Neuromusc. Disord., Vol.2, No. 5/6. pp. 351-359. 1992
096(}-8966/92 $5.00 + 0.00 ©1993 Pergamon Pros Ltd
Printed in Great Britain
POLYSACCHARIDE STORAGE MYOPATHY ASSOCIATED WITH RECURRENT EXERTIONAL RHABDOMYOLYSIS IN HORSES STEPHANIE J. VALBERG,*'[" GEORGE H . CARDINET
III,* GARY P. CARLSON~ a n d SALVATORE DIMAURC~
*Department of Anatomy and Cell Biology, and ~Department of Medicine, School of Veterinary Medicine, University of California, Davis, CA 95616; and §Neurological Institute, Columbia Presbyterian Medical Center, New York, NY, U.S.A.
(Received 27 May 1992, accepted 17 September 1992) Abstract--A polysaccharidestorage myopathy is described in nine Quarterhorses, Quarterhorse crossbreds, American Paints and Appaloosa horses which had a history of recurrent exertional rhabdomyolysis. Muscle biopsies were characterized by high muscle glycogen concentrations with up to 5% of type 2 muscle fibers containing inclusions which stained positively with the periodic acid Schiff (PAS) stain. The inclusions were classifiedas an acid mucopolysaccharide, based on their histochemicai staining characteristics. Ultrastructural studies revealed that the inclusions were composed of beta glycogen particles interspersed among arrays of filamentous material. In addition, many type 2 fibers contained multiple subsarcolemmal vacuoles. These vacuoles stained lightly with eosin and did not stain positively with PAS. Centrofascicular atrophy and necrosis of scattered type 2 fibers were present in biopsies from some horses. No glyco(geno)iytic enzyme deficiencies were identified using a biochemical screening test for anaerobic glycolysis. Attempts to measure branching enzyme activities in both affected and control samples were unsuccessful, employing methods developed for human muscle. The polysaccharide accumulation in these horses may represent a hereto yet undefined metabolic disorder of skeletal muscle.
Key words: Horse, muscle, rhabdomyolysis, polysaccharide, glycogen.
INTRODUCTION
Exertional rrryopathies are a common problem among working horses. At the turn of this century, recurrent exertional rhabdomyolysis was a common occurrence in draft horses, especially when they resumed exercise after a day of rest [1, 2]. This syndrome has been called Monday morning disease, azoturia, tying-up and paralytic myoglobinuria. Clinical signs occur shortly after exercise and include profuse sweating, stiffness, reluctance to move, myoglobinuria and in some cases recumbency and death. As race and pleasure horses have increased in popularity, a similar but often milder form of recurrent exertional rhabdomyolysis has frequently been reported [3-6]. In practice, the diagnosis of recurrent exertional rhabdomyolysis is made on the basis of clinical signs, myoglobinuria and increased activities of serum creatine kinase (CK), lactate
tAuthor to whom reprint requests should be sent.
dehydrogenase (LDH) and aspartate aminotransferase (AST) [7]. Muscle biopsies have not been routinely used to investigate the etiology of exertional rhabdomyolysis. In instances where muscle biopsies have been obtained, necrosis of type 2 fibers, increased glycogen concentrations and myofibrillar and mitochondrial degeneration have been described [5, 8-11]. Several etiologies for recurrent exertional rhabdomyolysis have been suggested including lactic acidosis, malignant hyperthermia, hypothyroidism, vitamin E and selenium deficiency, glycolytic enzyme abnormalities and electrolyte imbalances; however, definitive findings are lacking [12]. Since the diagnostic criteria for recurrent exertional rhabdomyolysis are only indications of muscle necrosis, it is likely that a variety of etiological factors may contribute to the development of the clinical syndrome of exertional., rhabdomyolysis. The purpose of this report is to describe the histochemical, biochemical and ultrastructural characteristics of skeletal muscle biopsies from nine horses which
351
352
S.J. V A L B E R G et al. Table I. The breed, sex, age, and maximum serum enzyme activities of horses with recurrent exertional rhabdomyolysis
Horse
Breed
Age at biopsy
Sex
Age of onset (yr)
(yr) I
Arab/Quarterhorse 2 Quarterhorse 3 Thoroughbred/ Quarterhor~ 4 Quarterhorse 5 Appaloosa 6 American Paint 7 Quarterhorse 8 Appaloosa 9 Quarterhorse Normal range:
Maximum serum enzyme activities CK AST LDH
(U I-')
(U I-')
(U I-')
G
5
4
8750*
13,124"
954
F F
10 and 13 9, 10 and 17
4 3
nd 1980
242 5454
453 559
F G F
3 4 and 9 4
2 2 3
nd 24,560 174,195
1965 >2400 >2400
925 nd >3200
F F F
4 3 8
2 2 5
3996 2661 * 200,000" 120-290
1088 3030* 12,609"
nd 2160" nd
140-400
160-410
F ~ female. G = gelding. • ~ 4 h after exercise test. nd = not determined.
