Dystrophin deficiency causes lethal muscle hypertrophy in cats

Journal of the Neurological Sciences, 110 (1992) 149-159 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00

149

JNS 03783

Dystrophin deficiency causes lethal muscle hypertrophy in cats F r 6 d 6 r i c P. G a s c h e n a, E r i c P. H o f f m a n b, j . R a f a e l M . G o r o s p e b, E l i z a b e t h W. U h l c, D a v i d F. S e n i o r a, G e o r g e H . C a r d i n e t , I I I d a n d L a u r i e K. P e a r c e a a Departmou of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, P.O. Box 100126, Gainesv~lle, FL 32610-0126, USA, h Departments of Molecular Genetics and Biochemistry, Human Genetics, and Pediatrics, BST W121!, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA, c Department of Comparative and Experimental Pathology, College of VeterinaryMedicine, Unil'ersity of Florida, P.O. Box 100145, Gainesville, FL 32610-0145, USA, and d Department of Anatomy, School of Veterinary Medicine, Unit,ersity of California, Davis, CA 95616, USA (Received 27 September, 1991) (Revised, received 10 January, 1992) (Accepted 19 January, 1992)

Key words': Dystrophin; Duchenne muscular dystrophy; Hypertrophic feline muscular dystrophy (HFMD); Neuromuscular disease

Summary Two 5-month-old male Domestic Shorthair iittermates showed general skeletal muscle hypertrophy, multifocal submucosal lingual calcification with lingual enlargement, and excessive salivation. Both cats had a reduced level of activity, walked with a stiff gait, and tended to "bunny hop" when they ran. These clinical features were similar to those of previously reported dystrophin-deficient cats. Using multiple dystrophin antibodies, we found that the cats described in this report also showed marked dystrophin deficiency. The histopathology was remarkable for hypertrophy and splitting of fibers, and progressive accumulation of calcium deposits within the muscle. There was little or no endomysiai fibrosis at 2 years of age. The natural history of dystrophin-deficiency in cats has not been described: both previous cats had been euthanized at 2 years of age prior to experiencing any life-threatening problems. At 6 months of age, one of the new cats developed megaesophagus because of severe progressive hypertrophy of the diaphragmatic muscles. The diaphragm completely occluded the esophagus, and the cat was euthanized for humane reasons. The second cat remained in good condition until age 18 months when it developed acute renal failure attributed to severe prolonged dehydration and hyperosmolality. The cat recovered after receiving supportive treatment but was unable to maintain fluid homeostasis. The ia~ufficient water intake was attributed to glossal hypertrophy and dysfunction. At age 2 years, the cat received regular subcutaneous injections of low-sodium fluids to maintain proper hydration. The clinical consequence of dystrophin deficiency in cats is lethal muscle hypertrophy. We have called the feline disease "hypertrophic feline muscular dystrophy" (HFMD).

Introduction Dystrophin-deficiency in humans results in Duchenne muscular dystrophy (DMD) (see Hoffman and Kunkel 1989; Rojas and Boffman 1991). DMD histopathology shows evidence of degeneration and regeneration of muscle fibers with progressive replacement of muscle by fibrous tissue, and with failure of

Correspondence to: Frederic P. Gaschen, Klinik fiir kleine Haustiere, Universifiit Bern, L[inggass-Str. 124&128, CH-3012 Bern, Switzerland.

muscle regeneration. Leg Weakness is present at age 4 or 5 years, and progressing muscle wasting leads to confinement to a wheelchair at age 11 years, and ventilatory support or death between 14 and 25 years. The high sporadic mutation rate has been attributed to the extremely large size of the dystrophin gene. To date, dystrophin-deficiency has also been documented in mice (Hoffman et al. 1987; Sicinski et al. 1990; Chapman et al. 1990), dogs (Cooper et al. 1988), and cats (Carpenter et al. 1989). Each of the dystrophin-deficient species expresses a different clinical disorder. Dystrophin, a large cytoskeletal protein, underlies the plasma membrane of all normal skeletal muscle fibers. It is also present in vascular and visceral smooth

150 muscle, a n d in the c e n t r a l n e r v o u s system ( H o f f m a n et al. 1988b; Byers et al. 1991). In skeletal muscle, dyst r o p h i n first a p p e a r s in fetal muscle fibers, w h e r e it r e m a i n s at a fairly c o n s t a n t level t h r o u g h a d u l t life ( P a t e l et al. 1988; B i e b e r et al. 1989). E v i d e n c e of tran sien t, localized p l a s m a m e m b r a n e instability in dystrophin-deficient fibers h a s b e e n a consistent f i n d i n g in b o t h m u r i n e a n d h u m a n diseases, early in d e v e l o p m e n t a n d in adult life ( B o d e n s t e i n e r a n d E n g e l 1978; R o w land 1980; Bertorini et al. 1984; M o r a n d i et al. 1990). T h e r e f o r e d y str o p h i n m a y stabilize the p l a s m a m e m b r a n e ( A r a h a t a et al. 1988; M e n k e a n d J o c k u s h 1991; H o f f m a n a n d G o r o s p e 1991). T h e n a t u r a l history o f d y s t r o p h i n deficiency in hum a n s , dogs, a n d mice has b e e n well c h a r a c t e r i z e d ( E n g e l 1986; V a l e n t i n e et al. 1988, 1 9 9 0 a ; C o u l t o n et al. 1988a, b). H o w e v e r , t h e previously r e p o r t e d two cat siblings ( C a r p e n t e r et al. 1989) did not e x p e r i e n c e any life-threatening p r o b l e m s b e f o r e being e u t h a n i z e d . W e have identified a n e w litter of d y s t r o p h i n - d e f i c i e n t cats w h o d e v e l o p e d potentially lethal c o m p l i c a t i o n s d u e to h y p e r t r o p h y of the d i a p h r a g m in o n e cat a n d o f the t o n g u e in the o th er .

