Calcium regulation by skeletal muscle membranes of horses with recurrent exertional rhabdomyolysis

OBJECTIVE: To determine whether an alteration in calcium regulation by skeletal muscle sarcoplasmic reticulum, similar to known defects that cause malignant hyperthermia (MH), could be identified in membrane vesicles isolated from the muscles of Thoroughbreds with recurrent exertional rhabdomyolysis (RER). SAMPLE POPULATION: Muscle biopsy specimens from 6 Thoroughbreds with RER and 6 healthy (control) horses. PROCEDURES: RER was diagnosed on the basis of a history of > 3 episodes of exertional rhabdomyolysis confirmed by increases in serum creatine kinase (CK) activity. Skeletal muscle membrane vesicles, prepared by differential centrifugation of muscle tissue homogenates obtained from the horses, were characterized for sarcoplasmic reticulum (SR) activities, including the Ca2+ release rate for the ryanodine receptor-Ca2+ release channel, [3H]ryanodine binding activities, and rate of SR Ca2+-ATPase activity and its activation by Ca2+. RESULTS: Time course of SR Ca2+-induced Ca2+ release and [3H]ryanodine binding to the ryanodine receptor after incubation with varying concentrations of ryanodine, caffeine, and ionized calcium did not differ between muscle membranes obtained from control and RER horses. Furthermore, the maximal rate of SR Ca2+-ATPase activity and its affinity for Ca2+ did not differ between muscle membranes from control horses and horses with RER. CONCLUSIONS AND CLINICAL RELEVANCE: Despite clinical and physiologic similarities between RER and MH, we concluded that RER in Thoroughbreds does not resemble the SR ryanodine receptor defect responsible for MH and may represent a novel defect in muscle excitation-contraction coupling, calcium regulation, or contractility.     

Myoplasmic calcium regulation in myotubes from horses with recurrent exertional rhabdomyolysis

OBJECTIVE: To determine whether alterations in myoplasmic calcium regulation can be identified in muscle cell cultures (myotubes) and intact muscle fiber bundles derived from Thoroughbreds affected with recurrent exertional rhabdomyolysis (RER). ANIMALS: 6 related Thoroughbreds with RER and 8 clinically normal (control) Thoroughbred or crossbred horses. PROCEDURES: Myotube cell cultures were grown from satellite cells obtained from muscle biopsy specimens of RER-affected and control horses. Fura-2 fluorescence was used to measure resting myoplasmic calcium concentration as well as caffeine- and 4-chloro-m-cresol (4-CMC)-induced increases in myoplasmic calcium. In addition, intact intercostal muscle fiber bundles were prepared from both types of horses, and their sensitivities to caffeine- and 4-CMC-induced contractures were determined. RESULTS: Myotubes of RER-affected and control horses had identical resting myoplasmic calcium concentrations. Myotubes from RER-affected horses had significantly higher myoplasmic calcium concentrations than myotubes from control horses following the addition of > or = 2mM caffeine; however, there was no difference in their response to 4-CMC (> or = 1 mM). Caffeine contracture thresholds for RER and control intact muscle cell bundles (2 vs 10mM, respectively) were significantly different, but 4-CMC contracture thresholds of muscle bundles from RER-affected and control horses (500 microM) did not differ. CONCLUSIONS AND CLINICAL RELEVANCE: An increase in caffeine sensitivity of muscle cells derived from a family of related RER-affected horses was detected in vitro by use of cell culture with calcium imaging and by use of fiber bundle contractility techniques. An alteration in muscle cell calcium regulation is a primary factor in the cause of this heritable myopathy.     