had recurrent exertional rhabdomyolysis and an abnormal intramuscular accumulation of polysaccharide forming intensely stained PAS positive inclusions within type 2 fibers. HORSES
The horses presented in this report were selected from biopsies of trunk and limb muscles from a total of 116 horses presenting to the Veterinary Medical Teaching Hospital (VMTH) at the University of California, Davis, over a 12 yr period. Some samples were also received from referring veterinarians. Out of 116 horses, 36 horses presented with a history of exertional rhabdomyolysis and 80 samples came from horses afflicted with a variety of other neuromuscular diseases. The breed, sex and age distribution of the horses in this study are shown in Table 1. Females were more prevalent than males, 7 : 2. The diagnosis of recurrent exertional rhabdomyolysis was made, in each case, on the basis of the history, clinical signs of recurrent exertional rhabdomyolysis and elevated serum CK, L D H and AST activities (Table I). Evaluation of horses presented to the VMTH also included a complete blood count, serum chemistry analyses for the enzymes alkaline phosphatase, gamma glutamyltransferase and sorbitol dehydrogenase and the concentrations of albumin, total protein, bilirubin, glucose, creatinine, blood urea nitrogen, sodium, potassium, calcium, chloride and phosphorus. All affected horses except horse 6 had normal values for these parameters. The horses first developed clinical signs at a young age shortly after they began training (Table 1). Clinical signs were similar to the other
27 horses that were biopsied because of exertional rhabdomyolysis, however, the development of clinical signs at an early age and the recurrence of rhabdomyolysis were more common features in the horses with polysaccharide storage myopathy. Episodes of rhabdomyolysis were reported to be intermittent in some horses (horses l, 3, 5-7) and frequently repeatable after light exercise in others (horses 2, 4, 8, 9). Three horses were subjected to exercise testing while hospitalized and in each case serum CK activities were notably elevated 4 h following exercise (Table 1). The clinical signs exhibited during and after exercise included, in order of their appearance; profuse sweating, increased respiratory rate, stiffness which particularly affected the hind limbs, and reluctance to move. Clinical signs of rhabdomyolysis usually abated within several hours, however, after some episodes muscle pain was still evident for up to 2 days. Seven horses (2-5, 7-9) have continued to have repeated episodes. Horse 1 was euthanitized because of the frequent recurrence of recurrent exertional rhabdomyolysis and no gross pathological abnormalities were found at post mortem. Horse 2 subsequently died following a severe attack ofrhabdomyolysis; however, a post mortem examination was not performed. Horse 6 was admitted to the VMTH after becoming recumbent from a severe episode of rhabdomyolysis. The heart rate~ respiratory rate, rectal temperature and appetite were normal on admission. Abnormalities in serum biochem/stry included elevated activities for CK, AST, L D H (Table 1), sorbitol dehydrogenase 14 U I -~ (normal < 3) and high blood glucose concentrations 2.21 g 1-~ (r~rmal 0.6-1.22). Myoglobin
Polysaccharide
Storage
Myopathy
353
T a b l e 2. T h e h i s t o p a t h o l o g i c a l f i n d i n g s o f l i g h t m i c r o s c o p i c e x a m i n a t i o n o f h o r s e s w i t h r e c u r r e n t exertional rhabdomyolysis Horse
Muscle biopsied
M u s c l e fiber
Necrosis I
Semitendinosus
Sarcoplasmic
Atrophy
Subsarcolemmal vacuoles
positive inclusions
PAS
Triceps
+ . +
+
+
+
2 2R
Semitendinosus Semitendinosus
+ +
-
+ +
+ +
3 3R I 3R2
Superficial gluteus Superficial gluteus Medial gluteus
+ -
+ -
+ + +
+ + +
4
Semitendinosus
-
-
+
+
5
Superficial gluteus Semitendinosus Biceps femoris Vastus lateralis Triceps Medial gluteus
+ + + . + +
-
+ + +
+ + -
-
+ +
+ +
Superficial gluteus Semitendinosus Triceps Cardiac Liver
+ + . +
+ + -
+ + +
+ -
-
-
-
7
Medial gluteus
-
-
+
+
S
Medial gluteus
-
-
+
+
9
Medial gluteus
+
-
+
+
Vastus lateralis
5R 6
+
changes
.
.
.
.
.
.
+
+
.
.
.
R ffi b i o p s y r e p e a t e d . + = present. - = absent.
was present in the urine. Treatment included intravenous fluid therapy and phenylbutazone, with physical assistance to stand provided by a body sling. Antibiotic therapy was initiated following a febrile episode. Serum CK, AST and L D H activities and blood glucose concentrations returned to normal over a 3 week period, however, the horse rem_ained weak and developed marked atrophy of the skeletal musculature. The owners elected to euthanitize the mare. Post mortem findings included a severe generalized myopathy, chronic interstitial pneumonia, acute multifocal hepatitis and chronic focal pyelonephritis. A group of healthy horses were used as controls and included one gelding and eight mares (four Arabians, two Quarterhorses and three thoroughbreds). Their mean age was 8.6 yr (range 3-19 yr) and their serum CK and AST activities were within normal limits.