Case reports

Two 5-month-old male Domestic Shorthair littermates were referred to the University of Florida Veterinary Medical Teaching Hospital for assessment of multifocal submucosal lingual calcification and muscle hypertrophy.Generalized muscle hypertrophy was first noticed when the kittens were about 3 months old. Ptyalism was present and the kittens' level of activity seemed reduced. Physical examination confirmed marked muscle hypertrophy involving all axial and appendicular muscles except those of the face in both kittens (Fig. I). The gait was stiff and they tended to "bunny hop" when they ran. Their tongues were enlarged and the tip often protruded from the mouth. Multiple white nodules were noticed at the lingual margins in both kittens (Fig, 2). Several complete blood counts (l for cat I and 3 for cat 2) were performed on each cat in the few weeks following initial presentation and appeared consistent with restraint-related stress and presence of geographically prevalent endoparasites. Serum biochemistry panels showed marked increases in creatine kinase (280-14784, normal 16-146 U/l), and moderately increased levels of aspartate aminotransferase (AST) (152-391, normal 5-30 U/l), alanine aminotran~ferase (ALT) (133-185, normal 19-61 U/l), and total cholesterol (179-242, normal 43-186 mg/dl). All other values were within normal limits for kittens of that age. Thoracic radiographs were normal. Abdominal radiographs and ultrasonography showed hepatosplenomegaly and mild peritoneal effusion in both cats, as described in detail elsewhere (Berry, C.R., F.P. Gaschen and N. Ackerman, submitted for publication). Electrocardiography performed on cat I revealed notching of the R wave. There appeared to be a slight increase in the left ventricular internal diameter on M-mode echocardiography, but normal values for young cats have not been established. Electromyographic (EMG) studies of axial and appendicular muscles of both cats showed bizarre high frequency discharges (also called complex repetitive discharges) interspersed with positive sharp waves in all muscles investigated. Motor nerve conduction velocity, assessed in the sciatic/tibial nerves, was normal. A second EMG was performed on cat 2 five months later with similar findings. Vastus lateralis muscle from both cats, as

Fig. 1. Generalized muscle hypertrophy. Cat 1 is shown at 5 months of age. There is generalized muscle hypertrophy and a tendency to keep the tip of the tongue protruding out of the mouth.

well as tongue from cat i, were biopsied under general anesthesia. Percussion myotonia ("dimpling") was observed in both cats while under isoflurane anesthesia. Cat I was seen again 3 weeks later for vomiting/regurgitation, excessive salivation and marked abdominal distention. Radiographs showed severe dilatation, megaesophagus and hepatomegaly. The diaphragm appeared irregularly shaped. An orogastric tube was placed; gas and 40-60 ml saliva were aspirated from the stomach. The owner was advised to feed many small meals a day and to maintain the cat in an upright position during and 15 rain after feeding. Four days later the signs recurred. The animal had lost nearly 20% of his body weight in the interval. An esophagogram revealed total lack of esophageal peristalsis. Food and barium remained in the esophagus and did not reach the stomach even though the animal was held upright for more than 10 rain. The signs were attributed to severe hypertrophy of the diaphragmatic musculature that impinged on and occluded the esophagus at the hiatus. Due to the grave prognosis, the kitten was euthanized. At necropsy there was severe generalized muscle hypertrophy and compression of the esophagus by a markedly thickened diaphragm (Fig. 3). Muscles of the tongue and larynx were also greatly enlarged. Numerous mineralized plaques were observed along the lateral margins of the tongue. The macroscopic appearance and the location of these lesions suggested that the calcifications may have been the result of repeated trauma of the markedly enlarged tongue against the teeth. There was thickening of both the right and left ventricular walls in the heart. Numerous small white mineralized plaques were seen on the endocardial surface of the left atrium and on intimai surface of the aorta above the semilunar valves. All other organs appeared normal.