Inheritance of recurrent exertional rhabdomyolysis in thoroughbreds

OBJECTIVE: To develop a diagnostic test for recurrent exertional rhabdomyolysis (RER) in Thoroughbreds that relied on in vitro contracture of muscle biopsy specimens and determine whether the inheritance pattern of RER diagnosed on the basis of this contracture test was consistent with an autosomal dominant trait. DESIGN: Clinical trial. ANIMALS: 8 adult horses with RER and 16 control adult horses for development of the contracture test; 23 foals for inheritance of RER. PROCEDURE: External intercostal muscle biopsy specimens from the 24 adult horses were tested for contracture in response to halothane and caffeine, and criteria for a positive test result were determined. These criteria were then applied to results for the 23 foals to determine whether they had RER. Simple segregation analysis was performed to determine whether results were consistent with a dominant pattern of inheritance. RESULTS: Results of the contracture test were positive for 5 of the 12 colts and 4 of the 11 fillies. Results of segregation analysis were consistent with an auto-. somal dominant pattern of inheritance. Two sires with RER produced colts with RER, supporting the hypothesis that RER had an autosomal, rather than an X-linked, inheritance pattern. In addition, in 1 instance, an unaffected colt was produced by 2 affected parents, which was not consistent with a recessive mode of inheritance. CONCLUSIONS AND CLINICAL RELEVANCE: Although the expression of the RER trait is influenced by sex, temperament, and diet, among other factors, results from the in vitro muscle contracture test and this breeding trial suggest that RER in Thoroughbreds can be modeled as a genetic trait with an autosomal dominant pattern of inheritance.     

Heritability of recurrent exertional rhabdomyolysis in Thoroughbred racehorses

OBJECTIVE: To determine the likely mode of inheritance and identify probable foundation horses for recurrent exertional rhabdomyolysis (RER) in Thoroughbred (TB) racehorses. ANIMALS: 4 families of TB racehorses with a high prevalence of RER, consisting of 3 to 53 horses/family, were used to determine mode of inheritance. Sixty-two TB horses with RER and 34 control TB racehorses without RER were used to identify probable foundation horses for the RER trait. PROCEDURE: RER was diagnosed by a veterinarian and verified by detecting high serum creatine kinase activity. Pedigrees dating from 1930 for all horses were entered into a database. Pedigrees of horses in 4 families were visually inspected for a pattern of inheritance and used for calculation of foundation horse contributions and inbreeding coefficients. The Markov chain Monte Carlo technique was used to analyze pedigrees of the 62 affected and 34 control horses for the conditional probability of foundation genotypes. A dominant mode of inheritance with variable expression model was used. RESULTS: Pedigree analysis supported an autosomal dominant mode of inheritance with variable expression. All affected horses from the 4 families shared a common ancestor. This ancestor and 5 other stallions had a conditional probability of 1.00 for being affected. All 6 stallions shared a common male ancestor within 3 to 5 generations. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of this study, the RER trait has been in TB racehorses for more than 70 years and may be inherited as an autosomal dominant trait with variable expression.     

Contracture test and histologic and histochemical analyses of muscle biopsy specimens from horses with exertional rhabdomyolysis

Biopsy specimens of the cutaneous omobrachialis muscle were obtained from 10 horses with a problem of myositis from mild exercise. One horse had been evaluated previously and malignant hyperthermia-like contractures developed in its muscle biopsy specimen during the contracture test. In this study, the halothane-caffeine contracture test and histologic and histochemical evaluations were performed on muscle biopsy specimens. In the contracture test, no muscle biopsy specimen developed contracture in the presence of 2 or 4% halothane alone. The mean (+/- SEM) caffeine-specific concentration in the presence of halothane was 5.23 +/- 0.5 mM for 2% halothane, and 4.46 +/- 0.6 mM for 4% halothane. The caffeine-specific concentration values were not significantly different. Contracture response for any muscle specimen did not resemble contracture associated with malignant hyperthermia. The cutaneous omobrachialis muscle was composed of type-II fibers, with type-I fibers seldom seen. For 9 of the 10 horses, overall fiber morphology was normal; 1 horse had necrotic fibers. Of the 10 muscle specimens, 9 had fibers that had positive reaction for alkaline phosphatase activity; 3 muscle specimens contained ringed myofibers. Three horses of this study were administered general anesthesia; 2 were research horses, anesthetized with halothane and succinylcholine, and 1 was a clinical case given halothane anesthesia plus a non-depolarizing muscle relaxant. One research horse developed a malignant hyperthermia-like reaction to anesthesia, with severe rhabdomyolysis evident after anesthesia, and an episode of muscle cramping in its stall 2 days after anesthesia. The other 2 horses had unremarkable postanesthetic periods.     

Prevalence and demographic characteristics of exertional rhabdomyolysis in horses in Australia

The proportion of the horses, of both sexes and of different ages, breeds and levels of activity, owned by a stratified random sample of Australian owners, which had suffered one or more episodes of exertional rhabdomyolysis during the previous 12 months was determined. The proportion in the general population was 1.9 per cent, and horses which exercised were significantly more likely to have experienced the condition than horses which did not. There were significant differences between the sexes and between certain breeds and activity groups in the proportions of horses affected.     