METHODS
Muscle biopsies (Table 2) were obtained using either open surgical techniques, a Bergstr6m biopsy needle or a modified Bergstr6m biopsy needle with a 5 mm outer diameter. All control samples were obtained percutaneously [13] from the gluteus medius muscle of unexercised horses on a similar ration. No standardization of diet or
exercise prior to biopsy was possible for patient horses. In several cases, muscle biopsies were repeated months to years following the initial biopsy in order to obtain a biopsy for biochemical analysis at a time when clinical rhabdomyolysis was not apparent (Tables 1 and 2). At post mortem, additional samples of several muscles were obtained from horses 1 and 6 and a cardiac and liver sample were obtained from horse 6, The biopsy specimens used for histopathological and histochemical examination were frozen in liquid freon cooled to -125"C with liquid nitrogen. Fresh frozen cryostat sections were then stained with a battery of tinctorial and histochemical staining procedures [14]. Specimens for electron microscopic examination from horses 1, 6-9 were prepared according to Tinling et al. [15]. All samples for biochemical analysis, except those of horses 2 and 4, were frozen shortly after biopsy in liquid nitrogen and stored at - 80"C for later analysis. The muscle biopsies from horses 2 and 4 were obtained by veterinarians in the field, shipped to the laboratory and were frozen within 24 h of collection. Muscle samples obtained at a time when minimal evidence of rhabdomyolysis was apparent histologically, were selected for biochemical analyses. Muscle glycogen, protein and hexokinase assays were performed on 1-2 mg
354
S.J. VALBERG et al.
samples of gluteus muscle or if not available semitendinosus muscle which had been freezedried and dissected free from blood, fat and connective tissue. Samples for glycogen analysis were boiled for 2 h in I M HCI and the glucose residues formed were measured fluorometrically according to Lowry and Passonneau [16]. Muscle samples for hexokinase and acid maltase enzyme analyses were homogenized ultrasonically in a 0.1 M potassium phosphate buffer solution (pH 7.3). The modified Lowry procedure was used to analyze protein content in muscle homogenates (Sigma Kit No. 690-A, St. Louis, MO). The activity of the enzyme hexokinase was measured fluorometrically according to Lowry and Passonneau [16]. Acid maltase activity was measured according to Engel and Gomez [17]. The activity of branching enzyme was measured according to Gambetti et al. [18]. A Student's ttest was used to compare glycogen, protein and enzyme activities between horses with recurrent exertional rhabdomyolysis and controls. The in vitro method of Layzer et al. [19] was modified for use as a biochemical screening test for anaerobic glyco(geno)lysis [20]. The glycogenolytic activity in situ was also assessed histochemically. Muscle sections 9/am thick were cut on a cryostat microtome and incubated for 10 min in 0.2% EDTA in 50 mM potassium phosphate buffer (pH 7.6). Subsequently, sections were incubated at 37°C for 5, 15, and 30 min in an incubation media [20] containing 1.5 mM AMP to provide an environment which would promote glycogenolysis [20]. No additional substrates were added to the incubation media. Sections incubated in phosphate buffer alone, as well as sections digested with 3.5% amylase, were used as negative and positive controls, respectively. RESULTS
The histopathological findings in the muscle biopsies from the horses with recurrent exertional rhabdomyolysis are summarized (Table 2). Extensive centrofascicular atrophy was found in the biopsies from horses 1 and 2 (Fig. 1). Necrosis of randomly scattered type 2A and 2B muscle fibers was evident in biopsies from several horses. Ultrastructurally, these fibers showed mild to severe myofibrillar lysis and degenerate mitochondria containing electron dense inclusions. In addition, scattered throughout many histological specimens were regenerating fibers which were small, basophilic and positively stained with alkaline phosphatase.
, ,,~ " t
\ ",-
~
i
* `'%`
'
"
¢,
-
.
i
/
1
/
.
Fig. I. Muscle biopsy of the semitendinosus muscle from horse 1 stained with hematoxylin and eosin. Note the extensive centrofascicular atrophy ( x 135).
Sections from horses with recurrent exertional rhabdomyolysis were more darkly stained with PAS (purple hue) than control sections (magenta hue). Electron microscopic examination revealed large accumulations of beta glycogen between myofibrils and under the sarcolemma. Single or multiple intensely PAS positive inclusions were present in a varying number of type 2A and 2B fibers in the gluteus, semitendinosus or triceps muscle of all horses (Table 2, Fig. 2) as well as macrophages in samples from horses 1 and 8. The staining characteristics o f PAS positive inclusions are summarized (Table 3). Many fibers with inclusions were grouped in a perifascicular location. Fibers with inclusions varied in size. Small fibers with inclusions frequently did not exhibit normal sarcoplasmic PAS staining (Fig. 2) while larger fibers tended to have normal PAS staining of the sarcoplasm surrounding the inclusions (Fig. 2). Electron microscopic examination revealed that the inclusions consisted of beta glycogen particles and arrays of short strands of fibrillar material (Fig. 3). In some fibers the accumulation of this material was so extensive that it disrupted the normal arrangement of the myofibrils.
Polysaccharide Storage Myopathy
Fig. 2. Periodic acid Schiff's stain of the gluteus muscle from horse 3. Note the subsarcolemmal distribution of inclusions in some fibers and the lack of a normal PAS positive staining background in the smaller fibers with extensive accumulation of inclusions ( x 350).