151 Cat 2 remained essentially healthy for the next year. At 7 months of age, his serum tested negative for FeLV antigen and FIV antibodies. EKG showed notching of the R waves. Two-dimensional echocardiography revealed moderate increase in left ventricular wall thickness, papillary muscle hypertrophy, and chamber dilation. Contractility was normal. The echogenicity of papillary muscles and myocardium suggested the presence of multiple mineralization foci. The cat was presented again at 18 months of age for weight loss and lethargy. Severe dehydration and renomegaly were found on physical examination. The initial serum chemistry profile showed marked increases in blood urea nitrogen (220.4, normal 20-32 mg/dl), creatinine (9.7, normal 0.7-1.8 mg/dl), phosphorus (22.0, normal 2.9-8.2 mg/dl), sodium (171.5, normal 150-157 meq./dl) and potassium (7.3, normal 4.0-5.4 meq./dl). The hemogram was normal. The cat was reportedly producing small amounts of urine, but none could be obtained before treatment was started. Treatment of acute renal failure with intravenous fluids led to improvement in the condition of the cat. Serum biochemical abnormalities also improved over the following 5 days. A needle renal biopsy was taken under general anesthesia with ultrasonographic guidance. Nonspecific histopathologic changes were especially prominent in the medulla and consisted of moderate multifocal interstitial nephritis and edema. A few days later, the cat appeared clinically recovered. The serum biochemistry values progressively returned to normal over the following weeks. Following the episode of renal failure, cat 2 was seen for regular

Fig. 3. Severe muscular hypertrophy of the diaphragm leading to compression of the esophagus. Diaphragm muscle of cat 1 is shown at necropsy. The severe hypertrophy of the diaphragm had occluded the esophagus, leading to functional starvation. While the hypertrophy of the diaphragm was the lethal event in the cat, nearly all muscles showed marked hypertrophy. rechecks of renal function. He was fed a diet selected for low protein and low sodium content (Feline k/d, Hill's Pet Products, Topeka, KS). However, the serum osmolality was consistently increased (380.9 +31.3, normal 290-305 mosmol/kg) with high values for serum sodium and blood urea nitrogen (Na:181.5+ 12.0, normal 150-157 meq/I; BUN: 51.2_+28.5, normal 20-32 mg/dl), indicative of dehydration and prerenal azotemia secondary to pure water loss. Extremely concentrated urine was produced in response to the deficient water intake (urine osmolality 1740-2520 mosmol/kg). Although the cat appeared thirsty and tried to drink water, he seemed incapable of lapping efficiently, presumably because of dysfunction of the hypertrophied lingual muscles. He was administered regular subcutaneous injections of low sodium fluids (2.5% dextrose and 1/2 strength lactated Ringer's solution, Baxter Healthcare Corporation, Deerfield, IL). Dehydration with hyperosmolar syndrome recurred twice when subcutaneous fluid therapy was interrupted. Cat 2 is currently maintained with 3 weekly parenteral fluid treatments with low sodium fluids. Between the ages of 18 and 24 months, increases in serum CK (between 2261 and 32650, normal 59-527 IU/I) and AST (207_+ 149, normal 12-50 IU/I) and ALT (86.4_+29.1, normal 30-100 IU/l) were consistently present.

Materials and methods Fig. 2. Lingual nodules and glossal hypertrophy. Cat 2 is shown at 5 months of age. Multiple white nodules are visible on the margins of the tongue. Glossal hypertrophy is easily noticeable.

Histopathological analyses Muscle specimens studied included necropsy specimens from a 2-year-old normal laboratory cat, a biopsy

152 from cat 1 taken at 5 months of age and necropsy specimen taken at 6 months, a biopsy from cat 2 taken at 2 years, a muscle biopsy from a mdx mouse, and a muscle biopsy from a 2-month-old Golden Retriever with CXMD. All muscle specimens were from the vastus lateralis, except for the mdx mouse specimen which was from the gastrocnemius. Both normal and Duchenne dystrophy-affected human muscle biopsies were processed in parallel. Muscle biopsies and necropsy specimens were flash-frozen and cryosections stained using standard techniques. Calcium was localized with the Von Kossa method (Thompson 1966), with alizarin red S (Thompson 1966), and with glyoxal-bis(2-hydroxyanilXGBHA) (Kashiwa and Atkinson 1963), as modified by Bodensteiner and Engel (1978) and Cornelio and Dones (1984). The fiber size distribution for 2-year-old normal cat vastus lateralis and age-matched cat 2 vastus lateralis were determined by measuring the least diameter of 118 fibers from photographs of H + E stained cryosections.

Dystrophin analyses Cryosections were processed for dystrophin immunofluorescence using affinity column-purified sheep IgG directed against the amino terminus (1-2a; Koenig and Kunkel 1990), central rod domain (30 kDa and 60 kDa; Hoffman et al. 1987) or carboxyl-terminus (dl0; Koenig and Kunkel 1990). The antibodies were diluted to 4 ~ g / m l , and anti-trpE antibodies absorbed by incubation with 250 /zg of insoluble bacterial trpE protein for 10 min at room temperature, followed by centrifugation and discarding of the pellet. Antibody incubations and washings were done as previously described (Bonilla et al. 1988; Hoffman et al. 1990). Visualization of fluorescent immune complexes was done on a Nikon FXA microscope using 10 x objective. All photographic exposures were kept at identical times in both the control and experimental biopsies. Dystrophin immunoblotting was done basically as previously described (Hoffman et ai. 1988a). The amount of dystrophin was densitometrically quantitated from immunoblots for the necropsied cat muscle,

Fig. 4. Comparison of muscle histologyof normal cat (panel A), cat 2 (panel B), and cat I (panel C). H + E stained cryosections of vastus lateralis muscle necropsy (panels A, C) or biopsy (panel B) specimens are shown. Panels A and B are from age-matched cats (2-yearold), while panel 3 is from cat I at the time of necropsy (6 months). Panel B shows the numerous foci of mineralization, wide variation in fiber size with markedly hypertrophied fibers, and central nuclei characteristic of the HFMD histopathology at 2 years. Minimal endomysial fibrosis is seen. Panel C shows the more variable pathology of cat I at necropsy, with focal connective tissue proliferation. Foci of mineralization were less frequent in the younger cat. Bar = 500/zm.

