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Keywords:

  • Anesthesia;
  • Calcium;
  • Pig;
  • Skeletal Muscle

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Background: The efficacy of intravenous (IV) administration of azumolene (Az), an analogue 30-fold more soluble than dantrolene, on pigs susceptible to malignant hyperthermia (MH) is incompletely understood.

Objective: To evaluate efficacy of Az on MH crisis in pigs.

Animals: Eight normal (MHN) and 7 susceptible to MH (MHS) pigs (Landrace × Large White × Pietran).

Methods: Prospective, laboratory trial. Hypermetabolic crisis was observed in MHS pigs, but not in MHN pigs, after a combined administration of inhaled halothane (1.5%) and IV injection of succinylcholine (SCh; 2.5 mg/kg). Susceptibility was confirmed using a caffeine and halothane contracture test. Az was administered 15 minutes after administration of SCh.

Results: Respiratory acidosis (pH 7.16 ± 0.02; Pco2, 46.2 ± 9.1 mmHg, HCO3, 22.5 ± 2.3 mmol/L), fever (38.2 ± 1.1°C), cardiac arrhythmias, and muscle contracture were observed in MHS pigs. MHS pigs (n = 5) treated with Az (2 mg/kg IV) survived the crisis with attenuation of signs (pH 7.30 ± 0.10; Pco2, 36.3 ± 4.5 mmHg; HCO3, 22.9 ± 2.3 mmol/L) and recovery of normal muscle tone and cardiac rhythm.

Conclusions and Clinical Importance: Az represents a possible substitute for dantrolene to reverse MH crisis in susceptible pigs.

Abbreviations:
Az

azumolene

BP

blood pressure

CHCT

caffeine halothane contracture test

DS

dantrolene sodium

ECG

electrocardiogram

HR

heart rate

MH

malignant hyperthermia

MHN

pigs not susceptible to malignant hyperthermia

MHS

pigs susceptible to malignant hyperthermia

RyR1

ryanodine receptor type 1

SCh

succinylcholine

SR

sarcoplasmic reticulum

Malignant hyperthermia (MH) is a pharmacogenetic disorder that is induced by volatile anesthetics, depolarizing muscle relaxants, or stress.1 MH occurs in humans and in many species of animals, including pig,2 horse,3 and dog.4 The crisis manifests clinically as a hypermetabolic state defined by hypercapnia, cyanosis, hyperthermia, acidosis, and skeletal muscle contracture. The crisis can aggravate intravascular coagulation, cardiac arrhythmias, and convulsion, resulting in a high-case fatality rate. MH syndrome is associated with mutations in the ryanodine receptor type 1 (RyR1), the sarcoplasmic reticulum (SR) Ca2+ channel of skeletal muscle. Currently >100 mutations have been associated with human MH.5 In contrast, pigs only have 1 point of mutation (R615C) associated with MH.1 In susceptible swine and humans,6,7 the mutations produce an abnormally high resting mioplasmatic Ca2+ concentration due to prolonged opening of the RyR1 receptor which increases Ca2+ induced Ca2+ release activity.8,9 The R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca2+ activation and store overload-induced Ca2+ release, while volatile anesthetics trigger MH by further reducing the Ca2+ threshold.10 Prior studies had suggested that dysfunction of Ca2+ homoeostasis associated with the MH phenotype is the result of interdomain interactions between regions 1 and 2 of the RyR1 from MHS.9

Dantrolene sodium (DS), 1-〈{[5-(4-nitrophenyl)-2-furfuryl]methylene}amino〉-2,4-imidazoleidinedione, is a skeletal muscle relaxant that inhibits excessive leak of Ca2+ by RyR1,11 and has been used to treat MH for almost 40 years. Despite the potency of DS, this agent is highly lipophilic and poorly water soluble, making its preparation difficult for clinical use. For intravenous (IV) administration of DS, it is necessary to adjust the pH of the solution (ie, 9.5), add mannitol, and protect the solution from light.11 An analogue of DS, azumolene sodium (1-{[(5-4-bromophenyl)-2-oxazolyl]-methylene} amino-2,4-imidazoleidinedione, Az),12 has a similar potency to DS in relaxing skeletal muscle in MHS human muscle.13 Az is 30-fold more soluble than DS, and as such, facilitates the preparation of the drug. Az inhibits store-operated Ca2+ entry into skeletal muscle fibers and in cultured cells expressing RyR1.14 Few studies have addressed the efficacy of Az in treating a MH crisis.