Membranous whorls were also found in areas adjacent to inclusions. A large number of fibers also contained subsarcolemma'l vacuoles in type 2 fibers which were unstained with PAS. These vacuoles were lightly eosinophilic with eosin (Fig. 4) and ultrastructural examination revealed that these vacuoles contained low density amorphous material, a sparse distribution of beta glycogen particles and degenerate mitochondria. The staining characteristics of these vacuoles are summarized (Table 3). The vacuoles were randomly located along the length of the fiber. The muscle biopsies from control horses showed no histopathological abnormalities. The mean muscle glycogen and protein concentrations and enzyme activities are summarized (Table 4). Muscle glycogen concentrations were significantly higher (p < 0.001) in muscle from horses with recurrent exertional rhabdomyolysis (range 647-1175 mmol kg-~ dw) than control horses (range 342-690). Notably, glycogen concentrations in samples shipped to the laboratory overnight were still high at 678 and 783 mmol kg-~. Protein concentrations and
355
the activities of hexokinase and acid maltase were similar between patient horses and controls. Enzyme activities were not diminished in the two shipped samples. The activity of branching enzyme could not be determined in either patient or control horses due to excessive background activity compared to human muscle samples. In retrospect, this was probably due to the high intramuscular glycogen concentrations in horse muscle compared to healthy human muscle [2 I]. Muscle from horses with recurrent exertional rhabdomyolysis were able to utilize the various substrates to produce lactate. However, the amount of lactate produced by horses with exertional rhabdomyolysis was less than controls (Fig. 5). When muscle sections were incubated in an anaerobic media or in amylase the staining intensity of the PAS positive inclusions was only slightly dimhaished in the horses with exertional rhabdomyolysis. The normal reticular PAS staining of muscle fibers disappeared in muscle sections incubated for 30 min in an anaerobic media in all control horses and all horses with recurrent exertional rhabdomyolysis except for horse 6. After 45 min of incubation in the media all reticular PAS stain was removed in horse 6. The muscle glycogen concentration in this horse was nearly twice the highest concentration in the control horses. DISCUSSION
The distinctive features of the horses with recurrent exertional rhabdomyolysis in this study were an elevated muscle glycogen cohtent accompanied by polysaccharide storage inclusions in type 2 fibers. Other studies have confirmed that horses susceptible to recurrent exertional rhabdomyolysis have high intramuscular glycogen concentrations. In 1932, Carlstrtm noted high muscle glycogen concentrations in work-horses that developed exertional rhabdomyolysis following a day of inactivity. He proposed that rhabdomyolysis was the result of an accumulation of intramuscular glycogen during a day of rest which, upon exercise, Was rapidly metabolized anaerobically causing a lactic acidosis which in turn caused myonecrosis [I]. Lactic acidosis, however, has not been demonstrated in this syndrome [6, 11]. The increases in serum CK following an exercise test coupled with the presence of numerous subsareolemmal vacuoles may indicate that repeated bouts of rhabdomyolysis occurred with exercise in these horses. We believe that the subsarcolemmal
356
S.J. VALBERG et al.
Fig. 3. Electron micrograph of several myofibriilar inclusions which stained positively with the periodic acid Schiff's stain from horse ! ( × 42,500). The inclusions consist ofbeta glycogen particles and arrays of filamentous material (insert × 74,000).
b
•
• :'tf
:4
Table 3. The histochemical staining characteristics of the subsarcolemmal vacuoles and the PAS positive inclusions in type 2A and 2B fibers from horses with recurrent exertional rhabdomyolysis Sareoplasmic changes Histochemical stains
Hematoxylin & eosin
•
"~'~
"~"~ ~...~\
Fig. 4. Numerous subsarcolemmal vacuoles in muscle fibers from horse 8 stained with hematoxylin-eosin. These vacuoles were not PAS positive and stained slightly with eosin ( x 350).
Modified trichrome Myosin ATPases pH 9.8 pH 4.5 pH 4.3 NADH-TR Esterase Acid phosphatase Alkaline phosphatase PAS PAS following amylase Lugols iodine Oil red O Myophosphorylaseiodine Toluidine blue Alcian blue Alcian blue following Hyaluronidase
Subsarcolemmal vacuoles
PAS positive inclusions
slightly eosinophilic grey
slightly eosinophilic light blue
us us us us us us us us us us us us
us us us us us us us magenta magenta brown us blue
us us us
us blue blue
us = unstained. NADH-TR = nicotinamide reductase. PAS = periodic acid Schiff.
adenine
dinucleotide
tetrazolium
Polysaecharide Storage Myopathy Table 4. The mean substrate concentrations and enzyme activities (4- S.D.) of horses with recurrent exertional rhabdomyolysis (RER) and control horses
Glycogen (retool kg-tdw) Protein g- 'dw) Hexokinase 0maol g-ldw rain-I)
Acid maltase (/maol g-t prot. rain -I)
RER horses mean ± S.D.
Controls mean 4- S.D.