153 and normal cat muscle relative to human muscle as follows. Immunobiots were subjected to 2-dimensional reflectance densitometry (BioRad Model 620 Video Densitometer), followed by 2-D to 1-D conversion of the digitized data over the complete areas of the dystrophin band at 400 kDa. The ratio of peak areas of the experimental biopsy relative to the two adjacent controls was calculated. The post-transfer Coomasie blue stained acrylamide gels corresponding to the quantitated blots were dried between sheets of dialysis membrane, and subjected to 2-dimensional transmission densitometry of the myosin heavy chain protein, followed by 2-D to 1-D conversion of the digital information as above. Again, a ratio of experimental to controls for the myosin heavy chain protein was calculated. Finally, the relative percentage of dystrophin in each experimental biopsy was adjusted for the muscle protein content of the lane by dividing the relative percentages of dystrophin from the immunoblot by the myosin heavy chain ratio from the post-transfer gel. The lanes quantitated were those shown in Fig. 8.

Results

Histopathological analysis. There was variation in fiber size and increased central nucleation in all biopsy and necropsy specimens (Fig. 4). The most remarkable feature was hypertrophy of individual myofibers. Serial sections showed much splitting of the hypertrophic fibers; one hypertrophied fiber often became 3 fibers of average size in adjacent sections. The myofiber hypertrophy and splitting seemed to account for the gross muscular hypertrophy. There were also small, basophilic regenerating fibers.

Fiber size distribution 25

Fig. 6. Focal fibrosis with failed myofiber regeneration in cat 1. Two different areas are shown of the necropsied vastus lateralis of cat I at 6 months of age. Foci of marked fibrosis containing small myofibers which appeared to have aborted attempts at regeneration. These isolated foci resemble human late-stage Duchenne muscular dystrophy histopathology. A single calcification is seen in panel B. Bar -- 500 /zm.

• Normal 20

•t

15

Number Fibers

10

-

5-

,I I~ -1

22

II

I

IJ,. " "

I

I

I

110

I

I

I

I

I

~'~0

I

I

[ "--T'-"T"

I

.

I

.

1

330

.

I

|a

iI

. I

I

I

I

I

i\i

55O66O

Least Fiber Diameter~m)

Fig. 5. Histogram of myofiber least diameter (n = 118) for cat 2 at 2

years of age (shaded bars), and an age-matched normal cat (solid bars).

The hypertrophic and regenerating fibers led to a broad size distribution of fibers (Fig. 5). In the necropsy specimen of cat 1 at 6 months, there were a few focal areas of endomysial fibrosis (Fig. 6), but these were not evident in the biopsy of cat 2 at 2 years of age. The fibrotic areas in cat 1 contained small angulated fibers (Fig. 6) that were often not basophilic and had peripheral nuclei, appearing atrophic rather

154 than regenerating. This was attributed to failed regeneration in the fibrotic areas. Particularly striking were the distinct foci of mineralization (Fig. 4). These deposits reacted with histochemical stains for calcium (Fig. 7). They were surrounded by many muscle fibers that did not show markedly increased intracellular calcium. The loci seemed to replace individual fibers, and were more numerous in the biopsy of cat 2 at 2 years (Fig. 4, panel B) than in the necropsy muscle sample from cat 1 at 6 months (Fig. 4, panel C; Fig. 6). Foci of calcium mineralization were also seen in the mdx mouse (Fig. 7) and cxmd dog (not shown) muscle, but not in human Duchenne dystrophy (not shown). The mineralization was less prominent in the mouse and dog than in the cat.

Dystrophin analyses Immunoblot analysis of necropsy muscle from cat 1 and from a muscle biopsy of cat 2 showed marked dystrophin deficiency (Fig. 8). The amount of dystrophin present in normal cat, cat 1, and normal human muscle was quantitated by densitometry from triplicate samples. The dystrophin content of cat 1 was 4.9 + 4.8% of that seen in normal cat muscle. Normal cat muscle contained 41 + 12.8% of the dystrophin

present in human muscle when normalized for myosin heavy chain content. Thus, cat 1 had 2% of the dystrophin seen in normal human muscle. The immunoblot results from cat 2 were similar, but were not quantified. In normal cat (Figure 9, panels A, B, C) and normal human muscle (not shown) all antibodies showed bright and continuous immunolabeling at the plasma membrane of all muscle fibers. In muscle from cat 2, the amino-terminal antibody showed a variable immunostaining pattern, with some fibers showing relatively bright and continuous immunofluorescence and others appearing partly positive or negative (Fig. 9, panel D). The immunostaining in cat 2 was largely negative with both the 30 kDa and dl0 antibodies (Fig. 9, panel E, F). This pattern was recapitulated with both the original Boston cat and the CXMD dog (Fig. 9, panels G through L): the amino-terminal antibody stained variably positive, while the central and carboxyl-terminai domains were largely negative. All samples showed some immunostaining of connective tissue, which was assumed to be non-specific. The dog showed a particularly intense and consistent immunostaining with the amino-terminal antibody (Fig. 9, panel J). Immunofluorescence analysis of cat 1 trapezius (necropsy) wasdone with the amino-terminal 60 kDa

