MALIGNANT hyperthermia (MH) is a genetic disease characterized by muscle hypermetabolism, and its pathophysiologic basis is considered to be defects in the calcium regulation metabolism within skeletal muscle. 1 In humans, this disorder has a heterogenetic origin. In the swine model, a single point mutation in the RYR1 gene is responsible for this phenotype. 2
Succinylcholine, a depolarizing muscle relaxant, is a potent trigger of MH. 1,3 Administration of succinylcholine to MH-susceptible (MHS) swine causes a severe arterial hypotension before the animals actually display hypermetabolism. 3 The hypotension has been observed to occur immediately (within 2 min of succinylcholine administration), even when clinically used doses of pancuronium are given as prior treatment. Thus, the cardiovascular depression was not considered to be caused by the effects of succinylcholine on the skeletal muscle itself (e.g., due to hyperemia and consecutive vasodilation after muscle fasciculations); rather, it was considered to be due to direct effects on the cardiovascular system. In other words, the MHS phenotypic response to succinylcholine is not easily explained by the genotypic defect of the skeletal muscle calcium release channel. Several studies have concluded that tissues other than the skeletal muscle may be altered in MH, 4-10 with described effects on the heart being controversial. 4,6,11,12 Therefore, it was of interest to reinvestigate the effects of succinylcholine on the cardiovascular system in vivo and in vitro. The purpose of the present study was to gain new insights into the hypotensive response mechanism caused by succinylcholine in MHS swine. To do so, both in vivo and in vitro experiments were performed. The latter were undertaken to determine whether observed effects of succinylcholine were due to either direct effects on the heart or arteries, or due to their responses to either a secondarily released substance or reflex mechanism.
Methods
In Vivo Effects of Succinylcholine
After obtaining approval from the Institutional Animal Care and Use Committee of the University of Minnesota, the following experiments were conducted on pure-bred Pietrain swine with the homozygeous mutation of arginine 615 in the ryanodine receptor (RYR1). These MHS animals, as well as nonsusceptible normal mongrel swine, were obtained from the University of Minnesota Rosemount Animal Facility (weight of MHS swine: 42.1 ± 5.1 kg; normals: 47.5 ± 7.6 kg, mean ± SD).
Malignant hyperthermia-susceptible swine (n = 10) and normal swine (n = 5) were initially anesthetized with 20 mg/kg intramuscular ketamine (KetaVed; Phoenix Scientific, Inc., St. Joseph, MO). An 18-gauge ear vein catheter (Jelco; Johnson & Johnson, Arlington, TX) was placed, and thiopental 20-25 mg/kg (Gensia Pharmaceuticals, Inc., Irvine, CA) was administered intravenously. The animals were intubated with a 6.5- or 7-mm ID endotracheal tube and mechanically ventilated with 65% N2O and 35% O2 to maintain an arterial carbon dioxide partial pressure (PaCO2) of 40 ± 2 mmHg. End-tidal carbon dioxide was monitored using a gas analyzer (Nellcor, Hayward, CA). No muscle relaxants were administered at this time. A thiopental infusion was then titrated to effect at a rate of approximately 100-200 mg/min. A femoral artery cannula (A. femoralis superficialis) was used for monitoring invasive blood pressure, and an axillary artery cannula was used for blood sampling. Arterial oxygen partial pressure (PaO2), PaCO2, p H, base excess, K+, and Ca2+ were determined from fresh arterial samples using a blood gas analyzer with temperature compensation (Model 1304; Instrumentation Laboratories, Inc., Lexington, MA). Mikro-Tip catheter transducers (5 French, Model MPC-500; Millar Instruments, Inc., Houston, TX) were placed in the right and left ventricles via access through the right external jugular vein and the right carotid artery, respectively. In addition, a balloon-tipped pulmonary artery thermodilution catheter (Swan-Ganz catheter model 93A-131-7F; American Edwards Laboratories, Santa Ana, CA) was inserted via the right external jugular vein and advanced into the pulmonary artery to measure pulmonary artery pressures, blood temperatures, and thermodilution cardiac outputs (Cardiac output monitor model 9520A; American Edwards Laboratory). A five-electrode lead configuration was used to monitor electrocardiograms (Spacelabs model 1020; SpaceLabs Inc., Chatsworth, CA). The common femoral artery and veins were surgically exposed, and femoral artery and vein blood flow was monitored using ultrasonic transit-time flow meters (Transonic Systems Inc., Ithaca, NY). In one MHS animal (MHS 3), flow artery data were not collected because of a technical problem. Core temperatures were measured using axillary, rectal, and pulmonary artery probes.
