• Open Access

Malignant Hyperthermia Associated with Ryanodine Receptor 1 (C7360G) Mutation in Quarter Horses

Authors


Corresponding author: Monica Aleman, MVZ, PhD, Diplomate ACVIM, Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Tupper Hall 2108, One Shields Avenue, Davis, CA 95616; e-mail: mraleman@ucdavis.edu.

Abstract

Background: Anesthetic-induced malignant hyperthermia (MH) has been documented in Quarter Horses with a single point mutation in the ryanodine receptor 1 gene (RyR1) at nucleotide C7360G, generating a R2454G amino acid substitution. However, there have been no reports of nonanesthetic manifestations of MH in horses with the C7360G mutation.

Objective: To describe clinical manifestations of Quarter Horses with the C7360G mutation.

Animals: Eleven Quarter Horses with the RyR1 C7360G mutation.

Methods: This prospective study included horses with suspected MH, undetermined etiology of sudden death, death within hours of onset of rhabdomyolysis, muscle rigidity, stiffness, intermittent sweating, and persistent increases in serum muscle enzyme activities. Whole blood in EDTA and skeletal muscle were processed for genetic and histochemical analysis. Medical records and pedigrees were collected when available.

Results: Both anesthetic- and non–anesthetic-associated myopathic manifestations of MH occurred in halter Quarter Horses with mutation of RyR1. The disease is inherited as an autosomal dominant trait. Clinical and laboratory abnormalities were similar in both forms. Rhabdomyolysis was a common finding in both groups of horses. Skeletal muscle histochemical findings were nonspecific and compatible with a noninflammatory myopathic process.

Conclusions and Clinical Importance: MH is a potentially fatal disease of Quarter Horses that could be triggered by halogenated anesthetics and other nonanesthetic factors that may include exercise, stress, breeding, illnesses, and concurrent myopathies.

Malignant hyperthermia (MH) is a life-threatening pharmacogenetic disorder of skeletal muscle elicited by halogenated anesthetics, depolarizing muscle relaxants, and stress.1 Six distinct loci for MH susceptibility (MHS) in humans have been identified.2 Specific single point mutations in the ryanodine receptor 1 (RyR1) and dihydropyridine receptor (CACNA1SA) genes have been identified in MHS individuals.2 The genetic basis of MH linked to RyR1 mutations has been reported in humans,3 pigs,4 dogs,5 and horses.6 Other myopathies such as central core disease and multiminicore disease have also been associated with RyR1 gene mutations in humans.7 Dysfunction of RyR1 (calcium release channel of the sarcoplasmic reticulum of skeletal muscle) results in excessive release of calcium into the myoplasm-triggering cascade events that lead to a hypermetabolic state and ultimately cell death.1 Clinical and laboratory manifestations include tachycardia, hyperthermia, muscle rigidity, rhabdomyolysis, respiratory and metabolic acidosis, and electrolyte derangements.1

The 1st report of the disease in horses occurred in 1975,8 and since then other cases have been documented.9–19 Reported breeds include Quarter Horse, Thoroughbred, Appaloosa, Arabian, and ponies. The clinical signs manifested on exposure to triggering agents are similar to those described in humans.16 A marked increase in muscle metabolism results in a rapid increase of body temperature that may exceed 43°C (109.4°F). Recently, a single missense point mutation (C7360G) in exon 46 of the RyR1 gene, generating an R2454G amino acid substitution, was identified in 2 Quarter Horse geldings.6 Several single missense point mutations have been identified in humans and are concentrated in 3 regions of the RyR1 gene.7,20 The pig has been the naturally occurring animal model for human disease.4 The C1843T mutation (R615C) in pigs is located at the N-terminal region,4 and is analogous to the C1840T (R614C) and C1841T (R614L) mutations in humans.3 The mutation in the horse is located in the central region of the gene and is analogous to 2 mutations in MHS in humans: the C7360T (R2454C) and the C7361A (R2454H) mutations.21,22 Among the known missense mutations in humans, almost half involve an arginine substitution.7 These residue substitutions in humans and horses occur between 2 different groups of amino acids with different physicochemical properties (arginine is a polar strongly basic amino acid whereas glycine is a nonpolar neutral amino acid) resulting in alterations of protein structure and function.6,7 Currently, there are no naturally occurring animal models for the C-terminal mutations of humans.

