In human skeletal muscles, the phosphodiesterase-III inhibitor enoximone induces in vitro contracture development, and it has been suggested that enoximone could trigger malignant hyperthermia (MH). In this study, the in vitro and in vivo effects of enoximone in MH-normal (MHN) and MH-susceptible (MHS) swine were investigated.
Malignant hyperthermia trigger-free general anesthesia was performed in MHS and MHN swine. Skeletal muscle specimens were excised for an in vitro contracture test with 0.6 mm enoximone. Thereafter, MHS and MHN swine were exposed to cumulative administration of 0.5, 1, 2, 4, 8, 16, and 32 mg/kg enoximone intravenously. Clinical occurrence of MH was defined by a Pco(2) greater than 70 mmHg, a pH less than 7.20, and an increase in body temperature of more than 2.0 degrees C.
Enoximone induced marked contractures in all MHS muscle specimens in vitro. In contrast, only small or no contracture development was observed in MHN muscle specimens, without an overlap in contractures between MHS and MHN muscles. However, in vivo, no clinical differences were found between MHS and MHN swine following cumulative enoximone doses. According to the defined criteria, none of the swine developed MH during the experiment. Furthermore, high enoximone doses induced progressive circulatory insufficiency, and after receiving 32 mg/kg enoximone, all animals died of cardiovascular failure.
The cumulative enoximone doses used in this study were 30- to 50-fold higher than therapeutic doses in humans. Enoximone does not trigger MH in genetically determined swine. However, enoximone might be useful for in vitro diagnosis of MH.
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 calcium metabolism within the skeletal muscle fiber and is usually triggered by volatile anesthetics and depolarizing muscle relaxants. 1,2Calcium homeostasis in skeletal muscles is regulated by a variety of intracellular second messenger systems. Alterations in second messenger systems (e.g. , serotonin system 3or inositolpolyphosphates 4) have been found to be associated with MH. Also, the cyclic AMP system seems to be affected in MH. In skeletal muscle cells from MH-susceptible (MHS) patients and animals, higher cyclic AMP concentrations were measured compared with MH-normal (MHN) individuals. 5–7
Phosphodiesterase-III inhibitors are substances with receptor-independent, positive inotropic effects on the cardiac muscle. Phosphodiesterase-III inhibitors act by decreasing the rate of cyclic AMP degradation. Cyclic AMP activates protein kinase A, which results in altered transport rates of different intracellular calcium channels. In cardiac muscle cells, phosphodiesterase-III inhibition increases the calcium release from the sarcoplasmic reticulum via the ryanodine receptor. 8,9
Although the mode of action of phosphodiesterase-III inhibitors in skeletal muscles is unknown, the phosphodiesterase-III inhibitor enoximone was shown to induce contracture development in human skeletal muscles in vitro . In previous studies, enoximone induced contracture development at lower concentrations in MHS muscles compared with MHN muscles. 10–12In these studies, even differentiation between MHS and MHN with an enoximone in vitro contracture test (IVCT) was possible.
Recently it was reported that a patient developed two MH episodes following cardiopulmonary bypass surgery during trigger-free general anesthesia. 13The MH episodes occurred concomitantly after the administration of enoximone, and treatment with dantrolene was successful both times. Thereafter, the patient was diagnosed as MHS by standard IVCT with halothane and caffeine. Furthermore, skeletal muscles from this patient developed marked contractures when an enoximone IVCT was performed. Therefore, enoximone was presumed to be the most probable trigger of MH in this case.
In light of the results of the in vitro contracture studies and the case report, it has been suggested that enoximone may be a trigger of MH. Hence, the purpose of the current study was to investigate the in vitro and in vivo effects of enoximone in MHS and MHN swine.