,i ¸'

tt

QI tm

i

B

Fig. 7. Calcium deposits in dystrophin-deficient cat and mouse muscle. Foci of mineralization Stained with two calcium stains in serial sections from dystrophin-deficient HFMD cat muscle (panels A, B) and dystrophin-deficient mdx mouse muscle (panels C, D) are shown. Panels A, C are stained with GBHA, and panels B, D with alizarin Red S. The loci of mineralization seen by H + E (Fig. 4) stain positively with all calcium stains, and also with the yon Kossa method (not shown). The foci are segmental, showing a limited longitudinal distribution.

155

i

DYSTROPHIN

. ~ - 4 2 7 kda

180

116 -<--84 •< -

58

.9(-- 4 8

MYOSIN

Fig. 8. Dystrophin immunoblot analysis in HFMD cat muscle. Solubilized muscle from a human with a disorder unrelated to Duchenne/ Becker dystrophy (normal human), normal cat muscle, and muscle from cat 1 (HFMD) is shown. The HFMD cat shows marked dystrophin deficiency. Quantitation of the above blot showed that the HFMD cat muscle contains 3% normal levels of dystrophin. The myosin heavy chain protein is also shown to indicate the amount of muscle protein loaded in each lane.

and carboxyl-terminal d l0 antibodies. The 60 kDa antibody was partially positive around most fibers (data not shown), and appeared qualitatively similar to the amino terminal 1-2a antibody results with cat 2 (Fig. 9). The all0 antibody results with cat 1 were qualitatively similar to the dl0 results with cat 2 (data not shown).

Discussion

Muscular dystrophy-like syndromes have been described in a few cats (Hulland 1970; Vos et al. 1986). Carpenter et al. (1989) documented dystrophin deficiency in littermate male kittens from Nantucket Island off the coast of Massachusetts. The male cats described here arose from a litter found in a supermarket parking lot in Gainesville, Florida. They are presumably unrelated to the previously reported cats. However, the new cats share the same genetic (X-linked), biochemical, histopathological and clinical features. They proba-

bly represent different alleles of the same nosological entity. Clinically, we have documented the potentially fatal outcome of muscular hypertrophy induced by dystrophin-deficiency in cats. In one animal, diaphragmatic hypertrophy led to complete occlusion of the esophagus. In the second cat, hypertrophy of the tongue prevented efficient fluid intake, resulting in severe dehydration, hyperosmolar syndrome, and acute renal failure. Based on the clinical features of the disease in cats, we propose the name "hypertrophic feline muscular dystrophy" (HFMD). Increases in the activity of serum CK and transaminases (AST, ALT) were comparable to those seen in DMD. Release of soluble cytoplasmic enzymes from myofibers may arise from either myofiber necrosis or leakage of cytoplasm due to generalized instability of the myofiber plasma membrane (Rowland 1980; Hoffman and Gorospe 1991). The paucity of overt necrosis in the 2-year-old cat, despite CK levels of 2261-32 650 IU/l (normal 59-527 IU/I) suggests that sublethal, transient leakage of muscle fibers is responsible for the high serum levels of sarcoplasmic enzymes. While the original Boston cats also had marked increases in serum ALT, these were thought to be of liver origin: the cats showed severe centrilobular hepathocellular disease attributed to congestive heart failure. The necropsied cat in our study showed no hepatic lesions, therefore the increased ALT serum level were likely of muscle origin. This conclusion is in agreement with studies of CXMD in Golden Retrievers (Valentine et al. 1990b). Histologically we documented marked hypertrophy of individual muscle fibers in cats. Myofiber hypertrophy is also characteristic of human Duchenne muscular dystrophy and CXMD muscle. It has been considered "compensatory hypertrophy", presuming that functional myofibers compensate for the loss of neighboring myofibers through hypertrophy. This hypothesis does not seem consistent with our observations in the HFMD cats, or reports on the mdx mouse. There is little or no muscle fiber loss or evident weakness in either cat or mouse, yet there is considerable muscular hypertrophy (Anderson et al. 1988; Coulton et al. 1988a, b; present study). In normal muscle, exercise results in CK elevations and subsequent muscular hypertrophy (Driessen-Kletter et al. 1990). Exercise may induce transient breaches of the myofiber membrane that result in the observed efflux of CK into the serum. Effiux of large proteins is most likely accompanied by influx of extracellular elements into the myofiber. This influx may signal the physiological adaptive response of myofiber hypertrophy. In dystrophin-deficient muscle, transient breaches of the membrane are a consequence of the primary biochemical defect but might simulate the effects of exercise at the cellular level. Thus, transient leakage may erroneously signal myofiber hyper-

156

(A)

