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Equine Malignant Hyperthermia
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This study confirms the association of a single missense mutation (C7360G) in a functional domain of the skeletal muscle ryanodine receptor 1 (RyR1) with equine malignant hyperthermia (EMH). Sequence-specific primers allow the rapid detection of susceptible individuals for the prevention of fatalities.
1. Introduction
Malignant hyperthermia (MH) is a life-threatening, pharmaco-genetic disease of skeletal muscle elicited by exposure to volatile anesthetics such as halothane, depolarizing muscle relaxants such as succinylcholine, and stress (in humans and pigs) [1]. The genetic basis of the disease has been confirmed in humans [2], pigs [3], and dogs [4]. Mutations in the ryanodine receptor 1 (RyR1) gene cause the dysfunction of the RyR1, also known as the calcium release channel of the sarcoplasmic reticulum in the skeletal muscle. This receptor is a key player in skeletal muscle excitation-contraction coupling. The dysfunction of this receptor results in the excessive release of calcium into the myoplasm, triggering a series of events that will result in a hypermetabolic state and/or death in many cases. Since the 1960s, when the use of inhalation anesthesia began in horses, cases of MH have been suspected but seldom reported [5-16]. In view of the low number of case reports, the incidence of MH in the horse seems to be low. However, incidence underestimation is possible because of the lack of triggering events and/or recognition. In addition, exposure of MH susceptible individuals to triggering agents does not always lead to a MH episode. Exertional rhabdomyolysis is a common cause of myopathy in the horse in which only a few etiologies have been recognized, such as recurrent exertional rhabdomyolysis (RER) [17] and polysaccharide storage myopathy (PSSM) [18]. In humans, chronic rhabdomyolysis has been described as a non-anesthetic manifestation of MH [19]. This raises the possibility that some rhabdomyolyses in horses may also be a non-anesthetic form of MH. Answers to several questions concerning MH in horses have been long overdue. The main objectives of this study were to investigate if mutations in the candidate gene, RyR1, are associated with equine MH (EMH) and to review the literature.
2. Materials and Methods
Literature Review
A PubMed search was performed with the key words malignant hyperthermia, in vitro contracture test (IVCT), and/or inhalation anesthesia-associated deaths in horses. From the initial search, 18 references were identified, but only 12 documented horses manifesting MH-like episodes [5-16].
Animals
Control Horses
A total of 86 healthy horses were selected as control horses (WTRyR1). Middle gluteal muscle samples were collected for DNA and RNA extraction from 24 horses. In addition, DNA from 62 horses was donated [a]. The study was performed following the guidelines of an animal care and use protocol required by the University of California at Davis.
Malignant Hyperthermia Horses
Muscle biopsies from the Neuromuscular Diseases Laboratory at the University of California at Davis from 2 Quarter Horses (MHRyR1) with a clinical diagnosis of MH were donated for the study. These horses developed fatal MH when anesthetized with halothane. The horses did not receive pre-medication (sedatives, injectable anesthetics), and were masked for induction and maintenance of anesthesia with halothane. Their clinical and laboratory parameters were hypercapnea, acidosis, profuse sweating, electrolytes abnormalities, hyperthermia, and muscle rigidity (Fig. 1) [16]. The muscle histochemical stains were examined by one of the authors (MA). Various different muscles were available for genetic analysis.
Figure 1. Quarter Horse gelding that developed a fatal MH episode while under halothane anesthesia. (A) One of the first signs observed was contracted pinnae (pointing caudally). (B) Note the profuse sweating, contracted pinnae, severe retraction of the eye ball, protrusion of the third eyelid, trismus, and flared nostrils. The horse developed hyperacute rigor mortis [16].
Muscle Biopsy Technique
The horses were sedated with detomidine HCl [b] (0.01 mg/kg IV) and locally infiltrated with lidocaine HCl [c] to obtain a muscle biopsy from the middle gluteal muscle as described by Lindholm and Piehl [20].
Genetic Analysis
DNA and RNA extractions from middle gluteal muscle were performed following the DNeasy blood/tissue kit [d] and RNeasy [e] handbook instructions, respectively. The Invitrogen SuperScript II RT [f] protocol was followed for cDNA preparation. Sixteen pairs of primers (forward and reverse) were designed to obtain the sequences of the regions where mutations have been reported for other species. The primers were generated from consensus sequences for the human, pig, and rabbit. The glyceraldehyde-3-phosphate dehydrogenase (GAPD) gene was used as the control sequence. A master mix was prepared by using Platinum Taq DNA polymerase [g]. Three different polymerase chain reaction (PCR) reactions were performed for each exon of each horse under study. The PCR products were run in a 2% agarose gel and visualized under ultraviolet light. Visualized bands with the expected size were extracted for sequencing. Exons with identified polymorphisms were cloned and digested with restriction enzymes. To validate the test, DNA from 62 horses was used for PCR and digestion assays. In addition, the expression of RyR1 gene was compared between WTRyR1 and MHRyR1 horses by Western blot.