Materials and Methods
Following approval by the animal care committee (University Hospital Hamburg-Eppendorf, Hamburg, Germany), 12 MHS Pietrain and 12 MHN Hampshire swine from a special breeding program at the Federal Breeding Center in Mölln, Germany, were investigated (male and female swine weighing 22.0–34.5 kg, aged 3–4 months). The MH genotype of the swine was determined by DNA analyses of ear tissue to check for the presence of the C1843T point mutation on chromosome 6 indicating MH susceptibility. 14Furthermore, MH phenotype was verified by an IVCT with halothane and caffeine according to the protocol of the European MH Group. 15
Anesthesia in all swine was started by administration of 4 mg/kg azaperone (Stresnil®; Janssen, Neuss, Germany) intramuscularly and 10 mg/kg metomidate (Hypnodil®; Janssen, Neuss, Germany) intraperitoneally. After insertion of an intravenous line into an ear vein, anesthesia was deepened by administration of 0.3 mg/kg etomidate (Hypnomidate®; Janssen, Boulogne, France) intravenously, and the trachea was intubated without any further administration of a muscle relaxant. The swine were mechanically ventilated, and anesthesia was maintained by continuous administration of 2.5 mg · kg−1· h−1etomidate and 50 μg · kg−1· h−1fentanyl (Fentanyl®; Janssen, Neuss, Germany) intravenously and 70% nitrous oxide in 30% oxygen. Monitoring included electrocardiography, pulse oxymetry, and rectal temperature measurement. Radiant heat application and warming blankets were used to maintain a stable body temperature. An arterial catheter was inserted into the femoral artery for blood pressure monitoring and to obtain blood samples. A multilumen central venous catheter was surgically placed in the right internal jugular vein for blood sampling, application of the test drug, administration of anesthetics, and infusion of fluid (5–10 ml · kg−1· h−1Ringer's solution). Muscle specimens were excised from the hind limb for the IVCT investigations.
In Vitro Experiments
All in vitro investigations were performed within a time period of 5 h after the muscle biopsy. Muscle bundles were excised carefully and were dissected into single strips (length, 15–25 mm; width, 2 to 3 mm; weight, 120–250 mg). The method and the test setup of the IVCT were in accordance with the European MH Group protocol. 15One viable muscle specimen with a twitch response to supramaximal stimulation greater than or equal to 10 millinewtons (mN) from each of the 12 MHS and 12 MHN swine was used for the IVCT with bolus administration of 0.6 mm enoximone as described previously. 12After a stable baseline tension of at least 10 min was reached, enoximone (Perfan®; Hoechst, Frankfurt, Germany) was added as a bolus directly to the tissue bath in order to obtain a bath concentration of 0.6 mm. The in vitro effects of enoximone in this concentration on contracture development and twitch response in the muscle specimens were continuously observed for at least 30 min.
In Vivo Experiments
The in vivo experiments were performed on six MHS and six MHN swine of the 24 animals investigated in this study. Prior to administration of the test substance, a steady state of all measured variables had to be achieved for at least 30 min. Enoximone (Perfan®) then was given intravenously in a bolus dose of 0.5 mg/kg. Subsequently, enoximone was given in increasing doses of 1, 2, 4, 8, 16, and 32 mg/kg every 20 min. The clinical occurrence of MH was defined by the presence of two of the following three conditions: Pco2greater than 70 mmHg, pH less than 7.20, and increase in body temperature greater than 2.0°C. In the event of a diagnosis of a MH crisis, further administration of enoximone was stopped. During the experiments, hemodynamic variables (heart rate, mean arterial pressure, central venous pressure), end-tidal carbon dioxide concentration, rectal body temperature (°C), blood gas concentrations (arterial oxygen saturation, Pco2, pH), and lactate concentrations were measured every 5 min. After all experiments were completed, the pigs were killed using magnesium chloride solution (10%).
Statistical evaluation was performed using a computer-based program (StatView 4.57; Abacus Concepts, Berkeley, CA). All in vitro data are presented as median and range, and the in vivo data are presented as mean ± SD. The effects of enoximone on contracture development in vitro were analyzed using the Mann–Whitney test. The data obtained during the in vivo experiments were compared using two-way analysis of variance for repeated measures followed by Mann–Whitney and Kruskall–Wallis tests. Results were considered significant if P values were less than 0.05.
In Vitro Experiments
Bolus administration of 0.6 mm enoximone in vitro induced marked contracture development in all MHS muscle specimens but no or only slight contractures in MHN muscles. Enoximone, 0.6 mm, induced a maximum contracture of 20.7 mN (range, 7.6–49.5 mN) in the MHS muscle specimens. Significantly smaller contractures were observed in the MHN group: only 7 of 12 MHN muscle specimens developed contractures of 0.2 mN (range, 0.0–1.0 mN). There was no overlap in maximum contracture between the MHS and MHN groups in the enoximone IVCT, enabling a clear differentiation between MHS and MHN. The individual data for maximum contractures are summarized in figure 1.
The muscle twitch recorded in both groups before the administration of enoximone was comparable: 49.6 mN (range, 11.0–154.3 mN) in the MHS muscles and 52.8 mN (range, 28.4–112.2 mN) in the MHN muscles. A significant decrease in muscle twitch was found in the MHS muscle preparations. The MHS muscle twitch decreased 15 min after enoximone administration to 14.0 mN (range, 7.5–94.5 mN) and after 30 min to 4.3 mN (range, 2.6–44.6 mN), whereas the muscle twitch in the MHN muscles remained unchanged in the enoximone IVCT. The muscle twitch data after administration of 0.6 mm enoximone are summarized in figure 2.