This study was performed to investigate the efficacy of Az in treating MHS pigs during crisis. Hemodynamic parameters and reversion of muscle contracture were evaluated after Az treatment.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Animals

The Ethics Committee for Animal Investigation and the Ethics Committee of the Universidade Federal do Rio de Janeiro approved the protocols used in this study.

Landrace × Large White × Pietran pigs (20–25 kg) from both sexes were obtained from the Departamento de Zootecnia of Universidade Federal de Viçosa. Pigs were genotyped for MH susceptibility and divided into 2 groups: normal (MHN, n = 8) and susceptible to MH (MHS, n = 7). The pig genotypic analysis was performed in the DNA, salt-extracted from white blood cells collected immediately after slaughter by a standard laboratory protocol of Laboratory of Animal Biotechnology of the Department of Animal Science, Universidade Federal de Viçosa, Brazil. The sequence of the RYR 1 gene that contains the substitution of cysteine for arginine 614, mutation responsible for triggering MH was amplified by PCR-RFLP.15

The animals were deprived of food and water 24 and 18 hours before treatments, respectively. An intramuscular injection of 0.75 mg/kg midazolama provided sedation of the animals before administration of 7.5 mg/kg thiopental sodium.b Anesthesia was maintained by continuous IV infusion of 150 μg/kg/min propofol.c The lungs of animals were ventilated with an enriched air mixture with oxygen with a Harvard mod. W051707 ventilator. The oxygen flow was regulated to maintain the blood oxygen saturation higher than 99%. Ventilation in all experiments was 15 mL/kg and respiratory rate of 24/min. Electrodes were placed in the thorax to record the electrocardiogram (ECG). Blood pressure (BP) was recorded in a polygraphd with a pressure transducer connected to a catheter placed in the iliac artery. Body temperature was measured through probe placed in the esophagus.e Azf was injected either in the cephalic or abdominal anterior veins.

Muscle Biopsies

The caffeine halothane contracture test (CHCT) was applied to biopsies of vastus lateralis muscle to phenotype MH susceptible pigs, confirming the previous genotype. Biopsies were obtained 30–60 minutes before challenge of the animal with halothane-succinylcholine (SCh). CHCT was performed simultaneously of the MH crisis protocol and at the end of experiment the phenotype was determined. Thus, the susceptibility to MH was recognized 2–3 hours after the beginning of experiment. For the CHCT smaller specimens of vastus lateralis (1.0–1.5 cm length and 3–5 mm width) from MHN and MHS pigs were dissected and prepared for isometric tension recording. One end of each muscle was attached to a force transducerg and the other to a fixed clamp. Chambers were filled with Ringer-Krebs solution (118.1 mm NaCl, 3.4 mm KCl, 0.8 mm MgSO4·7H2O, 2.5 mm CaCl2, 25 mm NaHCO3, 1.2 mm KH2PO4, 11.1 mm dextrose)16 and oxygenated with a carbogen mixture (95% O2, 5% CO2) at 37 ± 0.1°C, pH 7.40 ± 0.02. Muscular twitches were induced by field electrical stimulation (50 V for 2 ms at 0.2 Hz). The signals generated by the force transducer were conditioned by a Cyberamp,h digitalized,i and analyzed by Axoscope software.j CHCT was maintained during exposure of the muscle fragments to either increasing concentrations of caffeine (0.5–32 mm) or 3% halothane for 10 minutes. Pigs were considered MHS when the caffeine-induced contracture was greater than 0.2 g with 2 mm caffeine, when halothane-induced contracture was greater than 0.7 g.16

Protocol for MH Manifestation

After induction of anesthesia with propofol, MH crisis was induced in the pigs by inhalation of 1.5% halothane.k,l Fifteen minutes later, still under effect of halothane inhalation, 2.5 mg/kg SChm was IV injected followed by a single dose of Az (2.0 mg/kg) injected 15 minutes after SCh. All animals were sacrificed 30–40 minutes after Az administration immediately after blood sample collection. The BP and ECG were continuously recorded and data were collected 15 minutes after injection of SCh and Az.

