum of skeletal muscle. Anesthesiology. 1999, 91: 179-86. 10.1097/00000542-199907000-00026.View ArticlePubMedGoogle Scholar
- Xia R, Stangler T, Abramson JJ: Skeletal muscle ryanodine receptor is a redox sensor with a well defined redox potential that is sensitive to channel modulators. J Biol Chem. 2000, 275: 36556-61. 10.1074/jbc.M007613200.View ArticlePubMedGoogle Scholar
- MacLennan DH, Phillips MS: Malignant hyperthermia. Science. 1992, 256: 789-194.View ArticlePubMedGoogle Scholar
- Du GG, Ashley CC, Lea TJ: Effects of thapsigargin and cyclopiazonic acid on the saroplasmatic reticulum Ca2+ pump of skinned fibres from skeletal muscle. Pflugers Arch. 1994, 429: 169-175. 10.1007/BF00374309.View ArticlePubMedGoogle Scholar
- Anetseder M, Sixt S, Hartung E: Cyclopiazonic acid increases halothane induced contractures. Minerva Anestesiologica. 1994, 60: 59-64.Google Scholar
- Kurebayashi N, Ogawa Y: Discrimination of Ca2+ ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian skeletal muscle fibres: distinct effects of cyclopiazonic acid on the two ATPase activities. J Muscle Res Cell Motil. 1991, 12: 355-365. 10.1007/BF01738590.View ArticlePubMedGoogle Scholar
- Everts ME, Ørding H, Hansen O, Nielsen PA: Ca(2+)-ATPase and Na(+)-K(+)-ATPase content in skeletal muscle from malignant hyperthermia patients. Muscle Nerve. 1992, 15: 162-167. 10.1002/mus.880150206.View ArticlePubMedGoogle Scholar
- O'Brien PJ: Porcine malignant hyperthermia susceptibility: increased calcium-sequestering activity of skeletal muscle sarcoplasmic reticulum. Can J Vet Res. 1986, 50: 329-37.PubMedPubMed CentralGoogle Scholar
- Tong J, McCarthy TV, MacLennan DH: Measurement of resting cytosolic Ca2+ concentrations and Ca2+ store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca2+ release channels. J Biol Chem. 1999, 274: 693-702. 10.1074/jbc.274.2.693.View ArticlePubMedGoogle Scholar
- Conrescu M, Lopez JR, Medina P, Alamo L: Deficient function of the sarcoplasmic reticulum in patients susceptible to malignant hyperthermia. Muscle Nerve. 1987, 10: 238-241. 10.1002/mus.880100307.View ArticleGoogle Scholar
- Zucchi R, Ronca-Testoni S: The sarcoplasmic reticulum Ca2+ channel/ryanodine receptor: Modulation by endogenous effectors, drugs and disease states. Pharmacol Rev. 1997, 49: 1-51.PubMedGoogle Scholar
- Brody IA: Muscle contracture induced by exercise. A syndrome attributable to decreased relaxing factor. N Engl J Med. 1969, 281: 187-192.View ArticlePubMedGoogle Scholar
- Odermatt A, Taschner PE, Khanna VK, Busch HF, Karpati G, Jablecki CK, Breuning MH, MacLennan DH: Mutations in the gene-encoding SERCA1, the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+ ATPase are associated with Brody disease. Nat Genet. 1996, 14: 191-194. 10.1038/ng1096-191.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2253/5/8/prepub
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