Nonanesthetic triggered or “awake” MH episodes include exercise-induced and chronic rhabdomyolyses in humans.1,23–27 Association of MHS and other myopathies such as hyperkalemic periodic paralysis (HYPP) and hypokalemic periodic paralysis are rarely reported in humans.28,29 Similar to humans, nonanesthetic forms of MH such as porcine stress syndrome (PSS) and pale soft exudative (PSE) muscle have been documented in swine.30 These disorders can be induced by stress from fights, exertion, weaning, coitus, heat or extreme cold, branding, transportation, and preslaughter practices.30 Exertional and nonexertional rhabdomyolyses are common causes of myopathy and impaired performance in horses.31 However, only a few etiologies have been recognized.31 Nonanesthetic manifestations of MH and rhabdomyolysis have not been reported in horses. The purpose of this study was to describe the clinical and laboratory findings of Quarter Horses with the RyR1 C7360G mutation.

Materials and Methods

Case Selection

This prospective study included 11 of 20 Quarter Horses from 2005 to 2008 with suspected MH, or an undetermined etiology for 1 of the following: sudden death, death within hours of onset of rhabdomyolysis, or persistent or intermittent increase of serum muscle enzyme activity in conjunction with 3 or more of the following signs: “colic-like” episodes, muscle rigidity, stiffness, intermittent sweating, and persistent or intermittent increased rectal temperature. Tissue samples from selected horses submitted to the Neuromuscular Disease Laboratory (NDL) of the William R. Pritchard Veterinary Medical Teaching Hospital at the University of California at Davis were processed for genetic (MH, HYPP, and polysaccharide storage myopathy type 1 [PSSM type 1]32,33 testing) and histochemical analysis. Medical records, necropsy reports, and pedigrees were collected and reviewed when available. Signalment, clinical signs, onset of signs, clinicopathologic data, outcome, and pathologic diagnosis were recorded.

Sample Collection and Preparation

Whole blood in EDTA was processed for genomic DNA extraction according to the manufacturer's guidelines.a Identification of MHS horses was done by gDNA sequencing of RyR1 exon 46 as described elsewhere.6 Immediately after collection or arrival, skeletal muscle tissue was frozen in isopentane precooled in liquid nitrogen and stored at −80 °C until further processing. Available muscle specimens were processed for histochemical analyses according to NDL protocols, which have been described elsewhere.34

Results

Horses

Eleven Quarter Horses including 2 previously reported cases had the RyR1 C7360G mutation based on amplicon sequencing.6 There were 6 geldings, 2 mares, 2 colts, and 1 stallion. All 11 horses were heterozygous for the mutation. Pedigree analysis and genotyping the parents of 4 MHS horses indicated an autosomal dominant mode of inheritance. Six MHS horses shared common ancestors, and pedigrees were not available in 5 horses. All 11 Quarter Horses were of lineage within the halter horse discipline and were negative for the HYPP mutation. Muscle mass was markedly prominent in 10 horses. Nine horses died and 2 are alive. Five horses died during inhalation anesthesia and 4 died within a few hours after the onset of an episode of rhabdomyolysis. Based on the development of clinical signs and its association with anesthesia, horses were classified in 2 groups: (1) anesthesia-associated (n = 5) and (2) non–anesthesia-associated (n = 6).

