Ubiquitin immunostaining and inclusion body myositis: Study of 30 patients with inclusion body myositis

Distinction of inclusion body myositis (IBM) from other forms of inflammatory myopathy is significant from prognostic and therapeutic standpoints. This study retrospectively examines ubiquitin expression by paraffin immunohistochemistry in muscle biopsy material from 30 patients with IBM. Patients included 19 men and 11 women (ages 29 to 80 years; mean, 64 years). All biopsies were characterized by endomysial chronic inflammation, muscle fiber degeneration and regeneration, rimmed vacuoles, and angular atrophic esterase-positive muscle fibers. Ragged red fibers were identified in biopsies of five patients and a partial cytochrome C-oxidase deficiency by enzyme histochemistry in biopsies of 10 patients. Evidence of intranuclear or cytoplasmic tubulofilamentous structures confirming a diagnosis of IBM was observed in all 30 cases. Paracrystalline mitochondrial inclusions were noted in five patients. Discrete myocyte intranuclear ubiquitin-positive inclusions were noted in 14 patients (47%). Discrete intracytoplasmic ubiquitin-positive inclusions were noted in 24 (80%) patients. Positive staining of rimmed vacuoles by ubiquitin was observed in 25 (83%) patients. Diffuse staining of scattered muscle fibers was observed in 21 (70%) patients. In a control group including patients with polymyositis (n = 3), dermatomyositis (n = 3), necrotizing vasculitis (n = 1), and granulomatous myositis (n = 1), discrete intranuclear or cytoplasmic ubiquitin-positive inclusions were not observed. Rimmed vacuoles were not seen either by light microscopy or ubiquitin immunostaining in any of the eight cases. Occasional myofibers from all eight cases showed diffuse, positive muscle fiber staining. Although not present in all cases, evidence of ubiquitinpositive myocytic intranuclear or cytoplasmic inclusions or positive-staining rimmed vacuoles in the setting of an inflammatory myopathy may be suggestive of a diagnosis of inclusion body myositis. Use of ubiquitin immunohistochemistry may be useful in cases in which frozen tissue or tissue processed for electron microscopy is not available, and IBM is suspected. Light or electron microscopic evidence of mitochondrial abnormalities were noted in a significant subset of patients (13 of 30; 43%) of patients with IBM.     

Primary over‐expression of AβPP in muscle does not lead to the development of inclusion body myositis in a new lineage of the MCK‐AβPP transgenic mouse

The aim of this study is to determine whether primary over‐expression of AβPP in skeletal muscle results in the development of features of inclusion body myositis (IBM) in a new lineage of the MCK‐AβPP transgenic mouse. Quantitative histological, immunohistochemical and western blotting studies were performed on muscles from 3 to 18 month old transgenic and wild‐type C57BL6/SJL mice. Electron microscopy was also performed on muscle sections from selected animals. Although western blotting confirmed that there was over‐expression of full length AβPP in transgenic mouse muscles, deposition of amyloid‐β and fibrillar amyloid could not be demonstrated histochemically or with electron microscopy. Additionally, other changes typical of IBM such as rimmed vacuoles, cytochrome C oxidase‐deficient fibres, upregulation of MHC antigens, lymphocytic inflammatory infiltration and T cell fibre invasion were absent. The most prominent finding in both transgenic and wild‐type animals was the presence of tubular aggregates which was age‐related and largely restricted to male animals. Expression of full length AβPP in this MCK‐AβPP mouse lineage did not reach the levels required for immunodetection or deposition of amyloid‐β as in the original transgenic strains, and was not associated with the development of pathological features of IBM. These negative results emphasise the potential pitfalls of re‐deriving transgenic mouse strains in different laboratories.     

Involvement of clusterin and the aggresome in abnormal protein deposits in myofibrillar myopathies and inclusion body myositis

Myofibrillar myopathies (MM) are characterized morphologically by the presence of non-hyaline structures corresponding to foci of dissolution of myofibrils, and hyaline lesions composed of aggregates of compacted and degraded myofibrillar elements. Inclusion body myositis (IBM) is characterized by the presence of rimmed vacuoles, eosinophilic inclusions in the cytoplasm, rare intranuclear inclusions, and by the accumulation of several abnormal proteins. Recent studies have demonstrated impaired proteasomal expression and activity in MM and IBM, thus accounting, in part, for the abnormal protein accumulation in these diseases. The present study examines other factors involved in protein aggregation in MM and IBM. Clusterin is a multiple-function protein which participates in Abeta-amyloid, PrP(res) and a-synuclein aggregation in Alzheimer disease, prionopathies and a-synucleinopathies, respectively. gamma-Tubulin is present in the centrosome and is an intracellular marker of the aggresome. Moderate or strong clusterin immunoreactivity has been found in association with abnormal protein deposits, as revealed by immunohistochemistry, single and double-labeling immunofluorescence and confocal microscopy, in MM and IBM, and in target structures in denervation atrophy. Gamma-Tubulin has also been observed in association with abnormal protein deposits in MM, IBM, and in target fibers in denervation atrophy. These morphological findings are accompanied by increased expression of clusterin and gamma-tubulin in muscle homogenates of MM and IBM cases, as revealed by gel electrophoresis and Western blots. Together, these observations demonstrate involvement of clusterin in protein aggregates, and increased expression of aggresome markers in association with abnormal protein inclusions in MM and IBM and in targets, as crucial events related to the pathogenesis of abnormal protein accumulation and degradation in these muscular diseases.     