Effect of oral administration of dantrolene sodium on serum creatine kinase activity after exercise in horses with recurrent exertional rhabdomyolysis

OBJECTIVE: To determine the effect of oral administration of dantrolene sodium on serum creatine kinase (CK) activity after exercise in horses with recurrent exertional rhabdomyolysis (RER). ANIMALS: 2 healthy horses and 5 Thoroughbreds with RER. PROCEDURE: 3 horses received 2 doses of dantrolene (4, 6, or 8 mg/kg, p.o., with and without withdrawal of food) 2 days apart; 90 minutes after dosing, plasma dantrolene concentration was measured spectrofluorometrically. On the basis of these results, 5 Thoroughbreds with RER from which food was withheld received dantrolene (4 mg/kg) or an inert treatment (water [20 mL]) orally 90 minutes before treadmill exercise (30 minutes, 5 d/wk) during two 3-week periods. Serum CK activity was determined 4 hours after exercise. Plasma dantrolene concentration was measured before and 90 minutes after dosing on the first and last days of dantrolene treatment and before dosing on the first day of the inert treatment period, RESULTS: 90 minutes after dosing, mean +/- SEM plasma dantrolene concentration was 0.62 +/- 0.13 and 0 microg/mL in the dantrolene and inert treatment groups, respectively. Serum CK activity was lower in dantrolene-treated horses (264 +/- 13 U/L), compared with activity in water-treated horses (1,088 +/- 264 U/L). Two horses displayed marked muscle stiffness on the inert treatment. CONCLUSIONS AND CLINICAL RELEVANCE: In 5 horses with RER from which food had been withheld, 4 mg of dantrolene/kg administered orally provided measurable, though variable, plasma concentrations and significantly decreased serum CK activity after exercise in 4 of those horses.     

Effect of ration and exercise on plasma creatine kinase activity and lactate concentration in Thoroughbred horses with recurrent exertional rhabdomyolysis

OBJECTIVE: To determine the effects of 3 rations (low grain, fat, high grain) on plasma creatine kinase (CK) activity and lactate concentration in Thoroughbred horses with recurrent exertional rhabdomyolysis (RER). ANIMALS: 5 Thoroughbreds with RER and 3 healthy Thoroughbreds (control horses). PROCEDURES: Rations were formulated to meet (low-grain and fat rations) or exceed (high-grain ration) daily energy requirements. Each ration was fed to horses in a crossover design for 3 weeks. Horses were exercised on a treadmill Monday through Friday; maximum speed on Monday and Friday was 11 m/s (6% slope), on Tuesday and Thursday was 9 m/s, and on Wednesday was 4.5 m/s. Plasma CK activity and lactate concentration were determined before and after exercise. RESULTS: Horses with RER fed the high-grain ration had significantly greater CK activity and change in CK activity 4 hours after exercise, compared with those fed the low-grain ration. Horses with RER exercised at the trot or canter had significantly greater increases in CK activity, compared with those exercised at the gallop. Plasma lactate concentrations after exercise were similar in control and affected horses. Lactate concentration and CK activity were not correlated in horses with RER. CONCLUSIONS AND CLINICAL RELEVANCE: Rations high in grain and formulated to exceed daily energy requirements may increase episodes of rhabdomyolysis in thoroughbred horses susceptible to RER.     

Acute kidney injury mediated by oxidative stress in Egyptian horses with exertional rhabdomyolysis