Anesthesia-Associated Group

This group comprised 5 Quarter Horse geldings from 4 to 14 years of age. Historically, 1 horse had recurrent episodes of rhabdomyolysis of undetermined cause. The rest of the horses were reported to be healthy before the anesthetic procedure. Anesthesia was performed in these horses for research purposes (n = 2 apparently healthy horses) and elective orthopedic procedures (n = 3 clinical patients). Except for lameness in 3 horses, physical and laboratory variables were unremarkable in all horses before anesthesia. However, 2 horses had a history of intermittent mild increases of serum creatine kinase activity (CK < 1,000 IU/L; reference range 119–287 IU/L). Clinical signs were associated with or triggered by halothane (n = 3/5, minimal alveolar concentration [MAC] from 1.2 to 1.4) and isoflurane (n = 2/5, MAC up to 1.6). The most common clinical signs included tachycardia (sinus tachycardia or ventricular tachycardia leading to ventricular fibrillation), profuse sweating, rapidly increasing hyperthermia (40.0–45 °C [104 to 113°F]; reference range, 37.5–38.2 °C [99.5–100.7°F]), local muscle rigidity such as contracted pinnae and masseter muscle rigidity followed by generalized muscle rigidity that included protrusion of the third eyelid and severe retraction of the ocular globe, trismus, and death with hyperacute rigor mortis (n = 5/5). The most common clinical laboratory abnormalities during an MH episode included acidemia (pH 6.5–6.9; reference range, 7.35–7.42), respiratory acidosis (end tidal CO2 >60–140 mmHg; PaCO2 69–274 mmHg), metabolic acidosis (base deficit >8 mmol/L), and abnormal lung function despite mechanical ventilation with 100% oxygen (PaO2 < 122 mmHg; reference range, 400–500 mmHg). Hypertension also was identified (MAP >100–130 mmHg). All horses had increased hematocrits (52–65%; reference range, 32–45%), hyperproteinemia, hyperglycemia, azotemia, and electrolyte derangements that included hyperkalemia, hypochloremia, hyperphosphatemia, hypernatremia or hyponatremia, and hypercalcemia or hypocalcemia. Serum creatine kinase activity was mildly increased in all horses (843–2,300 IU/L) in samples collected before death. Serum myoglobin was measured in 2 horses and was found to be increased (88 and 98.4 ng/mL; reference range, 0–9 ng/mL).

Necropsy reports were available for 3 horses. Postmortem examination findings included hyperplastic adrenal glands because of cortical hyperplasia in the zona fascicularis, suggestive of a hypertensive state in all 3 horses. Brown discoloration of the urine was observed and confirmed to be caused by myoglobin. Liver vitamin E and selenium concentrations were available in 2 horses and found to be within reference range. Tissue vitamin E and selenium were not measured in the remaining horses. Skeletal muscle specimens collected during or immediately after a fatal MH episode were analyzed using formalin-fixed (n = 3) and fresh-frozen (n = 2) preparations. Myopathic alterations included mild to severe, multifocal to diffuse myolysis, interfascicular edema, hypercontraction, myofiber size variation, internal nuclei, ringbinden fibers (Fig 1A), and sarcoplasmic masses. On histochemical analysis (n = 2), there was evidence of glycogen depletion, decreased to absent myophosphorylase activity, and decreased oxidative activity by mitochondria as seen with nicotinamide adenine dinucleotide and succinic dehydrogenase reactions. Four horses were negative for the GYS1 mutation that causes PSSM type 1.32 DNA was not available for PSSM testing in 1 horse.

Figure 1.

 (A) Gluteus medius muscle on hematoxylin and eosin from affected horse. Note a ringbinden fiber (*) with a sarcoplasmic mass. Rhabdomyolysis is not shown. (B) Gluteus medius muscle with myophosphorylase reaction collected after fatal non–anesthesia-associated rhabdomyolysis. Note the absence of staining and contracted fibers (round appearance). (C) Gluteus medius muscle stained with periodic acid Schiff collected after fatal non–anesthesia-associated rhabdomyolysis. Note the decreased (pale magenta) glycogen staining. (D) Gluteus medius muscle stained with the succinic dehydrogenase reaction (oxidative activity within mitochondria) collected after fatal non–anesthesia-associated rhabdomyolysis. Note the diffuse decreased (pale black) staining.