A specially designed pressure bulb was positioned between the upper and lower incisors to assess the relative force output of the muscles of mastication, especially the masseter muscle. The signal from this unit was converted to voltage using a pressure transducer connected to the SpaceLabs monitor model 1020.
An additional two normal and three MHS animals were used for the isolated heart studies only and not for the in vivo protocol.
The following data were automatically collected using data acquisition hardware and LabVIEW software (National Instruments, Austin, TX): heart rates, arterial blood pressures, femoral artery and vein blood flows, and end-tidal carbon dioxide levels. Blood gas data, core temperatures, and cardiac outputs were recorded manually.
Epochs of the following data were collected at a sampling frequency of approximately 670 Hz by an AT-CODAS analog-to-digital data acquisition system (Dataq Instruments Inc., Akron, OH): pulmonary artery pressures, central venous pressures, right ventricular pressures, and left ventricular pressures. The first derivative of left and right ventricular pressures (dP/dt) was determined by off-line differentiation of the pressure signal. Maximum positive (+dP/dt) measurements were taken from the differentiated left and right ventricular pressure waves. Furthermore, systemic vascular resistance was calculated using the following simplified formula: systemic vascular resistance = mean arterial pressure/cardiac output.
For these investigations, all animals were maintained normothermic (38.05 ± 0.2°C) using convective-air warming as needed (Bair Hugger; Augustine Medical Inc., Eden Prairie, MN). The animals received NaCl 0.9% for intravenous fluid administration via the ear vein catheter (approximately 600-800 ml/h during the control periods).
After the control periods, (PaCO2 40 ± 2 mmHg, temperature 38 ± 0.5°C) and just before succinylcholine was given, all data were collected using the methods previously described (indicated as t = 0 min). Succinylcholine (Quelicin; Abbott Laboratories, North Chicago, IL), as a bolus dose of 2 mg/kg, was administered intravenously by injection into the central port of the pulmonary artery catheter; all data were sampled for a subsequent 15 min (manually recorded data were obtained at t = 1.5, 3, 5, 10, and 15 min). Five MHS animals and five normal swine did not receive any pretreatment (table 1). Two MHS animals received an intravenous pretreatment of 2.5 mg/kg dantrolene (Norwich-Eaton Pharmaceuticals, Norwich, NY) in 250 ml mannitol 1% 30 min before the bolus succinylcholine injection. Similarly, three MHS animals received a 1-mg/kg intravenous pretreatment with vecuronium (Norcuron; Organon Pharmaceuticals, West Orange, NJ) 10 min before succinylcholine was administered (table 1).
In pilot studies, two MH animals were pretreated with either: (1) 1 mg/kg ranitidine intravenously (Zantac; Glaxo Pharmaceuticals, Research Triangle Park, NC) and 1 mg/kg diphenhydramine intravenously (Elkins-Sinn, Inc., Cherry Hill, NJ); or (2) atropine 20 μg/kg intravenously (Elkins-Sinn, Inc.). The ranitidine/diphenhydramine and atropine were administered a minimum of 30 min before the bolus dose of succinylcholine was injected.
Treatment of severe, succinylcholine-induced arterial hypotension included intravenous fluids (NaCl 0.9%) as needed, initiated not before 5 min after the succinylcholine administration. If hypotension persisted beyond 5 min, administration of sympathicomimetic agents, such as epinephrine, was also provided (table 1).