The present study was carried out to evaluate the role of oxidative stress in the pathophysiologic process of acute renal failure associated with exertional rhabdomyolysis (ER) in Egyptian horses. ER was tentatively diagnosed in 31 Baladi horses based on case history, physical examination findings and confirmed by elevation of plasma creatine kinase (CK) and urine myoglobin concentrations. According to severity of the condition, the diseased horses were categorized into two main groups; the first group included 18 horses with minimal clinical signs and plasma CK <60 000 IU/L; whereas, the second group included 13 horses with overt clinical signs and plasma CK >100 000 IU/L). It was found that plasma creatol (CTL) was positively correlated (p < 0.01) with plasma malondialdehyde (MDA) (r = 0.775), nitric oxide (NO) (r = 0.768), methyguanididne (MG) (r = 0.995), CK (r = 0.768), urine glucose (r = 0.778), urine protein (r = 0.767), renal failure index (RFI) (r = 0.814) and urine sodium (r = 0.799) and negatively correlated (p < 0.01) with total antioxidant capacity (TAC) (r = −0.795), superoxide dismutase (SOD) (r = −0.815), glutathione peroxidase (GSH-Px) (r = −0.675), Vitamin C (r = −0.830), urine creatinine (r = −0.800), urine/plasma creatinine ratio (r = −0.827) and urine/plasma urea ratio (r = −0.807). The correlation between these biochemical variables might suggest a possible role of oxidative stress in renal injury associated with severe rhabdomyolysis in horses. It is suggested that exaggeration of oxidative stress associated with increased muscle membrane leakage plays a key role in acute kidney injury in Baladi horses with severe rhabdomyolysis.     

Plasma and urine electrolyte and mineral concentrations in Thoroughbred horses with recurrent exertional rhabdomyolysis after consumption of diets varying in cation-anion balance

OBJECTIVE: To determine whether plasma, urine, and fecal electrolyte and mineral concentrations differ between clinically normal horses and Thoroughbreds with recurrent exertional rhabdomyolysis (RER) after consumption of diets varying in cation-anion balance. ANIMALS: 5 Thoroughbred mares with RER and 6 clinically normal mixed-breed mares. PROCEDURE: Each of 3 isocaloric diets designated as low, medium, and high on the basis of dietary cation-anion balance (DCAB) values of 85, 190, and 380, respectively, were fed to horses for 14 days. During the last 72 hours, 3 horses with RER and 3 control horses had daily urine and fecal samples obtained by total 24-hour collection. Remaining horses had urine samples collected daily by single catheterization. RESULTS: For each diet, no differences existed between horses with RER and control horses in plasma pH, electrolyte concentrations, and creatine kinase activity or in urine pH and renal fractional excretion (FE) values. Plasma pH, strong ion difference, bicarbonate and total carbon dioxide concentrations, and base excess decreased and plasma chloride and ionized calcium concentrations increased with decreasing DCAB. Urine pH decreased with decreasing DCAB. The FE of chloride and phosphorus were greatest for horses fed the low diet. The FE values for all electrolytes exept magnesium did not differ between urine samples obtained by single catheterization and total 24-hour collection. Daily balance of calcium, phosphorus, sodium, chloride, and potassium did not differ significantly among horses fed the various diets. CONCLUSIONS: In clinically normal horses and in horses with RER, the DCAB strongly affects plasma and urine pH and the FE of sodium, potassium, chloride, and phosphorus.     

In vitro contractile responses and contracture testing of skeletal muscle from Quarter Horses with exertional rhabdomyolysis.

OBJECTIVE: To determine whether increased sensitivity to pharmacologic agents was a general property of equine exertional myopathies, including polysaccharide storage myopathy (PSSM) in Quarter Horses. ANIMALS: 5 adult Quarter Horses with exertional rhabdomyolysis and abnormal polysaccharide accumulation in skeletal muscle and 4 clinically normal adult Quarter or Quarter-type horses. PROCEDURES: Twitch time course measurements and contracture responses to various concentrations of caffeine and halothane for small bundles of intact external intercostal muscle fibers were measured in all horses. RESULTS: Caffeine contracture threshold of muscles from Quarter Horses with PSSM was not different from that of clinically normal horses (5 mM in both groups). Muscles from horses with PSSM and from clinically normal horses did not have contracture in response to up to 2% halothane. CONCLUSIONS AND CLINICAL RELEVANCE: Results were in contrast to the increased sensitivity to caffeine and halothane for muscles from Thoroughbreds with recurrent exertional rhabdomyolysis (RER). Although clinical signs of muscular stiffness after exercise are similar between Quarter Horses with PSSM and Thoroughbreds with RER, these breeds appear to have 2 distinct myopathies with different pathophysiologic bases. Unlike RER in Thoroughbreds, PSSM in Quarter Horses does not appear to be accompanied by a defect in regulation of muscle contraction.     