Blood Samples

Arterial blood samples were collected from the iliac artery for gas analysis before and 15 minutes after challenge with halothane and SCh, as well as 15 and 30 minutes after IV injection of Az. Changes in pH, Pco2, HCO3, and BE were detected.

Statistical Analysis

All data were expressed as means ± SEM. The differences among groups were considered statistically significant when P<.05 as determined by one-way analysis of variance (ANOVA) followed by posthoc Dunnet's test (to compare with control) or Newman-Keuls test (to compare MHN versus MHS). The data were compared with control (before the beginning of protocol) within groups and between groups (MHN versus MHS) for each time points.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Although the animals were previously genotyped, the expression of the mutant gene was confirmed using the CHCT. All MHN and MHS presented a level of muscle contraction in response to caffeine and halothane in the range accepted for normal or abnormal reaction as described by the North America Malignant Hyperthermia Group.17 For the in vivo experiments, MH crisis was induced by inhalation of halothane (1.5%) for 15 minutes, followed by injection of SCh (2.5 mg/kg). For the treated group, Az (2 mg/kg) was administered 15 minutes after SCh. Figure 1 shows typical recordings of BP and ECG from an MHS pig untreated (A) and treated (B) with Az. Cardiovascular collapse occurred in untreated pig (A) immediately after inhalation of halothane, and in this particular experiment the animal died before administration of SCh. In the experiment depicted in Figure 1B, severe cardiac arrhythmia occurred after administration of halothane and SCh that was completed reversed 5 minutes after Az.

image

Figure 1.  Blood pressure (BP) and electrocardiogram (ECG) recording from anesthetized MHS pigs. (A) MHS submitted to 1.5% halothane (Hal) inhalation. Note that the BP collapsed in 10 minutes after halothane inhalation. (B) MHS submitted to 1.5% halothane (Hal) inhalation during 15 minutes followed by IV administration of 2.5 mg/kg succinylcholine (SCh). Azumolene (Az) (2 mg/kg) was in bolus injected 15 minutes after SCh. Note instability of BP and ECG during MH crisis rapidly reversed by treatment with Az.

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Two of 7 MHS pigs challenged with halothane and SCh died before they were treated with Az. One untreated animal died few minutes after exposure to halothane (Fig 1A) and the other after exposure to halothane and SCh. The 5 MHS pigs treated with Az survived and their mean arterial pressure and ECG pattern normalized after the IV injection of Az.

Mean arterial blood pressure (MAP), heart rate (HR), temperature, and blood gas analysis were measured 15 minutes after inhalation of 1.5% halothane, 15 minutes after IV injection of 2.5 mg/kg SCh, and 15 and 30 minutes after IV injection of 2.0 mg/kg Az.

No difference on control MAP was detected between MHN and MHS groups (Table 1). Administration of halothane and SCh did not change the MAP in the MHN group; however, significant decrease (P<.05 versus control within the MHN group) was observed in the MHS animals after anesthesia with halothane (Table 1). Az did not significantly alter the MAP after 15 minutes of administration in either MHN and MHS but increased (P<.05 versus control within the MHS group) in the MHS after 30 minutes.

Table 1.   Mean arterial pressure (MAP), heart rate (HR), and temperature (T) from MHN and MHS groups submitted to MH crisis protocol and treatment with single dose of Az.
  MAP (mmHg)HR (bpm)T (°C)
  • The data represent the mean ± SEM from 8 MHN pigs and 5 MHS pigs.