Susceptibility to MH was verified by the initiation of MH in vivo (PaCO2 > 70 mmHg at constant ventilation) and/or by in vitro contracture testing. For the latter, bundles of rectus abdominis muscle were used for testing with the protocol recommended by the North American Malignant Hyperthermia Registry, on equipment previously described (table 1). 13,14
Effects on an Ex Vivo Isolated Heart Model
After median sternotomy was performed, a cardioplegia cannula (9 French double lumen, Medtronic, Minneapolis, MN) was introduced into the aorta, and refrigerated modified St. Thomas' cardioplegia (NaCl 110 mm, KCl 16 mm, CaCl2 1.2 mm, MgCl2 16 mm, NaHCO3 10 mm) was prepared for antegrade flow through the coronary vessels. A total of 10,000 USP units of heparin and 25 mg of adenosine were administered intravenously. Immediately after cardioplegia introduction, the excised hearts were placed in an iced saline slurry while transported to the apparatus, as well as during the (re)cannulation process. After removal of excess tissue and isolation of the great vessels, an aortic cannula (24 French) was directly inserted distal to the cardioplegia cannula. In addition, cannulas were inserted into the pulmonary artery (28 French), pulmonary vein (28 French), and directly into the right atrium (36 French). The cannulas were secured into place, with remaining apertures into the heart either tied or sutured closed. 15 The Millar pressure catheters were placed in vitro as they were used in the in vivo preparations described previously. The flow of cardioplegia was then stopped, and warm (38°C) perfusate (NaCl 118.0 mm, mannitol 16.0 mm, glucose 11.5 mm, NaHCO3 20.0 mm, EDTA 0.32 mm, KCl 4.5 mm, MgCl2 1.2 mm, NaH2PO4 1.2 mm, Na pyruvate 2.27 mm, CaCl2 2.5 mm, insulin 10 U/l) was supplied to the cardiac chambers. If there was no spontaneous atrial-ventricular rhythm, the heart was defibrillated using an electrocardiac monitor unit (9790C Vitatron, Medtronic). The heart was allowed to function in a Langendorff perfusion mode for recuperation from ischemia until atrial-ventricular rhythm stabilized and the heart became self sustaining. Both sides of the heart were then allowed to work by supplying fluid pressure heads into the preload and afterload chambers. 15 The preload was held constant throughout the experiment (mean right atrial pressure 11 mmHg, range 5-12 mmHg; mean left atrial pressure 14 mmHg, range 12-15 mmHg); the afterload was defined as the diastolic aortic root pressure (mean, 24 mmHg; range, 14-30 mmHg).
Isolated hearts from six MHS animals and five normals were investigated. In MHS hearts that had been exposed to succinylcholine in vivo, the time between in vivo and in vitro administration was 90, 150, and 200 minutes, respectively. From an additional three MHS and two normal animals, the hearts were removed without having been exposed to succinylcholine in vivo. For these MHS animals, which were therefore not used for in vivo effects of succinylcholine, MH susceptibility was verified by in vitro contracture testing as previously described.
After a control phase, i.e., stabilized function with a normal sinus rhythm, succinylcholine 2 mg/kg was added to the Krebs solution, and the effects on left and right ventricular pressures and contractility (dP/dt) were analyzed at 1.5, 3, 5, and 10 min.
