Mitochondrial Myopathy in a German Shepherd Dog
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Vet Pathol 40:507–511 (2003)
Mitochondrial Myopathy in a German Shepherd Dog
O. PACIELLO, P. MAIOLINO, G. FATONE, AND S. PAPPARELLA
Dipartimento di Patologia e Sanità Animale–Settore Anatomia Patologica (OP, PM, SP) and Dipartimento di Scienze
Cliniche Veterinarie–Sezione di Clinica Chirurgica (GF), Facoltà di Medicina Veterinaria, Università degli Studi di
Napoli Federico II, Naples, Italy
Abstract. A 9-month-old male German Shepherd dog was referred for evaluation of progressive exercise
intolerance. Clinical examination revealed a stiff, stilted gait and marked atrophy and hypotonia of skeletal
muscle. The dog had raised creatine kinase (181 U/liter), lactate dehydrogenase (510 U/liter), and aspartate
aminotransferase (123.6 U/liter) levels, suggesting a muscle disease. Histochemical evaluation of muscle bi-
opsies revealed the presence of subsarcolemmal oxidative activity, reduced nicotinamide adenine dinucleotide,
and succinate dehydrogenase, and the absence of cytochrome oxidase activity. Ragged red fibers were dem-
onstrated with Gomori trichrome stain. Ultrastructural examination of the muscle confirmed the presence of
subsarcolemmal accumulations of mitochondria and morphologically atypical mitochondria.
Key words: Dogs; histochemistry; mitochondrial myopathy.
Introduction in truncal and limb muscles, myalgia, and gait abnor-
Mitochondrial disorders are a heterogeneous group malities (stiff gait, thoracolumbar kyphosis, and bun-
of diseases with multisystem presentation.10,13 Because ny-hopping in pelvic limbs).
many metabolic processes take place in the mitochon- Orthopedic and neurologic examinations were un-
dria, the alteration of adenosine triphosphate produc- remarkable. Blood samples were taken for hematologic
tion due to diminished activity of the mitochondrial and serologic examination.
respiratory chain is the causative factor of the majority A diagnosis of skeletal myopathy of unknown eti-
of these diseases.5 The respiratory chain is embedded ology was made, and antibiotics (amoxycillin-clavu-
in the inner mitochondrial membrane and is composed lanic acid, 20 mg/kg bid po) and FANS (aspirin, 25
of four multi-subunit enzyme complexes encoded by mg/kg qid po) were given while waiting for results of
mitochondrial deoxyribonucleic acid (mtDNA) and the analysis. Five days later, the dog showed no im-
nuclear DNA. Therefore, mitochondrial diseases may provement.
arise from mutations in either mitochondrial or nuclear Routine hematology revealed no abnormalities. Bio-
genomes. Mutations in mtDNA are transmitted by ma- chemistry showed increased levels of creatine kinase
ternal inheritance and are generally heteroplasmic, that at 37 C (181 U/liter), lactate dehydrogenase (510 U/
is, wild-type and mutant mtDNAs coexist in the same liter), and aspartate aminotransferase (123.6 U/liter).
cell.5,7,9 In mitochondrial disorders, the most severely Radiographs of coxofemoral and stifle joints were
affected organs are those most dependent on oxidative taken, but no abnormalities were found.
metabolism, including brain, skeletal, and cardiac mus- Muscle biopsies were taken from the femoral biceps
cles; sensory organs; and kidney.8 muscle for histopathologic examination.
Few cases of mitochondrial diseases have been de-
Materials and Methods
scribed in veterinary medicine.2,3,4,11 We report the his-
tochemical and ultrastructural findings of mitochon- Muscle biopsy specimens from the femoral biceps muscle
drial myopathy in a German Shepherd dog. were frozen in isopentane precooled in liquid nitrogen. Sec-
tions were stained by the following histologic and histo-
Case History chemical techniques: hematoxylin and eosin (HE), modified
gomori trichrome, oil-red-O, periodic acid–Schiff (PAS), cy-
A 9-month-old male German Shepherd was referred
tochrome oxidase (COX), succinate dehydrogenase (SDH),
to the Department of Veterinary Clinical Science–Sur- and reduced nicotinamide adenine dinucleotide tetrazolium
gery Section, University of Naples, with a history of reductase (NADH-TR). We used a sample of biceps muscle
exercise intolerance, reluctance to move, and sponta- from a dog euthanatized for a neoplastic disease as a control
neous pain. Clinical signs had arisen 1 month earlier for histoenzymatic stains.