  • MHN, pigs not susceptible to malignant hyperthermia; MHS, pigs susceptible to malignant hyperthermia.

  • *

    P<.05 MHN versus MHS.

  • #

    P<.05 versus control.

ControlMHN90.7 ± 8.9107 ± 635.6 ± 0.8
MHS87.0 ± 14.8103 ± 1737.5 ± 0.4
15 minutes after HalMHN75.6 ± 15.9106 ± 535.0 ± 0.8
MHS61.3 ± 14.3#107 ± 2637.2 ± 0.7
15 minutes after SChMHN77.6 ± 12.5110 ± 734.3 ± 1.0
MHS72.6 ± 15.5151 ± 32*#38.2 ± 1.1*
15 minutes after AzMHN105.6 ± 15.386 ± 4.934.1 ± 1.2
MHS95.0 ± 12.8136 ± 98*#38.2 ± 0.9*
30 minutes after AzMHN79.8 ± 8.178 ± 1032.9 ± 1.2
MHS112.2 ± 5.6*180 ± 30*#38.0 ± 0.8*

The HR was not different in both MHN and MHS groups in the control, as well as after halothane inhalation (Table 1). However, after injection of SCh, HR increased in MHS pigs (P<.05 versus control within the MHS group and P<.05 MHN versus MHS after injection of SCh). Tachycardia was not observed in MHN animals and treatment with Az did not reduce the tachycardia induced in MHS pigs.

As shown in Table 2, no alteration in pH was observed when MHN pigs were treated with SCh or with Az. The combination of halothane and SCh induced acidosis, which was reversed 30 minutes after treatment with Az (P<.05 MHN versus MHS after injection of SCh). Po2 values measured in the control and after challenge with halothane and SCh were not significantly different between MHN and MHS groups. Pco2 values increased in the MHS group after treatment with halothane and SCh (P<.05 versus control within the MHS group and P<.05 MHN versus MHS after injection of SCh), indicating the presence of respiratory acidosis. HCO3 concentration in MHS pigs decreased (P<.05 versus control within the MHS group) after the halothane-SCh treatment, which was not altered by administration of Az.

Table 2.   Parameters of arterial blood gas (pH, PO2, PCO2, HCO3, and BE) measured from MHN (n = 8) and MHS (n = 5) pigs submitted to MH crisis protocol and followed by treatment with single dose of Az.
  pHPO2 (mmHg)PCO2 (mmHg)HCO3 (mmol/L)BE
  • The values represent the mean ± SEM.

  • MHN, pigs not susceptible to malignant hyperthermia; MHS, pigs susceptible to malignant hyperthermia.

  • *

    P<.05 MHN versus MHS.

  • #

    P<.05 versus control.

ControlMHN7.62 ± 0.05116.5 ± 20.227.3 ± 4.326.3 ± 0.76.8 ± 1.9
MHS7.53 ± 0.05127.1 ± 24.539.9 ± 5.529.5 ± 1.72.0 ± 2.5
15 minutes after HalMHN7.68 ± 0.0698.7 ± 19.426.7 ± 4.425.5 ± 1.26.3 ± 2.1
MHS7.43 ± 0.06113.5 ± 12.932.4 ± 5.426.9 ± 2.4−0.9 ± 3.0
15 minutes after SChMHN7.52 ± 0.12106.7 ± 15.532.2 ± 4.623.1 ± 2.11.2 ± 3.6
MHS7.16 ± 0.02*82.3 ± 9.846.2 ± 9.1*#22.5 ± 2.3#−6.9 ± 5.7
15 minutes after AzMHN7.52 ± 0.10114.4 ± 20.131.8 ± 5.124.1 ± 1.93.0 ± 2.9
MHS7.20 ± 0.14*109.3 ± 14.239.0 ± 4.5*20.5 ± 2.4#−7.8 ± 4.7
30 minutes after AzMHN7.54 ± 0.05139.9 ± 28.227.6 ± 1.623.2 ± 2.52.9 ± 3.4
MHS7.30 ± 0.10*117.7 ± 20.836.3 ± 4.5*22.9 ± 2.3#−6.6 ± 6.1