Non–Anesthesia-Associated Group

This group comprised 6 Quarter Horses: 2 colts, 1 stallion, 1 gelding, and 2 mares 9 months to 9 years of age. Four of the 6 horses died; 3 died within a few hours (4–12 hours) from the onset of rhabdomyolysis. One horse was found dead on pasture during an unusually hot day. This horse was last seen alive and sweating (as it had been observed to do previously) a few hours before death. Owners and referring veterinarians described that all 4 horses appeared rigid, unable to flex their limbs, and that the musculature became more prominent before death. Contracted pinnae and masseter muscle rigidity, tachycardia, tachypnea, hyperthermia (40.2–43.3°C [104.4–110°F]), and sweating was also recorded before death in 3 horses. Clinical pathology data were available before death in 3 horses. Abnormalities included increased hematocrit, leukocytosis, neutrophilia, hyperfibrinogenemia (in 1 yearling colt), hyponatremia, hyperkalemia, hypochloremia, hypocalcemia or hypercalcemia, hyperphosphatemia, hyperglycemia, azotemia, and increased serum creatine kinase (up to 283,812 IU/L) and aspartate aminotransferase activities (AST up to 21,702 IU/L; reference range, 168–494 IU/L). Blood gases and lactate concentration were available in 3 horses and indicated acidemia (pH 6.9–7.1), hypercapnia (PCO2 75–120 mmHg), and hyperlactatemia (12–30 mmol/L; reference range, <2 mmol/L). Historically, all 6 horses had mild to severe recurrent rhabdomyolysis, or colic-“like” episodes that could have occurred spontaneously or in association with exercise, stress, or breeding as in the case of 1 stallion. The first episode of recurrent rhabdomyolysis in a 9-month-old colt occurred at 3 months of age. Intermittent or persistent hyperthermia (up to 38.6°C [101.6°F]), hyperthermia during rhabdomyolysis (up to 40.6°C [105°F]), sweating, muscle rigidity, stiff gait, and exercise intolerance were also noted in these horses. All 6 horses (including 2 living horses) had persistent or intermittent increases in serum muscle enzyme activities (CK 600–36,000 IU/L; and AST up to 2,500 IU/L). Whole blood selenium and plasma vitamin E concentrations were measured in 5 horses and found to be within reference range. The remaining horse (yearling colt) had low vitamin E concentrations in liver tissue despite a diet considered adequate in vitamin E (alfalfa based) and additional PO supplementation. This colt was recently moved to a new facility for halter training and developed a bilateral nasal discharge a few days before fatal rhabdomyolysis.

Necropsy reports were available for 3 horses and indicated adrenal hyperplasia and generalized rhabdomyolysis. Skeletal muscle specimens collected during or immediately after fatal MH episodes were analyzed using formalin-fixed (n=3) and fresh-frozen (n=3) preparations. Myopathic alterations included segmental degeneration, myonecrosis, calcification (yearling colt), histiocytic infiltration within necrotic fibers (yearling colt), edema, and hypercontracted fibers. Other than histiocytic infiltration, there were no cellular (inflammatory) infiltrates in the skeletal muscle of the yearling colt. Ringbinden fibers also were observed in 2 horses. Histochemical analysis of skeletal muscle (n=3) revealed nonspecific abnormalities except for 1 mare that had periodic acid Schiff-positive inclusions resistant to amylase digestion, considered diagnostic for PSSM.32 Muscle glycogen, myophosphorylase, and oxidative activities appeared decreased in 1 fatal case (Fig 1B–D). Five horses were negative for the GYS1 mutation.32 DNA was not available for PSSM testing in the 1 mare with characteristic amylase-resistant periodic acid Schiff-positive inclusions diagnostic for PSSM in a muscle biopsy specimen.32

Discussion

This study described novel features of MH in Quarter Horses with the RyR1 C7360G mutation. These include identification of the mutation in Quarter Horses of halter lineage, an autosomal dominant mode of inheritance, and an association of isoflurane (in addition to halothane) with fatal MH episodes. This is also the first report of non–anesthesia-associated clinical manifestations of MH in horses, including rhabdomyolysis (exertional and nonexertional), hyperthermia, and the presence of concurrent myopathies such as PSSM and possible nutritional myodegeneration.

Quarter Horses from this study were of halter lineage and had a heavily muscled phenotype. However, halter Quarter Horses typically have marked muscular development and it is possible that this association may not necessarily represent a characteristic MH phenotype. In MHS humans, there is no distinct phenotype.2 The disease has been reported in certain breeds of pigs such as Duroc, Landrace, Petrain, Poland China, and Yorkshire.35,36 Typically, MHS pigs have greater muscular development than do non-MHS pigs.35 Similar to affected humans and dogs,3,5 the disease is inherited as an autosomal dominant trait in horses. The mode of inheritance differs in pigs in which the disease is inherited in an autosomal recessive mode.4 There were no MH homozygous horses in the present study. This observation may be due to the low number of cases and apparent low prevalence of the disease.6 Because the mutation is inherited in a dominant mode, disease caused by the RyR1 C7360G mutation could be observed in other breeds and Quarter Horse lineages (such as Paints and Appaloosas) if bred to affected horses.

