Comparison of Systemic Effects of 3,4-Methylenedioxymethamphetamine, of Ryanodex® Therapy and Uncoupling Protein 3 Expression in Malignant Hyperthermia Susceptible and Normal Swine
S. Becker1, M.U. Gerbershagen1, S. Burmester1, J.K. Schütte1, A. Starosse1, C. Hötzel1, U. Schäfer2, F. Wappler1
1Department of Anesthesiology and Intensive Care Medicine
2Institute for Research in Operative Medicine
University Witten/Herdecke,
Introduction
3,4-Methylendioxymethamphetamine (MDMA) is a synthetic amphetamine derivate commonly abused by young people in the rave movement beginning in the late 1980´s. In the last years increasing incidents of fatalities with MDMA abuse were reported. The side effects of MDMA are tachycardia, fasciculations, muscle pain and cramps, masseter muscle spasm, rhabdomyolysis and hyperthermia.
The MDMA induced hyperthermia seems to be the result of a mismatch in heat production and dissipation of heat. Recent studies showed a complex interaction between MDMA and the hypothalamic-pituitary-thyroid axis, sympathetic nervous system (SNS) and the activity of uncoupling proteins (UCP) (Mills et al., 2003; Spargue et al., 2003; Spargue et al., 2007).
In brown fatty tissue, found in animals and infants, uncoupling protein subtype 1 (UCP1) was first identified. This protein is able to discharge energy in form of heat. After application of low temperature there is an immediate hypothalamic activation of the SNS with an elevated release of norepinephrine. Norepinephrine binds at a1- and ß3-receptors, localized in adipocytes of brown fatty tissue, and stimulates the cAMP-depending lipolysis with a release of free fatty acids. Free fatty acids induce an activation of UCP1 which leads to a leakage in the inner mitochondrial membrane resulting in heat. Beside UCP1 further types of uncoupling proteins were characterized. UCP2 is expressed in macrophages, brain, splenic and pancreatic tissues. UCP3, first described in 1997, is primarily expressed in the skeletal muscle, but also found in heart and brown fatty tissue (Boss et al., 1997; Vidal-Puig et al., 1997).
Malignant hyperthermia (MH) is an uncommon autosomal dominant inherited pharmacogenetic syndrome of skeletal muscle cells. It is triggered by halogenated volatile anesthetics and depolarizing muscle relaxants leading to a hypermetabolic state. The clinical symptoms vary from mild to lethal forms including tachycardia, hypercapnia, hypoxemia, muscle rigidity and metabolic acidosis. The causive pathophysiology is an increased myoplasmic calcium concentration based on intense intracellular calcium release from the sarcoplasmatic reticulum (SR) through the ryanodine receptor calcium channel (RyR1).
Although MDMA intoxication and MH seem to be different, there is an increased activity of the sympathetic nervous system reported, evidenced by elevated concentration of norepinephrine in blood in both entities (Rothman et al., 2001). In a previous study MDMA has been shown to induce MH in genetically susceptible (MHS) swine (Fiege et al., 2003). In addition, interactions between MDMA and the ryanodine receptor have been disclosed in isolated sarcoplasmatic reticulum of the rat (Klingler et al., 2005).
Fiege et al. were able to demonstrate in MHS swine that dantrolene treatment of MDMA-induced MH-crisis partially counteracted the clinical signs of MH immediately.
While prompt dantrolene injection is the cornerstone of MH therapy, significant time is lost due to the difficult challenge of preparing and administering the commercially available dantrolene preparation (Dantrium®) for injection. Ryanodex® (
The aims of this study were (1) to re-evaluate the effect of MDMA in MHS- and MHN-swine in a modified study design in comparison to Fiege et al. (2003), (2) analyze the effectiveness of MH-treatment with Ryanodex® and (3) explore the role of UCP3 in the MDMA induced MH in swine.