and had progressively increased in severity. For transmission electron microscopy, additional muscle
The dog showed systemic muscle atrophy, mostly samples were fixed in 2.5% glutaraldehyde, postfixed in os-
507
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mium tetroxide, and embedded in low-viscosity Spurr resin. ing mtDNA can have several unique genetic charac-
Ultrathin sections were cut, counterstained with 0.5% uranyl teristics, including 1) maternal inheritance, 2) hetero-
acetate and lead citrate, and examined using a Zeiss Electron plasmy due to the high mutation rate of mtDNA and
Microscope 902. random segregation of mitochondria during cell divi-
Results sion, 3) threshold expression of phenotype, and 4) var-
iable clinical phenotype resulting from differences in
HE staining showed both single atrophic fibers and defects and the extent to which a tissue relies on mi-
fibers with a basophilic rim along the sarcolemma (Fig.
tochondrial energy production.7,8 Mitochondrial disor-
1). Sections stained with modified Gomori trichrome
ders due to mtDNA mutations are not always trans-
had many ‘‘red ragged fibers’’ (RRF) with subsarco-
mitted from an affected mother. Some cases may be
lemmal and intermyofibrillar deposits of reddish gran-
sporadic. Sporadic cases probably result from mtDNA
ular material (Fig. 2). PAS staining showed an increase
deletions during embryogenesis, which is favored in
in glycogen at the periphery of fibers (Fig. 3), but
development because deleted DNA reproduces more
staining with oil red O was unremarkable. SDH and
rapidly than its normal counterpart. Because most
NADH-TR stains demonstrated concentration of oxi-
dative activity at the periphery of the fibers (Fig. 4). mtDNA mutations either alter or eliminate transfer ri-
Many muscle fibers and all fiber types failed to stain bonucleic acid (tRNA) genes, thus altering biosynthe-
for COX activity (Fig. 5). Residual subsarcolemmal sis of most mitochondrial (mt)-encoded proteins, the
COX activity was seen in a few fibers. In contrast, diversity of the clinical and biochemical consequences
normal canine skeletal muscle showed punctate COX is puzzling. The pleiotropic effects of these diseases
staining distributed throughout the cytoplasm of a sub- suggest that additional factors may be important in de-
population of muscle fibers (Fig. 6). termining the clinical and biochemical phenotypes.8
Ultrastructural examination confirmed the presence The diagnosis of mitochondrial myopathy is based
of abnormal, large and giant mitochondria with bizarre on clinical presentation and on the following criteria:
shapes (e.g., ‘‘golf-club-shaped mitochondrion’’) in a evidence of mutations or deletions in mtDNA, defi-
subsarcolemmal position and near the nucleus (Figs. ciency of respiratory chain enzymes in fibroblasts or
7, 8). These mitochondria contained abundant matrix muscle, characteristic changes such as RRF or de-
and showed either disintegrating cristae or irregularly creased SDH or COX staining in muscle biopsy, evi-
undulating cristae (sinuous cristae). Many swollen mi- dence of abnormal mitochondrial structure, and a fam-
tochondria were observed. The mitochondrial matrix ily member with a proven mitochondrial disease. Un-
appeared vacuolated, sometimes containing osmio- fortunately, we had no data on the lineage of the ani-
philic, electron-dense bodies (Fig. 9). In a few mito- mal in the present case, but the owner knew no other
chondria, breaks in the wall were evident. similarly affected relative.
Muscle mitochondria were embedded in a sarco- Few cases of mitochondrial myopathies2,3,11 have
plasm containing a large amount of glycogen, as sug- been reported in domestic animals. Altered cyto-
gested by PAS staining. chrome c oxidase activity and reduced mt messenger
RNA (mRNA) was demonstrated, by enzyme assay, in
Discussion fibroblasts and skeletal muscle of an old English
DNA analysis has allowed better understanding of sheepdog.4 Some authors suggested that mt dysfunc-
the molecular bases of mitochondrial diseases that re- tion may be the causative factor of exertional meta-
sult in myopathies or encephalomyopathies.8 Mito- bolic myopathy with lactic acidosis in dogs.4,11
chondrial disorders associated with mutations involv- The muscle biopsy changes, although nonspecific,
←
Fig. 1. Skeletal muscle; dog. Atrophy of single-muscle fibers and fibers with a basophilic rim (arrows) along the
sarcolemma. HE stain. Bar 5 22 mm.
Fig. 2. Skeletal muscle; dog. RRF with subsarcolemmal and intermyofibrillar reddish deposits of granular material
(arrows). Modified Gomori trichrome stain. Bar 5 22 mm.
Fig. 3. Skeletal muscle; dog. Increase in glycogen at the periphery of the fibers (arrows). Bar 5 40 mm.
Fig. 4. Skeletal muscle; dog. Concentration of oxidative activity at the periphery of the fibers (arrow). SDH stain. Bar
5 40 mm.
Fig. 5. Skeletal muscle; dog. High proportions of muscle fibers and all fiber types failed to react for COX. Residual
COX activity is observable in a few fibers (arrows). COX stain. Bar 5 40 mm.
Fig. 6. Skeletal muscle; dog. Positive control that shows the normal distribution of COX activity. COX stain. Bar 5
40 mm.