MH is a potentially fatal disorder in horses with an estimated mortality rate of 34%.8,10,13,15–17 The disease has been induced by halothane alone or in combination with succinylcholine or painful stimuli.8,10,15,16 An additional case, a 12-day-old Quarter Horse filly with suspected HYPP developed mild hypercapnia, hyperthermia, and increased muscle enzyme activity while under isoflurane anesthesia.18 In our study, 5 horses with confirmed MHS developed clinical and laboratory abnormalities consistent with MH that resulted in death while under halothane or isoflurane anesthesia. All halogenated anesthetics (halothane, isoflurane, desflurane, and sevoflurane) can induce MH in susceptible humans and pigs, but most cases have been associated with halothane.1,37,38 Experimental reports in swine suggest a difference in the onset of MH crisis by volatile anesthetics as shown by halothane exposure, resulting in the fastest onset, followed by isoflurane, and desflurane.37 Desflurane and sevoflurane were recently introduced as anesthetics in equine practice but there have been no reports of anesthesia-induced MH episodes.

PSS and PSE muscle are other common non–anesthetic-induced forms of MH in certain breeds of pigs.35,36 The incidence of nonanesthetic MH is higher among pigs inbred for superior muscular development.35 Similar to anesthesia-induced MH, awake pigs can suffer from acidemia, muscle rigidity, hyperthermia, and death if exposed to stressful situations.30 Exertional rhabomyolysis as a nonanesthetic form of MH also occurs in humans,24–27 but MH most often results from exposure to inhalation anesthetics.1 Factors such as stress caused by transportation, foreign environment, onset of training, excitement from breeding, extreme heat, and concurrent illness may have contributed to clinical manifestations of muscle rigidity, stiff gait, sweating, rhabdomyolysis, recumbency, and death in susceptible horses in this study. Similar to anesthesia-induced MH, horses with the non–anesthesia-induced form of the disease had acidemia, hypercapnia, electrolyte derangements, and increases in serum creatine kinase activity during the MH episode. Six horses also had persistently increased serum muscle enzyme activities between episodes. Electrolyte derangements (hyperkalemia, hyponatremia, hypocalcemia, and hyperphosphatemia), acidemia, azotemia, hyperproteinemia, increased serum muscle enzyme activities, myoglobinuria, and arrhythmias can also be observed with other forms of severe rhabdomyolysis such as peracute cases of nutritional myodegeneration caused by selenium deficiency in neonatal foals.39 Therefore, other causes of rhabdomyolysis must be ruled out in affected horses.

The presence of concurrent illnesses or myopathies may contribute to the clinical manifestations of disease. Concurrent myopathies included nutritional myodegeneration caused by low vitamin E (yearling colt) and PSSM (1 mare). Studies in knock-in mice demonstrated that mutated RyR1 causes calcium leak, which drives increased generation of reactive oxygen and nitrogen species leading to increased mitochondrial lipid peroxidation.40 If similar in the horse, this could have contributed to the low hepatic vitamin E concentrations in the yearling colt due to consumption as an antioxidant. In addition, free radical formation can be increased in states of disease and stress making affected horses more susceptible to MH episodes. Because of the historical nasal discharge, immune-mediated myositis was ruled out as the cause of rhabdomyolysis and death in the yearling colt.41 It is unclear what specific role these concurrent myopathies play in the clinical manifestations and outcome of MHS in horses because many of the horses had no other apparent myopathy. Whether concurrent myopathies were altered by the presence of RyR1 mutation and resulted in death in most cases, or a combination of stressful events and concurrent myopathies induced the nonanesthetic manifestations of MH resulting in death, remains uncertain. There have been reports of MHS humans where positive MH status conferred predisposition to fatal rhabdomyolysis in response to exertion and high environmental temperatures.26,27,42 Studies of RyR1 mutated knock-in mice have demonstrated that episodes of muscle contractions, rhabdomyolysis, and death can be induced by increased environmental temperature alone.40 Furthermore, 2 specific mutations in humans (R401C and R614C) are associated with MH and environmental heat stroke.26,27