1- 2a

30 k d

d 10

NORMAL CAT

CAT2 (HFMD)

Fig. 9. Dystrophin immunofiuorescence in muscle from normal cat, cat 2 (HFMD), one of the original Boston cats (HFMD), and a biopsy from an affected Golden Retriever dog model of Duchenne dystrophy (CXMD). Arrows indicate the same fiber in adjacent cryosections. Antibodies directed against the amino-terminus (l-2a), central rod domain (30 kDa) and the carboxylterminal domains (dl0) were used for fluorescent immunostaining of dystrophin in adjacent sections. All experiments were done in parallel, and were photographed and printed using identical exposure times. The same myofiber~ ~re ~hown for the 3 cat muscles. Normal cat muscle (panels A, B, C) shows intense peripheral immunostaining on all muscle fibers with all three antibodies. Both the HFMD cats (panels D-.I) and the CXMD dog (panels .I, K, L) show positive immunostaining with the amino-terminal antibody, but are generally negative with the central and carboxyl-terminai antibodies. Isolated dystrophin-positive fibers ("revertants'; Hoffman et al. 1990) are seen in the dog muscle with the 30 kDa and dl0 antibodies. Bar = 500/zm. trophy. In dystrophin-deficient humans and dogs, the myofiber leakage also may signal wound repair mechanisms, leading to endomysial fibrosis followed by muscle wasting and death ( D ' A m o r e 1990; Hoffman and Gorospe 1991). Apparently, myofiber leakage in the dystrophin-deficient cat and mouse myofibers does not similarly induce the fibrotic replacement of muscle,

thereby allowing the successful regeneration and continued hypertrophy of the muscle. A n additional histopathological finding in our cats was the multifocal calcification in the muscle. This feature was also present in the other litter of H F M D cats (Carpenter et al. 1989). In the new cats, the calcium deposits seemed to accumulate, they were

157 (B)

1- 2a

3 0 kd

d 10

BOSTON CAT (HFMD)

DOG (CXMD)

Fig. 9. (continued).

much more prevalent at 2 years than at 6 months. While calcification is a frequent cellular reaction to repeated trauma in many tissues in most mammals, muscular calcification is not seen in human Duchenne dystrophy. Biochemically, by analogy to dystrophin studies in large numbers of DMD patients (Hoffman et al., 1988a, b; Nicholson et al. 1990), the degree of dystrophin deficiency found in the HFMD cats corresponds to the most severe DMD phenotype in humans. The low amount of dystrophin found in normal cat

muscle (about half the amount present in normal human muscle) may reflect a true quantitative difference in dystrophin, a difference in the amount of solubility of myosin heavy chain used as reference standard, or a difference in the specificity of the antibody used (antimouse 30 kDa dystrophin) for reline'vs, human muscle. We compared dystrophin immunostaining patterns in our litter with the previous Boston litter (Carpenter et al. 1989) and with the CXMD dog (Cooper et al. 1988) (Fig. 9). All three disorders stain with aminoterminal antibodies for dystrophiv, but are largely negative with

158

antibodies directed against the central rod domain and the carboxyl-terminal domains. There are two likely explanations for the lmmunoreactlwty with aminoterminal antibodies. The animals may all produce truncated dystrophin molecules as seen in a few human Duchenne patients (Bulman et al. 1991; Lupski et al. 1991). Alternatively, the strong amino acid homology between the amino-terminus of dystrophin and other proteins may result in cross-reactivity between the dystrophin antisera and other membrane proteins. By immunoblotting we found a low level (5%) of 400 kDa protein in the cats in this study (Fig. 8), and the CXMD dog (not shown). This small amount of 'normal' dystrophin probably corresponds to the rare dystrophin-positive fibers seen by immunofluorescence using carboxyl-terminal antibodies (Fig. 9, panels, K, L). •

•

•

These striking dystrophin-positive fibers p r o b a b l y represent genetic reversion of isolated m y o n u c l e i (Hoffm a n et al., 1990). Alternatively, m a n y anti-dystrophin antisera show some cross-reactivity to the "dystrophinrelated protein" ( D R P ) encoded on c h r o m o s o m e 6 ( H o f f m a n et al. 1989b; Love et al. 1989; K h u r a n a et al. 1990), and the small a m o u n t of 400 k D a p r o t e i n seen could represent this related protein. In conclusion, the clinical phenotype o f dystrophin deficiency in cats stands in marked contrast to the progressive muscle wasting characteristic of h u m a n D u c h e n n e dystrophy. Comparisons with the other dystrophin-deficient species reinforces the hypothesis of generalized muscle m e m b r a n e instability as the primary cellular defect, and misdirected proliferation of fibrotic tissue as the key secondary defect leading to muscle wasting and d e a t h in D u c h e n n e dystrophy. Further, this cat model provides an excellent opportunity to study the early cellular events leading to dystrophindeficiency-induced muscle fiber h y p e r t r o p h y and necrosis without the complicating variable of progressive fibrosis seen in both h u m a n s and dogs.

Acknowledgements Supported by grants to EPH from the March of Dimes, Muscular Dystrophy Association, and the National Institutes of Health. College of Veterinary Medicine, University of Florida, Journal Series #269.