In Vivo Experiments
No differences were found between six MHS and six MHN swine following the administration of cumulative enoximone doses. Furthermore, none of the swine developed signs of MH during the experiment, according to the defined criteria. Data on the course of Pco2, pH, and body temperature are summarized in figure 3. The Pco2of 43.0 ± 4.8 mmHg in the MHS swine and 40.8 ± 5.5 mmHg in the MHN swine before administration of enoximone remained unchanged after the lower doses of enoximone were administered. When the highest enoximone dose was given (32 mg/kg), the Pco2increased to 50.2 ± 12.7 mmHg in the MHS group and to 50.6 ± 1.2 mmHg in the MHN group. However, none of the animals had a Pco2greater than 70 mmHg. The starting pHs of the central venous blood samples from the two groups were comparable: 7.46 ± 0.04 in the MHS animals and 7.49 ± 0.04 in the MHN animals. A decrease in pH was detected in both groups after administration of the higher enoximone doses. After 32 mg/kg enoximone, the pH decreased to 7.26 ± 0.07 in the MHS group and to 7.33 ± 0.03 in the MHN group, but a pH less than or equal to 7.20 was not detected in any swine. Resting body temperatures of 36.9 ± 0.9°C in the MHS swine and 36.5 ± 0.5°C in the MHN swine remained unchanged during the cumulative administration of enoximone and reached 37.8 ± 1.1°C in the MHS group and 37.2 ± 1.0°C in the MHN group after 32 mg/kg enoximone. An increase in body temperature of more than 2.0°C was not observed in any of the swine. Therefore, the defined criteria for diagnosis of MH in this study were not fulfilled.
Before the administration of enoximone, the serum lactate concentrations in the two groups were comparable: 2.87 ± 0.98 mm in the MHS group and 2.40 ± 0.88 mm in the MHN group. In the MHS swine, an increase in lactate concentration to 4.70 ± 0.88 mm was observed at 8 mg/kg enoximone, and an increase to 8.9 ± 0.71 mm was observed at 32 mg/kg enoximone. In the MHN swine, the lactate concentration increased to 3.25 ± 0.65 mm at 8 mg/kg enoximone and to 5.45 ± 0.35 mm at 32 mg/kg enoximone.
Hemodynamic data of the MHS and MHN swine remained unchanged during the administration of the lower enoximone doses. However, high doses of enoximone induced progressive circulatory insufficiency. The heart rate in the MHS animals increased from 63.7 ± 15.5 beats/min at the start of the experiment to 101 ± 12.3 beats/min after administration of 4 mg/kg enoximone and to 157.5 ± 36.9 beats/min after 16 mg/kg enoximone. The increase was comparable in the MHN animals: the initial heart rate of 66 ± 6.8 beats/min increased to 112.5 ± 21.5 beats/min after administration of 4 mg/kg enoximone and to 160 ± 39.5 beats/min after 16 mg/kg enoximone. The mean arterial pressure remained stable after the administration of low doses of enoximone but decreased after high doses enoximone. In the MHS animals, the resting mean arterial pressure of 67.0 ± 7.3 mmHg decreased to 49.5 ± 5.8 mmHg after administration of 16 mg/kg enoximone. The initial mean arterial pressure in the MHN animals decreased from 62.5 ± 5.2 mmHg to 42.7 ± 13.1 mmHg after administration of 16 mg/kg enoximone. After the administration of 32 mg/kg enoximone, all animals developed progressive tachycardia with the decrease in mean arterial pressure and died of ventricular fibrillation.
Malignant hyperthermia is a myopathy that features an acute loss of control of intracellular calcium in human skeletal muscle following the administration of certain trigger substances. The site of the defect in MH is supposedly found in the complex of the dihydropyridine receptor (DHPR) and the ryanodine receptor (RYR1), located at the triad —the junction of the transverse tubules and the terminal cistern sacs of the sarcoplasmic reticulum. 1,2To date, more then 25 mutations in the genes encoding DHPR and RYR1 associated with MH have been reported. 16,17However, identification of mutations in the genes of DHPR or RYR1 failed in about 40% of European MH families. 16Therefore, it is possible that additional mechanisms of cellular calcium regulation are disturbed in the pathophysiology of MH.