The molecular basis of triggering events and associated phenotypic manifestations of MH are slowly emerging in humans.20 These are supported by pharmacologic and functional experimental studies for specific mutations in knock-in mice.43 The various phenotypes in humans appear to be attributed to specific mutations in different regions of the same gene.20 However, several of these mutations ultimately result in MHS when affected patients are exposed to halogenated anesthetics.20 In pigs, a single point mutation is responsible for all phenotypes reported in the literature, which include anesthesia-, PSS-, and PSE-induced fatal MH episodes.4 A similar situation exists in Quarter Horses with the RyR1 C7360G mutation. The 2 forms of MH described in this study may represent a single phenotype triggered by different events. For instance, horses with the nonanesthetic form did not have a history of inhalational anesthesia before clinical manifestations of the disease. Similarly, horses with the anesthetic form could have been subclinical and gone unrecognized before the fatal MH episode. In addition, if the disease in horses is similar to that in humans, exposure to triggering agents may not always lead to an MH episode.2 The presence of concurrent illnesses and myopathies could influence phenotypic manifestations as discussed previously.

Similar to MHS in humans and pigs, the skeletal muscle of affected horses did not show specific histologic abnormalities. Histologic evaluation of skeletal muscle in MHS horses revealed a mild nonspecific noninflammatory myopathy as described. Ringbinden fibers appear to be an uncommon (found in 2 of 229 equids in 1 study)44 and nonspecific histologic finding in horses.41,44–46 Ringbinden fibers were observed in 4 of 8 horses in the present study. Further, ringbinden fibers appeared to be a predominant histologic feature of suspected MHS horses in 2 studies.11,12 Ringbinden fibers have been described in humans with various myopathies and in muscles undergoing regeneration.47 Descriptions of histochemical alterations in confirmed fatal equine cases are limited.6,16 Common but nonspecific histochemical features during fatal episodes included multifocal interfascicular and myofiber edema, rhabdomyolysis, hypercontracted fibers, glycogen depletion, and decreased to absent myophosphorylase and oxidative activities.

In conclusion, both anesthetic- and nonanesthetic myopathic manifestations of MH occur in Quarter Horses of halter lineage with the RyR1 C7360G mutation. The disease is inherited as an autosomal dominant trait; therefore, other breeds and Quarter Horse lineages could be affected if bred to MHS horses. Hyperthermia, sweating, tachycardia, hypertension, tachypnea, muscle rigidity, rhabdomyolysis, hypercapnia, acidemia, and electrolyte derangements should raise suspicion of MH if inhaled anesthetics alone or in combination with succinylcholine are used in horses. Both halothane and isoflurane anesthetics could trigger fatal MH episodes in horses. Nonanesthetic manifestations may include rhabdomyolysis and “colic-like” episodes, muscle rigidity, stiffness, intermittent hyperthermia and sweating episodes. These may be induced or associated with exercise, stress, breeding, illnesses, or concurrent myopathies. Clinical and laboratory abnormalities are similar to those observed with anesthetic-induced MH if fatal. In addition, persistent or intermittent increases of serum muscle enzyme activity may be observed between episodes of rhabdomyolysis. Fatalities can occur with both forms of MH. Horses develop a hypermetabolic, catabolic state during an MH crisis, as reflected by clinical signs, laboratory abnormalities, and skeletal muscle histochemical alterations of glycogen depletion and decreased myophosphorylase and oxidative activities. Genotyping for the RyR1 mutation should be considered in Quarter Horses with unexplained or intermittent signs of rhabdomyolysis, muscle rigidity, stiffness, hyperthermia, sweating, and subclinical increases in muscle enzyme activity. Muscle rigidity or rhabdomyolysis followed by acute or peracute death of undetermined cause and myopathy or death associated with inhalation anesthesia should also prompt MH testing. Breeding of affected horses should be discouraged.

Footnote

aDNeasy tissue kit protocol, QIAGEN Inc, Valencia, CA

Acknowledgments

We thank referring veterinarians for submitting tissue samples, records, and pedigrees; and Dr Stephanie Valberg for performing the GYS1 genetic test.

This project was funded by the Comparative Gastroenterology Laboratory of the University of California at Davis. Genetic testing is available at the Neuromuscular Disease Laboratory at the William R. Pritchard Veterinary Medical Teaching Hospital, One Shields Avenue, University of California, Davis, CA 95616, Attention: Dr Monica Aleman.

Ancillary