After halothane and SCh administration, the body temperatures were higher in MHS pigs (P<.05 MHN versus MHS after injection of SCh) and were not affected by Az. The temperature did not increase significantly (P= .87) in MHN pigs. Additionally, all MHS pigs developed muscular rigidity during the MH crisis, which relaxed after Az treatment.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

This study demonstrated the efficacy of Az to reverse an MH crisis in MHS pigs, results consistent with the observation that Az reversed muscular contracture in pigs.18

The signs and clinical signs of MH syndrome included a rapid increase of muscle metabolism, which was manifested by an acidosis, increased HR, decreased BP, cardiac arrhythmias, and muscle contracture. In this study, all 5 of the MHS pigs treated with Az (2.0 mg/kg) survived the induced MH crisis with muscular rigidity, cardiac arrhythmias, and acidosis reversed by 30 minutes after Az administration. Similar results were observed in the early studies of DS to reverse MH crisis.19 However, recovery of tachycardia was not observed after administration of Az. This could be due to the absence of bicarbonate and antiarrhythmic drugs in the experimental protocol.

MH crisis in pigs is stereotypical. Within the first minutes of halothane inhalation, MHS pigs invariably develop increased skeletal muscle tone progressing to extreme rigidity. The core body temperature typically rises in excess of 42°C. Our results show that the body temperature did not increase as high as was expected. This could be explained by the experimental protocol used in which timing of administration of halothane and SCh did not produce such an increase. The protocol used was based on previous publication,19 in which the esophageal temperature of MHS pigs increased from 38.1 ± 1.2 to 38.5 ± 1.1°C 15 minutes after injection of SCh (2 mg/kg). Our data show that temperature increased from 37.5 ± 0.4 to 38.2 ± 1.1°C in MHS group. This incomplete MH crisis could be certainly explained by short time of exposure to halothane and SCh.

We hypothesize that Az reverses the effects of MH crisis by decreasing intracellular (Ca2+) in the skeletal muscle of MHS pigs.20 Alternatively, Az might reduce the opening rate of RyR1,n without altering Ca2+ uptake into the SR.21 Further studies are needed to elucidate the molecular mechanisms of Az; however, our functional evaluation shows that Az can revert an MH crisis in pigs.

In conclusion, the present study demonstrated that Az is effective to reverse an MH crisis in susceptible pigs and represents a possible compound in replacement to dantrolene to control a MH crisis.

Footnotes

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

a Midazolam, Produtos Roche Químicos e Farmacêuticos S.A., RJ, Brazil

b Thiopental sodium, Cristália Produtos Químicos e Farmacêuticos Ltda, SP, Brazil

c Propofol, Cristália Produtos Químicos e Farmacêuticos Ltda

d Grass mod. 7400, Quincy, MA

e Yellow Springs equipment, Yellow Springs, OH

f Azumolene, Cristália Produtos Químicos e Farmacêuticos Ltda

g Grass, FT-03

h Axon Instruments Inc, Foster City, CA

i Digidata 1322

j Axon Instruments Inc

k Halothane, Cristália Produtos Químicos e Farmacêuticos Ltda

l Tanohalo

m Succinylcholine, Ariston Indústria Química e Farmacêutica Ltda

n Mijares AJ, Gerardi A, López JR. EU-4093 does not modify the capacity of calcium uptake by the sarcoplasmatic reticulum. Biophysical Journal 1988;53:133 (abstract)

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

This work was supported by Financiadora de Estudos e Projetos (FINEP/MCT), Cristália Produtos Químicos e Farmacêuticos Ltda, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Universitária José Bonifácio (FUJB), Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and fellowships CNPq (MMT, PLC). Az was donated by Cristália Produtos Químicos e Farmacêuticos Ltda, Itapira, SP, Brazil.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References