The cytoplasmic calcium concentration is regulated by a variety of intracellular second messenger systems that, for the most part, directly or indirectly act at the RYR1. Alterations in second messenger systems (e.g. , serotonin system 3or inositolpolyphosphates 4,18) have been found to be associated with MH. However, the cyclic AMP system also seems to be altered in MH. A study of 33 MHS patients and 29 MHN individuals demonstrated a higher cytoplasmic cyclic AMP content and adenyl cyclase activity in the MHS patients. 5This observation was confirmed, and a lower cytoplasmic ATP content was observed in the MHS patients as well. 6In addition, during and after physical exercise, the cyclic AMP concentrations in blood serum increased more and were prolonged in the MHS patients compared with the MHN individuals. 19Furthermore, a higher cyclic AMP content in skeletal muscles was also found in MHS swine compared with MHN swine. 7However, whether the changes in the cyclic AMP system were causal mechanisms or secondary changes in the pathophysiology of MH could not be clarified by these studies.
Inhibition of phosphodiesterase-III is a common therapeutic principle in cardiovascular failure. 20,21In cardiac muscles, phosphodiesterase-III inhibition leads to an increased sarcoplasmic calcium release via the cardiac ryanodine receptor (RYR2) by elevated cytoplasmic cyclic AMP content and consecutive activation of protein kinase A. Furthermore, phosphodiesterase-III–inhibiting compounds can sensitize the myofibrils to calcium and enhance the cardiac calcium release channel. In this way, phosphodiesterase-III inhibitors are receptor-independent, positive inotropic agents. Alteration in calcium metabolism resulting from phosphodiesterase-III inhibition is not restricted to cardiac muscles. For example, phosphodiesterase-III inhibitors are also potent vasodilators mediated by increased cytoplasmic calcium content.
In human skeletal muscle specimens, the selective phosphodiesterase-III inhibitor enoximone induced a dose-dependent increase in muscle twitch and contracture development. 10–12However, the in vitro effects of enoximone was more intense in the skeletal muscles of MHS patients compared with MHN individuals. 10–12MHS 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 with MHN muscle preparations. The assumed cytoplasmic calcium–enhancing effect of enoximone also was confirmed in porcine MHS skeletal muscles by the IVCT with enoximone performed in this investigation. A clear discrimination between MHS and MHN porcine muscle preparations was possible and enabled diagnosis of MH by the IVCT with bolus administration of 0.6 mm enoximone. In light of the results of these in vitro studies, it was tempting to speculate that enoximone may be a trigger of MH under in vivo conditions.
This assumption was supported by a case report. 13A patient developed clinical MH signs twice in association with the administration of the phosphodiesterase-III inhibitor enoximone. Interactions with other known triggers of MH, such as volatile anesthetics, succinylcholine, or 4-chloro-m-cresol, could be ruled out in this case. The patient was diagnosed as MHS by the halothane and caffeine IVCT; furthermore, surplus muscle specimens developed distinctive contractures in an IVCT with enoximone. The diagnosis was supported by the detection of a G7300A mutation, which is known to be associated with MH. 22
The swine in this study initially received a dose of enoximone comparable to that administered therapeutically in humans. The enoximone doses then were increased in short intervals; the cumulative enoximone doses in this study were at least 30- to 50-fold higher than therapeutic doses used in humans. 23None of the swine developed clinical signs of a MH crisis according to the defined criteria. An increase in the serum lactate concentration was observed in all swine after the administration of high enoximone doses and could be attributed to increasing cardiovascular insufficiency. However, the serum lactate concentration increased more distinctly in the MHS swine compared with the MHN swine. It could be speculated whether enoximone induced a specific skeletal hypermetabolism in the MHS swine, but all of the swine developed rapid cardiovascular failure, resulting in death. This is a plausible explanation for the failure of enoximone to induce MH in MHS swine.
In this study, enoximone did not trigger MH in swine that were homozygous carriers of the C1843T mutation. In contrast, MH in humans is a genetically heterogeneous disease, most often with a heterozygous carrier status. Given the genetic differences between MH in swine versus humans, the specific effects of enoximone in MHS muscles in vitro , and the case report, 13the potential of phosphodiesterase-III inhibitors to trigger MH in humans could not be ruled out in this study. Therefore, clinical use of phosphodiesterase-III inhibitors should be avoided in MHS patients. Furthermore, if patients develop clinical signs of MH during therapy with phosphodiesterase-III inhibitors, a specific MH therapy should be considered, and a diagnosis of MH susceptibility should be made. In vitro diagnosis of MH may be improved by a contracture test with enoximone.
The authors thank Michael Agurski (Radiometer GmbH, Willich, Germany) for the provision of an ABL 625 blood gas analyzer for the study period. The authors also thank Jens Dimigen, D.V.M. (Chair, Department of Veterinary Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany), and his team for their excellent help and experimental animal care.