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often provide the initial diagnostic clue and prompt
further investigations to discover the underlying de-
fects. The morphologic hallmark of mitochondrial my-
opathies, due either to nuclear or mtDNA mutation
involving the respiratory chain, is the presence of RRF.
They are irregular in outline and show reddish sub-
sarcolemmal and intermyofibrillar granular deposits
with the modified Gomori trichrome stain. The sar-
coplasm appears fragmented; changes resemble arti-
factual cracking. This appearance represents the ac-
cumulation of mitochondria beneath the sarcolemma
and between myofibrils. However, RRF also can occur
in adult acid maltase deficiency and as a nonspecific
isolated finding in various myopathies. In normal hu-
man muscles, their frequency increases with age,
reaching a maximum of about 0.4% by the eighth de-
cade. This probably reflects the age-related accumu-
lation of abnormal mitochondria with DNA damage.1
RRF are most commonly seen with primary muta-
tions in mtDNA, usually in the transfer RNA genes,
and rarely with mutations in nuclear DNA.3
SDH, which indicates abnormally increased num-
bers of mitochondria, has been frequently used as a
qualitative histochemical marker for RRF in skeletal
muscles. SDH is the complex II of the respiratory
chain, embedded in the inner mitochondrial mem-
brane, and is specifically involved in the oxidation of
succinate. It carries electrons from flavin adenine di-
nucleotide (reduced form) to coenzyme Q. SDH is the
only enzyme in the respiratory chain complex that is
entirely encoded by the nuclear genome.12 Either par-
tial deficiency of SDH activity or increased SDH ac-
tivity may be seen in mitochondrial myopathy.12
In our case, a peculiar finding was the failure of
muscle fibers to stain histochemically for COX activ-
ity. COX-negative fibers are invariably present in hu-
man patients with progressive external ophthalmople-
gia, Kearns-Sayre syndrome, and benign reversible
form of cytochrome c oxidase deficiency, but they of-
ten occur in other mitochondrial encephalomyopathies
and in patients with myopathy alone. Recent studies
showed that mitochondria with deleted mtDNA are al-
most exclusively concentrated in segments of the mus-
cle fibers that lack COX activity.8 These studies also
have shown that most deleted mtDNAs are transcrip-
tionally active, but the overabundant mt mRNAs,
which are mainly transcribed from partially deleted ge-
nomes, are not translated into proteins.8 Translational
failure in ragged red, COX-negative fiber segments has
Fig. 7. Skeletal muscle; dog. Electron micrograph. Ab-
←
normal and large mitochondria at the subsarcolemmal posi-
tion and near the nucleus. Bar 5 2.5 mm. Fig. 9. Electron micrograph. Skeletal muscle; dog.
Fig. 8. Electron micrograph. Skeletal muscle; dog. A gi- Swollen mitochondria with cavitation of the matrix and dis-
ant golf-club–shaped mitochondrion. Bar 5 6 mm. ruption of the cristae. Bar 5 8 mm.
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been attributed to lack of indispensable tRNA genes 3 De Vivo DC: The expanding clinical spectrum of mito-
eliminated by the deletions or depletion of wild-type chondrial disease. Brain Dev 15:1–22, 1993
mtDNA or interference with expression of near-normal 4 Di Mauro S, Moraes C: Mitochondrial encephalomyopa-
levels of wild-type mtDNA by an overabundance of thies. Arch Neurol 50:1197–1208, 1993
5 Ghadially NF, ed.: Ultrastructural Pathology of the Cell
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and Matrix, 4th ed., vol. 1, pp. 195–327. Butterworths-
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dicating a more widespread distribution of abnormal 6 Houlton JEF, Herrtage ME: Mitochondrial myopathy in
mitochondria than was expected on the basis of the the Sussex spaniel. Vet Rec 106:206, 1980
frequency of the RRF. 7 Moraes C, Shanske S, Tritschler HJ, et al: MtDNA de-
This finding was confirmed in our case by ultra- pletion with variable expression: a novel genetic abnor-
structural examination, which showed large numbers mality in mitochondrial disease. Am J Hum Genet 48:
of structurally abnormal muscle mitochondria. Ultra- 492–501, 1991
structural changes in mitochondria are nonspecific and 8 Morgan-Hughes JA: Mitochondrial Disease in Myology,
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Acknowledgement 11 Vijayasarathy C, Giger U, Prociuk U, Patterson DF,
We thank Mr. R. Ilsami for technical assistance. Breitschwerdt EB, Avadhani NG: Canine mitochondrial
myopathy associated with reduced mitochondrial mRNA
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Request reprints from Dr. O. Paciello, Dipartimento di Patologia e Sanità Animale–Settore Anatomia Patologica, Facoltà
di Medicina Veterinaria, Università degli Studi di Napoli Federico II, Via F. Delpino, 1,80137 Naples (Italy).
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