Malignant hyperthermia (MH) is a potentially lethal myopathy that is often inherited as an autosomal dominant trait . It is widely accepted that susceptibility to MH is caused by abnormal Ca2+ metabolism within the skeletal muscle fibre [2,3]. Ca2+ homoeostasis in skeletal muscles is regulated by a variety of intracellular second messenger systems. Alterations in some of the second messenger systems, for example, serotonin  or inositolpolyphosphates  have been found to be associated with MH. Also the cyclic adenosine monophosphate (cAMP) system seems to be affected in MH. In skeletal muscle cells from MH susceptible (MHS) patients and animals higher cAMP concentrations were measured compared to MH normal (MHN) [6-8].
Phosphodiesterase-III (PDE-III) inhibitors exert receptor-independent, positive inotropic effects on the cardiac muscle by decreasing the rate of cAMP degradation (Fig. 1). The cAMP activates proteinkinase A, which results in altered transport rates of various intracellular Ca2+ channels. In cardiac muscle cells, PDE-III inhibition increases the Ca2+ release from the sarcoplasmic reticulum via the ryanodine receptor (RYR) [9,10].
The specific PDE-III inhibitor enoximone has been shown to induce contracture development in human skeletal muscles in vitro. In previous studies, enoximone-induced contracture developed at lower enoximone concentrations in MHS compared to MHN muscles [11-14]. In these studies, even differentiation between MHS and MHN with an enoximone-induced in vitro contracture test was possible.
Regarding the results of studies with enoximone, it is tempting to speculate that on the one hand the in vitro effects in skeletal muscle preparations were specific for the substance enoximone or on the other hand could be explained in general by inhibition of PDE-III, respectively. Therefore, the purpose of the current study was to investigate the in vitro effects of the PDE-III inhibitor amrinone in MHS and MHN swine.
Materials and methods
After approval by the animal research committee of the University Hospital Hamburg-Eppendorf, eight MHN swine (male and female German Landrace pigs, weighting 19-21 kg, aged 2-3 months) and eight MHS swine (male and female Pietrain pigs, weighting 14-22 kg, aged 2-3 months) from a special breeding farm (Research Station Thalhausen, Technical University Munich, Munich, Germany) were investigated. Prior to the study in all animals genomic DNA was isolated from blood preserved in ethylenediaminetetraacetic acid to check the presence of the Arg615-Cys point mutation on chromosome 6 indicating MH susceptibility .
Swine were fasted overnight with free access to water. Trigger-free general anaesthesia was induced by administration of ketamine 10 mg kg−1 intramuscularly (Ketavet®; Pharmacia & Upjohn, Erlangen, Germany). After insertion of a venous line into an ear vein, anaesthesia was deepened with propofol 10 mg kg−1 (Disoprivan® 2%; Astra-Zeneca, Wedel, Germany) and fentanyl 10 μg kg−1 (Fentanyl-Janssen®; Janssen-Cilag, Neuss, Germany) intravenously. After tracheotomy and intubation the lungs were mechanically ventilated with an air/oxygen-mixture (FiO2 0.4). Anaesthesia was maintained with propofol 10 mg kg−1 h−1 and fentanyl 50 μg kg−1 h−1. Neuromuscular blocking drugs were not administered. Muscle specimen were excised from a hind limb for the in vitro contracture tests.
All in vitro investigations were performed within a time period of 5 h after the muscle biopsy. Muscle bundles were excised carefully and immediately placed in Krebs-Ringer solution (constituents (mmol): NaCl, 118.1; KCl, 3.4; CaCl2, 2.5; MgSO4, 0.8; KH2PO4, 1.2; NaHCO3, 25.0; glucose, 11.1) equilibrated with carbogen (5% carbon dioxide: 95% oxygen). The muscle was dissected into strips (length: 15-25 mm; width: 2-3 mm; weight: 120-250 mg) and only viable muscle specimens with a twitch response ≥10 mN to supramaximal stimulation were used for in vitro contracture test according to the protocol of the European MH Group . Each muscle sample was secured with silk sutures to a fixed point and connected to a force displacement transducer (Lectromed, Welwyn Garden City, UK). The specimens were suspended in a 20 mL tissue bath perfused with Krebs-Ringer solution continuously bubbled with carbogen. Temperature was held constant at 37°C, and pH was 7.4. The muscles were stimulated electrically with square waves to achieve a supramaximal response using an HSE Stimulator Type 215/I (Hugo Sachs Elektronik, March, Germany) with a stimulus duration of 1 ms and a frequency of 0.2 Hz. Contracture curves were displayed on a Linseis L2200 II recorder (Linseis, Selb, Germany) and analysed 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−1) to 150 ± 10% of the resting length.