In Vitro Effects on Femoral Artery Rings
The common femoral arteries (opposite side from where the femoral cannula was placed) were removed from normal (n = 6) and MHS (n = 4) swine before succinylcholine exposure. Subsequently, no ischemia in the limbs was observed. The femoral arteries were prepared under a stereo-microscope as vessel rings without removing the endothelium. Care was taken to remove as much surrounding tissue as possible without damaging the intimal surface. The presence of endothelium was later verified by histologic assessment of selected preparations. The vessel rings (15 MH, 32 normals) were approximately 0.5-1 mm in diameter and weighed between 10 and 20 mg. The experiments were performed in organ chambers (42 ml) containing Krebs solution (NaCl 118.3 mm, KCl 4.7 mm, MgSO4 1.2 mm, KH2PO4 1.2 mm, CaCl2 2.5 mm, NaHCO3 25 mm, glucose 11.1 mm, Ca2+ EDTA 0.026 mm) that was gassed with 95% O2/5% CO2. Temperature of the bathing solution was kept constant at 37°C. The vessel rings were suspended between a fixed clip and a force transducer by two stainless-steel wires inserted into the lumen. Isometric tensions were recorded continuously. After this procedure, the preparations were allowed to equilibrate at their optimal length for approximately 30 min before drug administration. After a stable baseline reading was obtained, the following agents were studied: (1) phenylephrine added as incremental doses (0.5 μm per dose, up to 2 μm); (2) succinylcholine at a final bath concentration of 0.06 mm; and (3) acetylcholine at a 0.1-μm concentration (Sigma Chemical Co., St. Louis, MO). Vessel rings that did not elicit a response to phenylephrine or acetylcholine were excluded.
Statistical Analysis
Statistical analyses were performed using either repeated measures analysis of variance with a Bonferroni multiple comparison posttest or the two-tailed Mann-Whitney test, as appropriate. A P value < 0.05 was considered significant. The data were presented on an animal-by-animal basis or as mean ± SD unless otherwise indicated.
Results
In Vivo Study
The MH phenotype was verified by in vivo triggering (PaCO2 > 70 mmHg at constant minute ventilation) and/or by in vitro muscle contracture testing as previously described (table 1).
Hemodynamic Data
Succinylcholine (2 mg/kg intravenously) caused a significant, severe decrease in arterial blood pressure in the MHS animals, including those pretreated with dantrolene (fig. 1A). In two MHS animals, epinephrine was necessary for blood pressure support 5 min after succinylcholine administration; for statistical comparisons between groups, hemodynamic data were provided only at 0, 1.5, and 3 min. In animals pretreated with 1 mg/kg vecuronium (n = 3) and in normal swine, severe arterial hypotension was not observed (fig. 1B). However, one animal in this group developed a fatal hypotension 10 min after succinylcholine administration, probably caused by an air embolism, and died at 20 min; no acute effects of succinylcholine on the cardiovascular system were detected.
Another MHS animal without pretreatment died shortly after succinylcholine administration during the hypotensive period (t = 5 min). Neither of these animals displayed signs of a MH episode (PaCO2 > 70 mmHg at constant ventilation); their MH susceptibility was confirmed by in vitro contracture testing.
In swine with the MH mutation, the mean arterial pressure just before succinylcholine administration was 96 ± 11 mmHg and subsequently decreased to 35 ± 7 mmHg at 1.5 min (P < 0.001; intragroup comparison), and to 41 ± 25 at 3 min (P < 0.01). In normal swine, the mean arterial pressure before succinylcholine administration was 89 ± 20 mmHg, and was 84 ± 15 mmHg and 86 ± 15 mmHg at 1.5 and 3 min posttimes, respectively. Importantly, the mean arterial pressures were statistically different between the genotypes at both 1.5 min (P < 0.001) and 3.0 min (P < 0.01; intergroup comparison). Interestingly, pretreatment with vecuronium abolished the succinylcholine-induced hypotension completely (fig. 1B).
In three of the seven MHS animals, heart rates increased; however, these effects were relatively inconsistent and thus not significant (fig. 1C). No changes in heart rates were observed in MHS animals pretreated with vecuronium or in normals (fig. 1D).
Cardiac output decreased for only the MH animals (fig. 1E) after succinylcholine administration (from 4.1 ± 1 l/min to 1.7 ± 1.3 l/min at 2 min;P < 0.01 vs. normals and P < 0.05 vs. MHS pretreated with vecuronium;fig. 1F).
The succinylcholine-induced arterial hypotension in the MH genotype was associated with decreased left ventricular contractility (+dP/dt) at 1.5 min as well as 3 min after succinylcholine administration (P < 0.01 vs. normals and MHS with vecuronium;fig. 2A). Pretreatment with vecuronium abolished such decreases in left ventricular contractility in the MHS animals. Contractility was stable throughout in normals (fig. 2B). Associated with the abnormal changes in contractility in the MHS animals were concomitant and significant changes in left ventricular pressures (both at 1.5 and 3 min postadministration;P < 0.05) compared with preadministration values (fig. 2C); no contractility changes were observed in MHS swine pretreated with vecuronium or in normals (fig. 2D).