Methods
With approval by the local ethics committee 6 MHN- and 6 MHS-Pietrain swine were examined. After premedication anesthesia was induced with fentanyl and propofol intravenously. Following endotracheal intubation via tracheotomy, controlled ventilation was conducted with an O2/N2O mixture (FiO2 = 0.45). PaCO2 was set between 38-42 mmHg. Anesthesia was maintained by continuous intravenous administration of fentanyl, propofol and flunitrazepame. ECG, pulseoxymetric and capnometric monitoring were conducted. Normothermia (esophageal temperature 38.0 – 38.7 °C) was achieved by elevated room temperature. After achieving steady state conditions (T0), intravenous MDMA was administered in doses of 2 mg/kg (T0), 2 mg/kg (T1), 4 mg/kg (T2) and 4 mg/kg (T3) MDMA with a time interval of 48 min between each administration. Once any two of the following MH-criteria, pH = 7.20, pCO2 = 75 mmHg and increase of body core temperature = 2.0°C were met, standardized MH therapy was initiated and FiO2 was set to 1.0, respiratory minute volume was doubled, 2 mmol/kg sodiumbicarbonate and Ryanodex® (5 mg/kg) were administered intravenously (T4). Administration of Ryanodex® (5 mg/kg) was repeated after 30 min. MH-therapy data were monitored for further 72 min (T5-T8).
Biopsies were obtained at four measuring points from the musculus adductor longus dexter: steady-state (T0) after 96 min (MDMA 4 mg/kg) (T2) after 192 min (MDMA 12 mg/kg) (T3) upon determination of clinically defined MH-crisis (T4) and 72 min after the 2nd Ryanodex® dose (T8). The expression of UCP3-mRNA in the muscle sample was determined by semi-quantitative rt-PCR. For quantification of UCP3 expression in flush-freezed skeletal muscle tissue RNA was isolated. The integrity of RNA was proofed. The isolated RNA was transcripted to DNA. After transcription quantification of UCP expression was verified with RT-PCR.
Data are shown as mean and standard deviation. Changes in test series were analysed with the t-test. The level of significance was set at p < 0.05.
Results
The courses of pH, PaCO2 and temperature showed a significant increase in comparison to the steady-state conditions in both analysed groups, so that in all tested animals the defined MH-criteria were achieved (figures 1-3). However, no intergroup differences were monitored. After onset of standardized MH-therapy plus administration of Ryanodex® a normalization of pH and PaCO2 levels were recorded. After therapy the body temperature remained stable in MHS- and MHN-swine (figures 1-3).
Further, a significant increase of UCP3-expression 96 min after 4 mg/kg MDMA was measured in MHS- as well as MHN-swine. At T4 in comparison to T0 the UCP3-expression was increased in both groups and additional in the MHN- was significantly higher than in the MHS-group. After therapy UCP3-expression remained stable in both groups (figure 4).
Figure 1: Course of arterial pH in MHS and MHN swine. Data are mean ± SD.
# MHN significant intragroup difference in comparison to T0; § MHS significant intragroup difference in comparison to T0 (p<0.05).
# MHN significant intragroup difference in comparison to T0; § MHS significant intragroup difference in comparison to T0 (p<0.05).
Figure 3: Course of temperature in MHS and MHN swine. Data are mean ± SD.
# MHN significant intragroup difference in comparison to T0; § MHS significant intragroup difference in comparison to T0 (p<0.05).
Figure 4: Course of UCP 3 expression in MHS and MHN swine. Data are mean ± SD.
* Intergroup differences, # MHN significant intragroup difference in comparison to T0; § MHS significant intragroup difference in comparison to T0 (p<0.05).
Conclusions
(1) We showed that administration of MDMA induces an intensified hypermetabolism in both groups, but we could not reconfirm a significant difference in the MHS- compared to the MHN-group in this modified study design. Fiege et al. examined two different races, MHS-pietrain and MHN-german landrace swine. It is tempting to speculate that MHN-german landrace swine in comparison to MHN-pietrain swine present a weaker metabolic response to MDMA intoxication. Furthermore, in the present study the MHN-swine were exposed longer to each MDMA challenge, than in the study design of Fiege et al. A slightly weaker reaction to MDMA might have been compensated in this manner.
A direct effect of MDMA on the RYR1 in the sarcoplasmatic reticulum in rats in vitro was ruled out (Klingler et al., 2005). So we conclude that, in swine also, the mutation of the RYR1 seems to have no influence on the MDMA induced hypermetabolism.
(2) Standard MH therapy in combination with Ryanodex® seems to be partly effective in both groups with a normalisation of PaCO2 and pH. Since the treatment did not include physical cooling of the animals, a decline of body temperature was not expected during the monitored
period of 72 minutes.
(3) The MDMA-dependent role of UCP3 in thermoregulation in rats was previously shown. We demonstrated that, in swine as well, UCP3 expression is upregulated by exposure to MDMA and hence, UCP3 seems to be a mediator in the MDMA induced hyperthermia in swine. However, a causal relationship between expression of UCP3 and MH-disposition was not found.
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