A viable muscle specimen from each of the eight MHS and eight MHN swine was used for an in vitro contracture test with cumulative administration of amrinone. After a stable baseline tension of at least 10 min, amrinone (Sigma Chemical, St. Louis, Missouri, USA) was added cumulatively every 5 min in order to obtain organ bath concentrations of 20, 40, 60, 80, 100, 150, 200 and 400 μmol L−1. The in vitro effects of these amrinone concentrations on contracture development and twitch response in the muscle specimens were continuously recorded for at least 40 min.
Statistical evaluation was performed by using a computer-based program (StatView 4.57; Abacus Concepts, Berkeley, CA, USA). The data are presented as mean ± standard deviation (SD). The effects of amrinone on contracture development were analysed with the U-test. Results were considered significant if P-values were less than 0.05.
Skeletal muscle preparations of eight MHS and eight MHN pigs were examined in a cumulative amrinone in vitro contracture test. Amrinone induced concentration-dependent contractures in all muscle preparations, however, they were higher and started at lower concentrations in the MHS compared to the MHN muscles. Typical original in vitro contracture test tracings of a MHS and a MHN muscle following cumulative administration of amrinone are shown in Figure 2.
Contracture development started in three MHS muscle specimens at 20 μmol L−1 amrinone (Fig. 3). With an organ bath concentration of 80 μmol L−1 amrinone all MHS muscles had developed a contracture of 6.4 ± 8.3 mN. The contractures increased in the MHS muscles in a concentration-dependent fashion. At a concentration of 400 μmol L−1 the maximum contracture reached 17.2 ± 17.5 mN.
The MHN muscle preparations also showed concentration-dependent contracture development, but in comparison with the MHS muscles contractures started at higher concentrations of amrinone (Fig. 3). With a bath concentration of 80 μmol L−1 amrinone a contracture of 0.8 mN was detectable in one MHN muscle. At an amrinone bath concentration of 150 μmol L−1 the contractures were found to be 2.6 ± 3.2 mN. Only with 400 μmol L−1 amrinone all MHN muscles developed contractures of 9.3 ± 6.1 mN.
In the beginning of the experiment MHS muscle twitches of 117.2 ± 88.1 mN were not statistically different from the MHN group with twitches of 169.1 ± 115.2 mN due to the high SD (Fig. 4). Initially the cumulative administration of amrinone did not lead to any modification of the muscle contraction force in both groups. With a bath concentration of 400 μmol L−1 the muscle twitch decreased in the MHS muscle preparations to 47.8 ± 34.6 mN. The muscle twitch in the MHN muscle preparations at this concentration was measured as 87.8 ± 42.0 mN.
MH is a genetically determined myopathy, characterized by an acute loss of control of intracellular Ca2+ homeostasis in the human skeletal muscle following administration of certain trigger substances such as volatile anaesthetics and depolarizing muscle relaxants [1,2]. The site of the defect in MH is supposed to be the Ca2+ release mechanism of the sarcoplasmic reticulum in skeletal muscles, namely the complex of the dihydropyridine receptor and muscle RYR1 [3,17]. To date, more than 25 mutations in the genes encoding for the dihydropyridine and RYRs associated with MH have been published [17,18]. However, attempted identification of mutations in these receptors were not possible in about 40% of the European MH families . Therefore, other mechanisms of cellular Ca2+ regulation might be altered in the pathophysiology of MH.
Phosphodiesterases of cyclic nucleotides hold a key position in metabolism of cAMP and guanosine monophosphate. Many cellular functions are regulated by activation of adenyl- and guanylcyclase, leading to an increase of cAMP and cyclic guanosine monophosphate. Phosphodiesterases lower the effects of these intracellular messengers by enhancing the degradation of cyclic nucleotides . Various isoenzymes of phosphodiesterases exist, divided into 5 subgroups (I-V)  and with varying distribution in the tissues.