Calcium sensitivity of force production and myofibrillar ATPase activity in muscles from Thoroughbreds with recurrent exertional rhabdomyolysis

OBJECTIVE: To determine whether the basis for recurrent exertional rhabdomyolysis (RER) in Thoroughbreds lies in an alteration in the activation and regulation of the myofibrillar contractile apparatus by ionized calcium. ANIMALS: 4 Thoroughbred mares with RER and 4 clinically normal (control) Thoroughbreds. PROCEDURES: Single chemically-skinned type-I (slow-twitch) and type-II (fast-twitch) muscle fibers were obtained from punch biopsy specimens, mounted to a force transducer, and the tensions that developed in response to a series of calcium concentrations were measured. In addition, myofibril preparations were isolated from muscle biopsy specimens and the maximal myofibrillar ATPase activity, as well as its sensitivity to ionized calcium, were measured. RESULTS: Equine type-I muscle fibers were more readily activated by calcium than were type-II muscle fibers. However, there was no difference between the type-II fibers of RER-affected and control horses in terms of calcium sensitivity of force production. There was also no difference between muscle myofibril preparations from RER-affected and control horses in calcium sensitivity of myofibrillar ATPase activity. CONCLUSIONS AND CLINICAL RELEVANCE: An alteration in myofibrillar calcium sensitivity is not a basis for pathologic contracture development in muscles from RER-affected horses. Recurrent exertional rhabdomyolysis in Thoroughbreds may represent a novel heritable defect in the regulation of muscle excitation-contraction coupling or myoplasmic calcium concentration.     

Epidemiologic analysis of factors influencing exertional rhabdomyolysis in Thoroughbreds

OBJECTIVE: To determine incidence, effect on performance, and management practices associated with exertional rhabdomyolysis (ER) in Thoroughbreds. SAMPLE POPULATION: Medical records for 984 Thoroughbreds and a survey of trainers of horses with and without ER. PROCEDURES: Medical records for 984 Thoroughbreds stabled at a midwestern racetrack were examined to determine the incidence of ER during the 1995 racing season. A retrospective questionnaire was administered to trainers to determine management practices associated with ER in 59 Thoroughbreds with ER and 47 control Thoroughbreds in training. Multiple logistic regression was used to determine management factors associated with ER. RESULTS: ER affected 48 of 984 (4.9%) Thoroughbreds. Two-year-old females were most frequently affected, and 36 of 96 (37.5%) trainers had > or = 1 horse with ER. Horses with ER were more likely not to race during the racing season, compared with control horses. For horses that raced, differences were not found with respect to racing performance between ER and control horses. Exertional rhabdomyolysis developed frequently in susceptible horses that had > or = 1 day of rest prior to exercise and that galloped during exercise. Horses with ER were commonly fed > 4.5 kg of grain daily. Nervous and extremely nervous horses were 5.4 times more likely, and horses with some form of lameness were 4.2 times more likely, to have ER. CONCLUSIONS AND CLINICAL RELEVANCE: Exertional rhabdomyolysis is common in Thoroughbreds, and ER can be affected by temperament, sex, age, diet, exercise routines, and lameness. Management that minimizes excitability, particularly when tailored to each horse, may be most effective for controlling ER.     

Incidence of exertional rhabdomyolysis in polo horses in the USA and the United Kingdom in the 1999/2000 season

A questionnaire was used to determine the incidence of exertional rhabdomyolysis and the factors that might have contributed to its occurrence among 423 polo horses in 11 yards. The yards were selected at random, six of them with 111 horses, in north-eastern USA, and five with 312 horses in south-east England. During the 1999/2000 season the incidence of the condition was 7.3 per cent (9 per cent in the USA and 6.7 per cent in England). The incidence in mares was similar in the two countries, 8.4 per cent in the USA and 8.0 per cent in England, but the incidence in geldings in the USA (10 per cent) was much higher than in England (1.5 per cent). A more excitable than average temperament was reported in 71 per cent of the horses with the condition, and most cases occurred after a game (chukka) in which the horse was perceived to be not fit enough for the level of play demanded. Sixty-four per cent of the cases occurred early in the season and on average each episode resulted in the loss of 7.6 training days.     

A review of exertional rhabdomyolysis in wild and domestic animals and man.

Exertional rhabdomyolysis is a condition arising in several species of newly captured wild animals after some form of physical exertion and stress. It is characterized by muscle necrosis and myoglobinuria. Death may result from secondary renal failure, acute or chronic heart failure and progressive emaciation.