3. Results
Literature Review
Seventeen horses with MH-like episodes were identified. The affected breeds included Quarter Horse (n = 5), Thoroughbred (n = 4), pony (n = 4), Appaloosa (n = 1), Arab (n = 1), and unknown (n = 2) [5-16]. Females and males seemed to be equally affected. The age range was from 12 days old to 14 yr old. The most common clinical signs under inhalation anesthesia were hyperthermia (39°C to >42°C), profuse sweating, tachycardia, tachypnea, and muscle rigidity. Complete blood parameters were not available in every case, but the most common abnormalities were hypercapnea, acidosis, hypertension, electrolyte disorders, increased creatine kinase, and myoglobinuria. The disease in these horses was triggered by halothane (n = 8), halothane in combination with succinylcholine (n = 6), and halothane and nerve stimulation (n = 3). Treatment of MH consisted of rapid discontinuation of anesthesia, continuation of oxygen, cold therapy that included IV fluids and local ice packs, non-steroidal anti-inflammatory drugs (NSAIDs), and muscle relaxants such as dantrolene [7-10,12]. The outcome was reported for 13 horses in which the mortality associated with MH was 36%.
MH Muscle Biopsy
Abnormal findings in muscle biopsies from MH horses were fiber size variation, central nuclei, mild necrosis, areas of glycogen depletion, and hypercontracted fibers [21]. Sarcoplasmic masses and ringbinden fibers were observed in 1 horse. The abnormalities were found in all fiber types, except for the sarcoplasmic masses that were only present in type 2B myofibers [21].
MH Genetic Analysis
The RyR1 gene was partially sequenced and cloned in the MHRyR1 group and compared with WTRyR1 horses. Three polymorphic sites were detected in the N-terminal region (exons 15, 17, and 46) in both groups of horses [21]. Amino acid derivation of the polymorphic nucleotides did not generate a different amino acid. An additional polymorphic site that generated an amino acid change was identified in exon 46 in the MHRyR1 group [21]. Heterozygosity was identified as a double peak at nucleotide 7360 (SGC, S = C or G) in the MHRyR1 horses, whereas, in the WTRyR1 horses, the sequence was CGC. The nucleotide substitution would give rise to a residue substitution (glycine for arginine) [21]. A screening test with restriction enzymes was also developed for the faster detection of heterozygous and homozygous individuals. Sequence specific primers (SSP) were designed for a faster and specific detection of affected heterozygous and homozygous individuals [21]. There was no difference in RyR1 gene expression between WTRyR1 and MHRyR1 horses [21].
4. Discussion
The diagnosis of EMH has been based on clinical manifestations triggered by volatile anesthetics such as halothane or an in vitro contracture test that is not always available. Horses and ponies with suspected MH show hyperthermia, hyperhidrosis, tachycardia, dysrhythmias, tachypnea, hypercapnea, acidosis, muscle rigidity, rhabdomyolysis, myoglobinuria, and death with acute rigor mortis. The first EMH case was documented in 1975 [5]. The reported breeds are Quarter Horse, Thoroughbred, Appaloosa, Arab, and ponies [5-16]. Most MH-susceptible people and swine show no specific muscle histochemical abnormalities. Central core disease (CCD) is a congenital myopathy closely associated with MH in 50% of humans [22]. Histochemical analysis in CCD patients revealed fibers with a core devoid of mitochondria and oxidative enzyme activity. A mild non-inflammatory myopathy was observed in our MHRyR1 horses with no evidence of central cores. Reports in MH-suspect horses had mild myopathic changes with the presence of ringbinden fibers as a predominant feature [11,13]. In our study, ringbinden fibers were only observed in 1 horse [21]. Ringbinden fibers have been described in humans with all types of muscular dystrophy, various myopathies, and in muscle undergoing regeneration. Their significance in horses has not been studied.
MH in these Quarter Horses is associated with a single missense mutation resulting in an amino acid change (R2454G) [21] in a codon reported to be mutated in two of the five exon 46 mutations in human MH [23,24]. The mutation found is located in a conserved region for the WTRyR1 in horses and other species. Genotypically, the MHRyR1 horses were heterozygous [21] but presented an MH phenotype when challenged with halothane, suggesting a dominant susceptibility. Anesthetic mortality in horses has always been considered higher than other species, and it is attributed to their size, body mass, and unique cardiopulmonary physiology. However, retrospective studies in horses revealed that mortality related to anesthesia was about 1.5% [25]. Screening tests and preventive measures in susceptible individuals and early recognition of the disease may decrease fatalities in the future. The present study is the first to identify a mutation within the RyR1 gene in the horse. However, it is unknown if this mutation is responsible for all cases of EMH. A practical benefit of this discovery is the creation of a faster [h] and direct screening test, a PCR with allele specific oligonucleotides [21], to identify susceptible horses to gain insight into the pathophysiology, prevention, and treatment of MH in the horse [h].
This project was supported by the Center for Equine Health (Edwin J. Gregson Memorial Fellowship), the Hart Scholarship, and the Neuromuscular Diseases Laboratory.
Footnotes
- DNA was courtesy of Dr. Johanna Watson, University of California at Davis, Davis, CA 95616.
- Dormosedan, Pfizer Animal Health, Exton, PA 10017.
- Lidocaine HCl, Abbott Laboratories, North Chicago, IL 60064.
- DNeasy, QIAGEN Inc., Valencia, CA 91355.
- RNeasy, QIAGEN Inc., Valencia, CA 91355.
- SuperScript II RT, Invitrogen, Carlsbad, CA 92008.
- Platinum Taq, Invitrogen, Carlsbad, CA 92008.
- The test is available at the Neuromuscular Diseases Laboratory, Attention: Monica Aleman, Veterinary Medical Teaching Hospital, 1 Garrod Avenue, University of California at Davis, Davis, CA 95616.
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- Get unlimited access to books, proceedings and journals.
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1. Akira S. Mammalian Toll-like receptors. Curr Opin Immunol 2003; 15:5-11.
2. Akira S and Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004; 4:499-511.
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