752:t: 177"
5204- 102
4864- 40
454 4- 68
I 1.7 4-4.2
10.2 4- 2.2
3.1 4-1.3
3.54-1.8
*Significantly different from controls (p
096(}-8966/92 $5.00 + 0.00 ©1993 Pergamon Pros Ltd
Printed in Great Britain
POLYSACCHARIDE STORAGE MYOPATHY ASSOCIATED WITH RECURRENT EXERTIONAL RHABDOMYOLYSIS IN HORSES STEPHANIE J. VALBERG,*'[" GEORGE H . CARDINET
III,* GARY P. CARLSON~ a n d SALVATORE DIMAURC~
*Department of Anatomy and Cell Biology, and ~Department of Medicine, School of Veterinary Medicine, University of California, Davis, CA 95616; and §Neurological Institute, Columbia Presbyterian Medical Center, New York, NY, U.S.A.
(Received 27 May 1992, accepted 17 September 1992) Abstract--A polysaccharidestorage myopathy is described in nine Quarterhorses, Quarterhorse crossbreds, American Paints and Appaloosa horses which had a history of recurrent exertional rhabdomyolysis. Muscle biopsies were characterized by high muscle glycogen concentrations with up to 5% of type 2 muscle fibers containing inclusions which stained positively with the periodic acid Schiff (PAS) stain. The inclusions were classifiedas an acid mucopolysaccharide, based on their histochemicai staining characteristics. Ultrastructural studies revealed that the inclusions were composed of beta glycogen particles interspersed among arrays of filamentous material. In addition, many type 2 fibers contained multiple subsarcolemmal vacuoles. These vacuoles stained lightly with eosin and did not stain positively with PAS. Centrofascicular atrophy and necrosis of scattered type 2 fibers were present in biopsies from some horses. No glyco(geno)iytic enzyme deficiencies were identified using a biochemical screening test for anaerobic glycolysis. Attempts to measure branching enzyme activities in both affected and control samples were unsuccessful, employing methods developed for human muscle. The polysaccharide accumulation in these horses may represent a hereto yet undefined metabolic disorder of skeletal muscle.
Key words: Horse, muscle, rhabdomyolysis, polysaccharide, glycogen.
INTRODUCTION
Exertional rrryopathies are a common problem among working horses. At the turn of this century, recurrent exertional rhabdomyolysis was a common occurrence in draft horses, especially when they resumed exercise after a day of rest [1, 2]. This syndrome has been called Monday morning disease, azoturia, tying-up and paralytic myoglobinuria. Clinical signs occur shortly after exercise and include profuse sweating, stiffness, reluctance to move, myoglobinuria and in some cases recumbency and death. As race and pleasure horses have increased in popularity, a similar but often milder form of recurrent exertional rhabdomyolysis has frequently been reported [3-6]. In practice, the diagnosis of recurrent exertional rhabdomyolysis is made on the basis of clinical signs, myoglobinuria and increased activities of serum creatine kinase (CK), lactate
tAuthor to whom reprint requests should be sent.
dehydrogenase (LDH) and aspartate aminotransferase (AST) [7]. Muscle biopsies have not been routinely used to investigate the etiology of exertional rhabdomyolysis. In instances where muscle biopsies have been obtained, necrosis of type 2 fibers, increased glycogen concentrations and myofibrillar and mitochondrial degeneration have been described [5, 8-11]. Several etiologies for recurrent exertional rhabdomyolysis have been suggested including lactic acidosis, malignant hyperthermia, hypothyroidism, vitamin E and selenium deficiency, glycolytic enzyme abnormalities and electrolyte imbalances; however, definitive findings are lacking [12]. Since the diagnostic criteria for recurrent exertional rhabdomyolysis are only indications of muscle necrosis, it is likely that a variety of etiological factors may contribute to the development of the clinical syndrome of exertional., rhabdomyolysis. The purpose of this report is to describe the histochemical, biochemical and ultrastructural characteristics of skeletal muscle biopsies from nine horses which
351
352
S.J. V A L B E R G et al. Table I. The breed, sex, age, and maximum serum enzyme activities of horses with recurrent exertional rhabdomyolysis
Horse
Breed
Age at biopsy
Sex
Age of onset (yr)
(yr) I
Arab/Quarterhorse 2 Quarterhorse 3 Thoroughbred/ Quarterhor~ 4 Quarterhorse 5 Appaloosa 6 American Paint 7 Quarterhorse 8 Appaloosa 9 Quarterhorse Normal range:
Maximum serum enzyme activities CK AST LDH
(U I-')
(U I-')
(U I-')
G
5
4
8750*
13,124"
954
F F
10 and 13 9, 10 and 17
4 3
nd 1980
242 5454
453 559
F G F
3 4 and 9 4
2 2 3
nd 24,560 174,195
1965 >2400 >2400
925 nd >3200
F F F
4 3 8
2 2 5
3996 2661 * 200,000" 120-290
1088 3030* 12,609"
nd 2160" nd
140-400
160-410
F ~ female. G = gelding. • ~ 4 h after exercise test. nd = not determined.