References Anderson, J.E., B.H. Bressler and W.K. Ovalle {1988) Functional regeneration in the hindlimb skeletal muscle of the mdx mouse. J. Muscle Res. Motil., 9: 499-515. Arahata, K., S. lshiura, T. Ishiguro, T. Tsukahara, Y. Suhara, C. Eguchi, T. Ishihara, !. Nonaka, E. Ozawa and H. Sugita (1988) lmmunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide. Nature, 333: 861-863. Bertorini, T.E., F. Cornelio, S.K. Bhattacharya, G.M.A. Palmieri, I. Dones, F. Dworzak and B. Brambati (1984) Calcium and magne-

slum content in fetuses at risk and prenecrotic Duchenne muscular dystrophy. Neurology, 34: 1436-1440. Bieber, F.R., E.P. Hoffman and J. Amos (1989) Dystrophin analysis in Duchenne muscular dystrophy: Use in fetal diagnosis and in genetic counseling. Am. J. Hum. Genet., 45: 362-367. Bodensteiner, J.B. and A.G. Engel (1978) Intracellular calcium accumulation in Duchenne dystrophy and other myopathies: a study of 567,000 muscle fibers in 114 biopsies. Neurology, 28: 439-446. Bonilla, E., C.E. Samitt, A.F. Miranda, A.P. Hays, G. Salviati, S. DiMauro, L.M. Kunkel, E.P. Hoffman and L.P. Rowland (1988) Duchenne muscular dystrophy: deficiency of dystrophin at the muscle cell surface. Cell. 54: 447-452. Bulman, D.E., E.G. Murphy, E.E. Zubrzycka-Gaarn, R.G. Worton and P.N. Ray (1991) Differentiation of Duchenne and Becket muscular dystrophy phenotypes with amino- and carboxy-terminal antisera specific for dystrophin. Am. J. Hum. Genet. 48: 295-304. Byers, T.J., L.M. Kunkel and S.C. Watkins. (1991). The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle. J. Cell Biol., 115: 411-421. Carpenter, J.L., E.P. Hoffman, F.C.A. Romanul, L.M. Kunkel, R.K. Rosales, N.S.F. Ma, J.J. Dasbach, J.F. Rae, F.M. Moore, M.B. McAfee and L.K. Pearce (1989) Feline muscular dystrophy with dystrophin deficiency. Am. J. Pathol., 135: 909-919. Chapman, V.M., D.R. Miller, D. Armstrong, and C.T. Caskey (1989) Recovery of induced mutations for X chromosome-linked muscular dystrophy in mice. Proc. Natl. Acad. Sci. USA, 86: 1292-1296. Cooper, B.J., N.J. Winand, H. Stedman, B.A. Valentine, E.P. Hoffman, L.M. Kunkel, M.O. Scott, K.H. Fischbeck, J.N. Kornegay, R.H. Avery, J.R. Williams, R.D. Schmickel and J.E. Syivester (1988). The homologue of the Duchenne locus is defective in X-linked muscular dystrophy of dogs. Nature, 334: 154-156. Cornelio, F. and I. Dones (1984) Muscle fiber degeneration and necrosis in Muscular Dystrophy and other muscle diseases: cyto- ' chemical and immunocytochemical data. Ann. Neurol., 16: 694701. Coulton, G.R., J.E. Morgan, T.A. Partridge and J.C. Sloper (1988a) The mdx mouse skeletal muscle myopathy: !. A histological, morphometric and biochemical investigation. Neuropathol. Appl. Neurobiol., 14: 53-70. Coulton, G.R., M.A. Curtin, J.E. Morgan and T.A. Partridge (1988b) The mdx skeletal muscle myopathy: 11. Contractile properties. Neuropathol. Appl, Neurobiol., 14: 299-314. D'Am:,re, P.A. (1990) Modes of FGF release in vivo and in vitro. Cat~er Metastasis Rev., 9: 227-238. Driessen-Kletter, M.F., G.J. Amelink, P.R. Bar and J. van Gijn (1990) Myoglobinis a sensitive marker of increased muscle membrane vulnerability. J. Neurol., 237: 234-238. Engel, A.G. (1986) Duchenne Dystrophy• In: A.G. Engel and B.Q. Banker (Eds.), Myology: Basic and Clinical, McGraw-Hill, New York, pp. 1185-1240. Hoffman, E.P., R.H. Brown and L.M. Kunkel (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell, 51: 919-928. Hoffman, E.P., K.H. Fischbeck, R.H. Brown, H. Johnson, R. Medori, J.D. Loike, J.B. Harris, R. Waterston, M. Brooke, L. Specht, W. Kupsky, J. Chamberlain, C.T. Caskey, F. Shapiro and L.M. Kunkel (1988a) Dystrophin characterization in muscle biopsies from Duchenne and Becker muscular dystrophy patients. New Engi. J. Med., 318: 1363-1368. Hoffman, E.P., M. Hudecki, P. Rosenberg, C. Pollina and L.M. Kunkel (1988b) Cell and fiber-type distribution of dystrophin. Neuron, 1: 411-420. Hoffman, E.P. and L.M. Kunkel (1989) Dystrophin abnormalities in Duchenne/Becker muscular dystrophy. Neuron, 2: 1019-1029. Hoffman, E.P., L.M. Kunkel, C. Angelini, A. Clarke, M. Johnson and J.B. Harris (1989a) Improved diagnosis of Becker muscular dystrophy by dystrophin testing. Neurology, 39: 1011-1017.