Inhibition of PDE-III is a common therapeutic principle in cardiovascular failure [21,22]. In cardiac muscles, PDE-III inhibition leads to an increased sarcoplasmic Ca2+ release via the cardiac RYR2 by elevated cytoplasmic cAMP content and consecutive activation of protein kinase A. In addition, PDE-III inhibiting compounds can sensitize the myofibrils to Ca2+. By this means, PDE-III inhibitors are receptor-independent, positive inotropic agents. Alteration in Ca2+ metabolism by PDE-III inhibition is not restricted to cardiac muscles. For example, PDE-III inhibitors are also potent vasodilatators mediated by increased cytoplasmic Ca2+ concentration.
The cytoplasmic Ca2+ concentration is regulated by a variety of intracellular second messenger systems, mostly directly or indirectly acting at the muscle RYR. Alterations in these systems, for example, serotonin  or inositolpolyphosphates [5,23], have been found to be associated with MH. Also, the cAMP system seems to be altered in MH. A study of 33 MHS patients and 29 MHN individuals demonstrated a higher cytoplasmic cAMP content and adenylcyclase activity in the MHS patients . This observation was confirmed and in addition a lower cytoplasmic adenosine triphosphate (ATP) content in MHS patients was found, as well . Additionally, during and after physical exercise the cAMP concentrations in blood serum increased more and were prolonged in MHS compared to MHN patients . Furthermore, a higher cAMP content in skeletal muscle was also found in MHS compared to MHN swine . However, whether the changes in the cAMP system were causal MH-mechanisms or secondary changes cannot be clarified yet.
In human skeletal muscle specimens the selective PDE-III inhibitor enoximone induced a dose-dependent increase of muscle twitch and contracture development [11-14], indicating a cytoplasmic Ca2+ elevation in skeletal muscle cells. However, the in vitro contractures following administration of enoximone were more intense in skeletal muscles of MHS compared to MHN patients and swine. MHS skeletal muscles showed an increase followed by a marked decrease in muscle twitch, indicating muscle fatigue, and contracture development at significantly lower enoximone concentrations compared to MHN muscle preparations.
According to our current knowledge it was unclear, whether the in vitro effects in MHS skeletal muscle preparations were specific for the substance enoximone or could be attributed in general to inhibition of PDE-III. Amrinone-induced contracture development in all skeletal muscle preparations in this study. This could be assumed to be an indirect indicator for release of Ca2+ of the sarcoplasmic reticulum in the skeletal muscle cell. Contracture development started at lower amrinone concentrations in the MHS compared to the MHN muscles. These observations are in accordance with prior results of studies with enoximone [11-14]. In contrast to these enoximone studies discrimination between MHS and MHN was not possible with the amrinone in vitro contracture test. Therefore, amrinone is probably not helpful for in vitro diagnosis of MH.
The results of this study support the assumption, that the clinical use of PDE-III inhibitors in MH patients might be dangerous. On the one hand this assumption is based on differences between MHS and MHN skeletal muscle preparations in enoximone-induced contractures in vitro [11-13]. On the other hand a patient has been reported who developed clinical MH signs twice in association with the administration of the PDE-III inhibitor enoximone . The patient was diagnosed MHS by the halothane and caffeine in vitro contracture test, furthermore, surplus muscle specimens developed distinctive contractures in in vitro contracture test with enoximone. However, in MHS swine enoximone failed to induce a clinical MH syndrome before the animals developed cardiovascular failure, even if skeletal muscle hypermetabolism was detectable . Regarding the genetic differences between MH in swine and human beings, the possibility of PDE-III inhibitors to trigger MH in human beings could not be ruled out. Therefore, clinical use of PDE-III inhibitors should be avoided in MHS patients.
In conclusion amrinone-induced contracture development in porcine skeletal muscles of MHS swine in vitro. Taking the studies with enoximone into account, induction of skeletal muscle contractures seems to be a class effect of PDE-III inhibitors. Whether the skeletal mode of action is comparable within the cardiac muscle (Fig. 1) or is based upon a non-specific release of sarcoplasmic Ca2+ needs to be examined in further studies. However, phosphodiesterases and the cAMP system seem to play a role in the pathophysiology of MH.
Support for this study was provided solely from departmental sources.
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