had recurrent exertional rhabdomyolysis and an abnormal intramuscular accumulation of polysaccharide forming intensely stained PAS positive inclusions within type 2 fibers. HORSES
The horses presented in this report were selected from biopsies of trunk and limb muscles from a total of 116 horses presenting to the Veterinary Medical Teaching Hospital (VMTH) at the University of California, Davis, over a 12 yr period. Some samples were also received from referring veterinarians. Out of 116 horses, 36 horses presented with a history of exertional rhabdomyolysis and 80 samples came from horses afflicted with a variety of other neuromuscular diseases. The breed, sex and age distribution of the horses in this study are shown in Table 1. Females were more prevalent than males, 7 : 2. The diagnosis of recurrent exertional rhabdomyolysis was made, in each case, on the basis of the history, clinical signs of recurrent exertional rhabdomyolysis and elevated serum CK, L D H and AST activities (Table I). Evaluation of horses presented to the VMTH also included a complete blood count, serum chemistry analyses for the enzymes alkaline phosphatase, gamma glutamyltransferase and sorbitol dehydrogenase and the concentrations of albumin, total protein, bilirubin, glucose, creatinine, blood urea nitrogen, sodium, potassium, calcium, chloride and phosphorus. All affected horses except horse 6 had normal values for these parameters. The horses first developed clinical signs at a young age shortly after they began training (Table 1). Clinical signs were similar to the other
27 horses that were biopsied because of exertional rhabdomyolysis, however, the development of clinical signs at an early age and the recurrence of rhabdomyolysis were more common features in the horses with polysaccharide storage myopathy. Episodes of rhabdomyolysis were reported to be intermittent in some horses (horses l, 3, 5-7) and frequently repeatable after light exercise in others (horses 2, 4, 8, 9). Three horses were subjected to exercise testing while hospitalized and in each case serum CK activities were notably elevated 4 h following exercise (Table 1). The clinical signs exhibited during and after exercise included, in order of their appearance; profuse sweating, increased respiratory rate, stiffness which particularly affected the hind limbs, and reluctance to move. Clinical signs of rhabdomyolysis usually abated within several hours, however, after some episodes muscle pain was still evident for up to 2 days. Seven horses (2-5, 7-9) have continued to have repeated episodes. Horse 1 was euthanitized because of the frequent recurrence of recurrent exertional rhabdomyolysis and no gross pathological abnormalities were found at post mortem. Horse 2 subsequently died following a severe attack ofrhabdomyolysis; however, a post mortem examination was not performed. Horse 6 was admitted to the VMTH after becoming recumbent from a severe episode of rhabdomyolysis. The heart rate~ respiratory rate, rectal temperature and appetite were normal on admission. Abnormalities in serum biochem/stry included elevated activities for CK, AST, L D H (Table 1), sorbitol dehydrogenase 14 U I -~ (normal < 3) and high blood glucose concentrations 2.21 g 1-~ (r~rmal 0.6-1.22). Myoglobin
Polysaccharide
Storage
Myopathy
353
T a b l e 2. T h e h i s t o p a t h o l o g i c a l f i n d i n g s o f l i g h t m i c r o s c o p i c e x a m i n a t i o n o f h o r s e s w i t h r e c u r r e n t exertional rhabdomyolysis Horse
Muscle biopsied
M u s c l e fiber
Necrosis I
Semitendinosus
Sarcoplasmic
Atrophy
Subsarcolemmal vacuoles
positive inclusions
PAS
Triceps
+ . +
+
+
+
2 2R
Semitendinosus Semitendinosus
+ +
-
+ +
+ +
3 3R I 3R2
Superficial gluteus Superficial gluteus Medial gluteus
+ -
+ -
+ + +
+ + +
4
Semitendinosus
-
-
+
+
5
Superficial gluteus Semitendinosus Biceps femoris Vastus lateralis Triceps Medial gluteus
+ + + . + +
-
+ + +
+ + -
-
+ +
+ +
Superficial gluteus Semitendinosus Triceps Cardiac Liver
+ + . +
+ + -
+ + +
+ -
-
-
-
7
Medial gluteus
-
-
+
+
S
Medial gluteus
-
-
+
+
9
Medial gluteus
+
-
+
+
Vastus lateralis
5R 6
+
changes
.
.
.
.
.
.
+
+
.
.
.
R ffi b i o p s y r e p e a t e d . + = present. - = absent.
was present in the urine. Treatment included intravenous fluid therapy and phenylbutazone, with physical assistance to stand provided by a body sling. Antibiotic therapy was initiated following a febrile episode. Serum CK, AST and L D H activities and blood glucose concentrations returned to normal over a 3 week period, however, the horse rem_ained weak and developed marked atrophy of the skeletal musculature. The owners elected to euthanitize the mare. Post mortem findings included a severe generalized myopathy, chronic interstitial pneumonia, acute multifocal hepatitis and chronic focal pyelonephritis. A group of healthy horses were used as controls and included one gelding and eight mares (four Arabians, two Quarterhorses and three thoroughbreds). Their mean age was 8.6 yr (range 3-19 yr) and their serum CK and AST activities were within normal limits.