There was a significant decrease in systemic vascular resistance in MHS swine only at 5 min after succinylcholine injection, but not at 1.5 or 3 min (when excluding one animal pretreated with dantrolene;P < 0.05, intragroup comparison;table 2).
No genotype-specific changes were observed in either right ventricular pressures or contractilities (table 2). The mean pulmonary artery pressures before succinylcholine administration did not change significantly and were 22 ± 4 mmHg in normals and 26 ± 3 mmHg in MHS animals; at 1.5 min, they were 25 ± 5 mmHg in normals and 29 ± 4 mmHg in MHS animals; and at 3 min, they were 24 ± 5 mmHg in normals and 24 ± 9 mmHg in MHS animals. In particular, pulmonary diastolic pressures (table 2) and central venous pressures (data not shown) remained stable for both genotypes after succinylcholine was administered.
In pilot studies, pretreatment with either atropine or diphenhydramine/ranitidine did not prevent immediate cardiovascular depression or MH in susceptible animals (data not shown).
In Vivo Study: Metabolic Data
No significant changes of K+ and Ca2+ were detected during the hypotensive period in MHS swine (table 2). The mean time to trigger significant episodes of MH in susceptible swine (i.e., PaCO2 > 70 mmHg, fixed ventilation) was 13.5 min (table 1). Significant decreases in p H were observed at 5 and 10 min after succinylcholine administration (P < 0.05). Statistically significant changes in mean PaCO2 did not occur within the first 10 min postsuccinylcholine, but a clear trend of increased PaCO2 was observed (table 3). Changes in individual end-tidal carbon dioxide values are presented in figures 3A and B. Core temperatures were significantly elevated postsuccinylcholine in these MH animals, but not until several minutes later. For example, the esophageal temperatures before succinylcholine were 37.9 ± 0.3°C in susceptible swine (n = 4), and at 10 min after succinylcholine administration, they increased to 38.3 ± 0.3°C (P < 0.05, intragroup comparison;table 3).
Table 3 |
Fig. 3 |
The initial administration of succinylcholine did not induce episodes of MH in animals pretreated with either dantrolene (n = 2) or vecuronium (n = 3). However, MH was triggered subsequently in two animals pretreated with vecuronium and in one MHS animal pretreated with dantrolene. The readministration of succinylcholine in one vecuronium animal 90 min after the first dose induced a cardiovascular reaction very similar to that without pretreatment; the mean arterial pressure decreased from 95 to 45 mmHg (table 1).
The maximum change in masseter force after succinylcholine administration (within 90 s) was 92 mmHg in MHS swine (n = 5) and 2 mmHg in normal animals (n = 4;P < 0.05). No change in masseter force was observed in MHS animals with vecuronium pretreatment (n = 3;table 1).
Femoral artery blood flow of MHS animals decreased at 3-4 min after succinylcholine administration and increased significantly at 8-11 min (P < 0.05, intragroup comparison, fig. 4). In the susceptible swine, a similar decrease of venous blood flows at 3 and 4 min and significant increases were observed at 9, 10, 11, and 12 min (P < 0.05, analysis of variance). Arterial and venous flows were stable throughout in normals and in the vecuronium-pretreated MH animals.
Ex Vivo Isolated Heart Studies
Administration of succinylcholine at an equivalent dose to 2 mg/kg to the Krebs perfusate had no effect on either right and left ventricular contractility or pressures, or on heart rates in hearts isolated from either genotype. The left ventricular contractility and left ventricular pressures of isolated MH and normal swine hearts are shown in figure 5.
Isolated Femoral Artery Rings
The results from these investigations are summarized in figure 6. The baseline force was 0.88 ± 0.32 g in rings isolated from normal swine and 0.91 ± 0.3 g from the MHS swine. After