159 Hoffman, E.P., A.H. Beggs, M. Koenig, L.M. Kunkel and C. Angelini (1989b) Cross-reactive protein in Duchenne muscle. Lancet, ii: 1211-1212. Hoffman, E.P., J.E. Morgan, S.C. Watkins, H.S. Slayter and T.A. Partridge (1990) Somatic reversion/suppression of the mouse mdx phenotype in vivo. J. Neurol. Sci., 99: 9-25. Hoffman, E,P, ~.nd J.R. Gorospe (1991) The animal models of Duuhenne r~scular dystrophy: windows on the pathophysiologieal consequences of dystrophin deficiency. In: J. Morrow and M. Mooseker (Eds.), Topics in Membranes, Vol. 38, Academic Press, New York, pp. 113-154. Hulland, T.J. (1970) Muscle. In: K.V.F. Jubb and P.C. Kennedy (Eds.), Pathology of Domestic Animals, Academic Press, New York, pp. 453-494. Kashiwa, H.K. and W.B. Atkinson (1963) The applicability of a new Schiff base, glyoxal-bis-(2-hydroxyanil) for the cytochemical localization of ionic calcium. J. Histochem. Cytochem., 11:258-264. Khurana, T.S., E.P. Hoffman and L.M. Kunkel (1990) Identification of a chromosome 6 encoded dystrophin related protein. J. Biol. Chem., 265: 16717-16720. Koenig, M. and L.M. Kunkel (1990) Detailed analysis of the repeat domain of dystrophin reveals 4 potential hinge regions that may confer flexibility. J. Biol. Chem., 265: 4560-4566. Love, D.R., D.F. Hill, G. Dickson, N.K. Spurr, B.C. Byth, R.F. Marsden, F.S. Walsh, Y.H. Edwards and K.E. Davies (1989) An autosomal transcript in skeletal muscle with homology to dystrophin. Nature, 339: 55-58. Lupski, J.R., C.A. Garcia, H.Y. Zoghbi, E.P. Hoffman and R.G. Fenwick (1991) Discordance of muscular dystrophy in monozygotic female twins: evidence supporting asymmetric splitting of the inner cell mass in a manifesting carrier on Duchenne dystrophy. Am. J. Med. Genet., 40: 354-364. Menke, A. and H. Jockush (1991) Decreased osmotic stability of dystrophin-less muscle cells from the mcLx mouse. Nature, 349: 69-71. Morandi, L., M. Mora, E. Gussoni, S. Tedeschi and F. Cornelio

(1990) Dystrophin analysis in Duchenne and Becker muscular dystrophy carriers: correlation with intracellular calcium and albumin. Ann. Neurol., 28: 674-679. Nicholson, L.V.B., K. Davison, M.A. Johnson, C.R. Slater, C. Young, S. Bhattacharya, D. Gardner-Medwin and J.B. Harris (1989) Dystrophin in skeletal muscle. II. Immunoreactivity in patients with Xp21 muscular dystrophy. J. NeuroI.Sci., 94: 137-146. Partridge, T. (1991) Animal models of muscular dystrophy: what can they teach us? Appl. Neurobiol. Neuropath., in press. Patel, K., T. Volt, M.J. Dunn, P.N. Strong and V. Dubowitz (1988) Dystrophin and nebulin in the muscular dystrophies. J. Neurol. Sci., 87: 315-326. Rojas, C.V. and E.P. Hoffman (1991) Recent advances in dystrophin research. Curr. Opinion Neurobiol., 1: 420-429. Rowland, L.P. (1980) Biochemistry of muscle membranes in Duchenne muscular dystrophy. Muscle Nerve, 3: 3-20. Sicinski, P., Y. Geng, A.S. Ryder-Cook, E.A. Barnard, M.G. Darlison and P.J. Barnard (1989) The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science, 244: 1578-1580. Thompson, S.W. (1966) Selected Histochemical and Histopathological Methods, Charles C Thomas, Springfield, IL. Valentine, B.A., B.J. Cooper, A. de Lahunta, R. O'Quinn and J.T. Blue (1988) Canine X-linked muscular dystrophy; An animal model of Duchenne muscular dystrophy: clinical studies. J. Neurol. Sci., 88: 69-81. Valentine, B.A., B.J. Cooper, J.F. Cummings and A. de Lahunta (1990a) Canine X-linked muscular dystrophy: morphologic lesions. J. Neurol. Sci., 97: 1-23. Valentine, B.A., F.T. Blue, S.M. Shelley and B.J. Cooper (1990b) Increased serum alanine aminotransferase activity associated with muscle necrosis in the dog. J. Vet. Int. Med., 4: 140-143. Vos, J.H., J.S. Van der Linde-Sipman and S.A. Goedegebuurer (1986) Dystrophy-like myopathy in the cat. J. Comp. Pathol, 96: 335-341.