METHODS
Muscle biopsies (Table 2) were obtained using either open surgical techniques, a Bergstr6m biopsy needle or a modified Bergstr6m biopsy needle with a 5 mm outer diameter. All control samples were obtained percutaneously [13] from the gluteus medius muscle of unexercised horses on a similar ration. No standardization of diet or
exercise prior to biopsy was possible for patient horses. In several cases, muscle biopsies were repeated months to years following the initial biopsy in order to obtain a biopsy for biochemical analysis at a time when clinical rhabdomyolysis was not apparent (Tables 1 and 2). At post mortem, additional samples of several muscles were obtained from horses 1 and 6 and a cardiac and liver sample were obtained from horse 6, The biopsy specimens used for histopathological and histochemical examination were frozen in liquid freon cooled to -125"C with liquid nitrogen. Fresh frozen cryostat sections were then stained with a battery of tinctorial and histochemical staining procedures [14]. Specimens for electron microscopic examination from horses 1, 6-9 were prepared according to Tinling et al. [15]. All samples for biochemical analysis, except those of horses 2 and 4, were frozen shortly after biopsy in liquid nitrogen and stored at - 80"C for later analysis. The muscle biopsies from horses 2 and 4 were obtained by veterinarians in the field, shipped to the laboratory and were frozen within 24 h of collection. Muscle samples obtained at a time when minimal evidence of rhabdomyolysis was apparent histologically, were selected for biochemical analyses. Muscle glycogen, protein and hexokinase assays were performed on 1-2 mg
354
S.J. VALBERG et al.
samples of gluteus muscle or if not available semitendinosus muscle which had been freezedried and dissected free from blood, fat and connective tissue. Samples for glycogen analysis were boiled for 2 h in I M HCI and the glucose residues formed were measured fluorometrically according to Lowry and Passonneau [16]. Muscle samples for hexokinase and acid maltase enzyme analyses were homogenized ultrasonically in a 0.1 M potassium phosphate buffer solution (pH 7.3). The modified Lowry procedure was used to analyze protein content in muscle homogenates (Sigma Kit No. 690-A, St. Louis, MO). The activity of the enzyme hexokinase was measured fluorometrically according to Lowry and Passonneau [16]. Acid maltase activity was measured according to Engel and Gomez [17]. The activity of branching enzyme was measured according to Gambetti et al. [18]. A Student's ttest was used to compare glycogen, protein and enzyme activities between horses with recurrent exertional rhabdomyolysis and controls. The in vitro method of Layzer et al. [19] was modified for use as a biochemical screening test for anaerobic glyco(geno)lysis [20]. The glycogenolytic activity in situ was also assessed histochemically. Muscle sections 9/am thick were cut on a cryostat microtome and incubated for 10 min in 0.2% EDTA in 50 mM potassium phosphate buffer (pH 7.6). Subsequently, sections were incubated at 37°C for 5, 15, and 30 min in an incubation media [20] containing 1.5 mM AMP to provide an environment which would promote glycogenolysis [20]. No additional substrates were added to the incubation media. Sections incubated in phosphate buffer alone, as well as sections digested with 3.5% amylase, were used as negative and positive controls, respectively. RESULTS
The histopathological findings in the muscle biopsies from the horses with recurrent exertional rhabdomyolysis are summarized (Table 2). Extensive centrofascicular atrophy was found in the biopsies from horses 1 and 2 (Fig. 1). Necrosis of randomly scattered type 2A and 2B muscle fibers was evident in biopsies from several horses. Ultrastructurally, these fibers showed mild to severe myofibrillar lysis and degenerate mitochondria containing electron dense inclusions. In addition, scattered throughout many histological specimens were regenerating fibers which were small, basophilic and positively stained with alkaline phosphatase.
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Fig. I. Muscle biopsy of the semitendinosus muscle from horse 1 stained with hematoxylin and eosin. Note the extensive centrofascicular atrophy ( x 135).
Sections from horses with recurrent exertional rhabdomyolysis were more darkly stained with PAS (purple hue) than control sections (magenta hue). Electron microscopic examination revealed large accumulations of beta glycogen between myofibrils and under the sarcolemma. Single or multiple intensely PAS positive inclusions were present in a varying number of type 2A and 2B fibers in the gluteus, semitendinosus or triceps muscle of all horses (Table 2, Fig. 2) as well as macrophages in samples from horses 1 and 8. The staining characteristics o f PAS positive inclusions are summarized (Table 3). Many fibers with inclusions were grouped in a perifascicular location. Fibers with inclusions varied in size. Small fibers with inclusions frequently did not exhibit normal sarcoplasmic PAS staining (Fig. 2) while larger fibers tended to have normal PAS staining of the sarcoplasm surrounding the inclusions (Fig. 2). Electron microscopic examination revealed that the inclusions consisted of beta glycogen particles and arrays of short strands of fibrillar material (Fig. 3). In some fibers the accumulation of this material was so extensive that it disrupted the normal arrangement of the myofibrils.
Polysaccharide Storage Myopathy
Fig. 2. Periodic acid Schiff's stain of the gluteus muscle from horse 3. Note the subsarcolemmal distribution of inclusions in some fibers and the lack of a normal PAS positive staining background in the smaller fibers with extensive accumulation of inclusions ( x 350).
Membranous whorls were also found in areas adjacent to inclusions. A large number of fibers also contained subsarcolemma'l vacuoles in type 2 fibers which were unstained with PAS. These vacuoles were lightly eosinophilic with eosin (Fig. 4) and ultrastructural examination revealed that these vacuoles contained low density amorphous material, a sparse distribution of beta glycogen particles and degenerate mitochondria. The staining characteristics of these vacuoles are summarized (Table 3). The vacuoles were randomly located along the length of the fiber. The muscle biopsies from control horses showed no histopathological abnormalities. The mean muscle glycogen and protein concentrations and enzyme activities are summarized (Table 4). Muscle glycogen concentrations were significantly higher (p < 0.001) in muscle from horses with recurrent exertional rhabdomyolysis (range 647-1175 mmol kg-~ dw) than control horses (range 342-690). Notably, glycogen concentrations in samples shipped to the laboratory overnight were still high at 678 and 783 mmol kg-~. Protein concentrations and
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the activities of hexokinase and acid maltase were similar between patient horses and controls. Enzyme activities were not diminished in the two shipped samples. The activity of branching enzyme could not be determined in either patient or control horses due to excessive background activity compared to human muscle samples. In retrospect, this was probably due to the high intramuscular glycogen concentrations in horse muscle compared to healthy human muscle [2 I]. Muscle from horses with recurrent exertional rhabdomyolysis were able to utilize the various substrates to produce lactate. However, the amount of lactate produced by horses with exertional rhabdomyolysis was less than controls (Fig. 5). When muscle sections were incubated in an anaerobic media or in amylase the staining intensity of the PAS positive inclusions was only slightly dimhaished in the horses with exertional rhabdomyolysis. The normal reticular PAS staining of muscle fibers disappeared in muscle sections incubated for 30 min in an anaerobic media in all control horses and all horses with recurrent exertional rhabdomyolysis except for horse 6. After 45 min of incubation in the media all reticular PAS stain was removed in horse 6. The muscle glycogen concentration in this horse was nearly twice the highest concentration in the control horses. DISCUSSION
The distinctive features of the horses with recurrent exertional rhabdomyolysis in this study were an elevated muscle glycogen cohtent accompanied by polysaccharide storage inclusions in type 2 fibers. Other studies have confirmed that horses susceptible to recurrent exertional rhabdomyolysis have high intramuscular glycogen concentrations. In 1932, Carlstrtm noted high muscle glycogen concentrations in work-horses that developed exertional rhabdomyolysis following a day of inactivity. He proposed that rhabdomyolysis was the result of an accumulation of intramuscular glycogen during a day of rest which, upon exercise, Was rapidly metabolized anaerobically causing a lactic acidosis which in turn caused myonecrosis [I]. Lactic acidosis, however, has not been demonstrated in this syndrome [6, 11]. The increases in serum CK following an exercise test coupled with the presence of numerous subsareolemmal vacuoles may indicate that repeated bouts of rhabdomyolysis occurred with exercise in these horses. We believe that the subsarcolemmal
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Fig. 3. Electron micrograph of several myofibriilar inclusions which stained positively with the periodic acid Schiff's stain from horse ! ( × 42,500). The inclusions consist ofbeta glycogen particles and arrays of filamentous material (insert × 74,000).
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Table 3. The histochemical staining characteristics of the subsarcolemmal vacuoles and the PAS positive inclusions in type 2A and 2B fibers from horses with recurrent exertional rhabdomyolysis Sareoplasmic changes Histochemical stains
Hematoxylin & eosin
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Fig. 4. Numerous subsarcolemmal vacuoles in muscle fibers from horse 8 stained with hematoxylin-eosin. These vacuoles were not PAS positive and stained slightly with eosin ( x 350).
Modified trichrome Myosin ATPases pH 9.8 pH 4.5 pH 4.3 NADH-TR Esterase Acid phosphatase Alkaline phosphatase PAS PAS following amylase Lugols iodine Oil red O Myophosphorylaseiodine Toluidine blue Alcian blue Alcian blue following Hyaluronidase
Subsarcolemmal vacuoles
PAS positive inclusions
slightly eosinophilic grey
slightly eosinophilic light blue
us us us us us us us us us us us us
us us us us us us us magenta magenta brown us blue
us us us
us blue blue
us = unstained. NADH-TR = nicotinamide reductase. PAS = periodic acid Schiff.
adenine
dinucleotide
tetrazolium
Polysaecharide Storage Myopathy Table 4. The mean substrate concentrations and enzyme activities (4- S.D.) of horses with recurrent exertional rhabdomyolysis (RER) and control horses
Glycogen (retool kg-tdw) Protein g- 'dw) Hexokinase 0maol g-ldw rain-I)
Acid maltase (/maol g-t prot. rain -I)
RER horses mean ± S.D.
Controls mean 4- S.D.
752:t: 177"
5204- 102
4864- 40
454 4- 68
I 1.7 4-4.2
10.2 4- 2.2
3.1 4-1.3
3.54-1.8
*Significantly different from controls (p