Ocular myopathies

Ocular myopathies are manifested by primary and progressive involvement of extraocular muscles. In most cases of involvement of extra-ocular muscles a biopsy from somatic muscles studied by histochemistry and electron microscopy permits to make the diagnosis of the underlying condition. The two main clinico-pathological types of ocular myopathies are the oculocraniosomatic syndrome (Kearns-Sayre syndrome) and oculopharyngeal muscular dystrophy. The oculocraniosomatic syndrome is a multisystemic disorder and its histopathological hallmark is the presence of ragged-red muscle fibres which contain aggregates of abnormal mitochondria, often with paracrystalline inclusions. In the oculopharyngeal muscular dystrophy are observed muscle fibres with rimmed vacuoles and intranuclear tubular filamentous inclusions about 8.5 nm in external diameter. The rimmed vacuoles may occur in other muscle diseases but the intranuclear inclusions appear to be specific for oculopharyngeal muscular dystrophy. Their nature is unknown.     

Dose-dependent, prion protein (PrP)-mediated facilitation of excitatory synaptic transmission in the mouse hippocampus

Disruption of both alleles of the prion protein gene, Prnp, has been shown repeatedly to abolish the susceptibility of mice to developing prion diseases. However, conflicting results have been obtained from phenotypic analyses of prion protein (PrP)-deficient (Prnp0/0) mice lines. To explore the possible neurophysiological properties associated with expression or absence of the normal isoform of the cellular prion protein (PrPC), we used conventional in vitro extracellular field potential recordings in the hippocampal CA1 area of mice from two independently-derived Prnp0/0 strains. Basal synaptic transmission and a short-term form of synaptic plasticity were analysed in this study. Results were compared with animals carrying a wild-type mouse PrP transgene to investigate whether PrP expression levels influence glutamatergic synaptic transmission in the hippocampus. There was a clear correlation between excitatory synaptic transmission and PrP expression; i.e. the range of synaptic responses increased with the level of PrPC expression. On the other hand, the probability of transmitter release, as assessed by paired-pulse facilitation, appeared unchanged. Interestingly, whereas the overall range for synaptic responses was still greater in older mice over-expressing PrPC, this effect in these animals appeared to be due to better recruitment of fibres rather than facilitation of synaptic transmission per se. Taken together, these data are strong evidence for a functional role for PrPC in modulating synaptic transmission.     

Physiopathology and molecular diagnosis for prion diseases

Prion diseases, including Creutzfeldt-Jakob disease (CJD), are infectious neurodegenerative disorders. The etiological agent, prion, is postulated to consist mainly of a proteinase K-resistant isoform of prion protein (PrPSc) which is generated by post-translational conversion from the proteinase K-sensitive normal version (PrPC) physiologically expressed on the surface of neuronal and glial cells. The constitutive conversion results in the tremendous accumulation of PrPSc in the prion-infected brain. Homozygous disruption of the Prnp gene encoding PrPC renders mice resistant to prion, and the animals are no longer capable of generating PrPSc, indicating an essential role for PrPSc in the pathogenesis of prion diseases. The PrP-null mice (Ngsk Prnp0/0) revealed progressive ataxia due to the degeneration of cellebellar Purkinje cells at old ages. Successful rescue of Ngsk Prnp0/0 mice from neurodegeneration by a transgene encoding the normal mouse PrPC has indicated that the functional loss of PrPC is essential for this phenotype. Moreover, we detected aberrant mRNAs chimeric between Prnp exon 1-2 and a novel gene encoding PrP-like protein (PrPLP). These results suggested that, in addition to the functional loss of PrPC, ectopic expression of the PrPLP in the brain of Ngsk Prnp0/0 mice could be associated with Purkinje cell degeneration.     

Lectin binding and desmin expression during necrosis, regeneration, and neurogenic atrophy of human skeletal muscle

Changes in the cytoplasm of skeletal muscle fibres during necrosis, regeneration, and neurogenic atrophy have been studied in a wide range of human neuromuscular diseases with a panel of eleven biotinylated lectins and by immunohistochemical staining for the cytoskeletal protein desmin. Increased binding of several lectins was observed in both necrotic and regenerating fibres, with Concanavalin A the most consistently positive lectin. Staining for desmin was strong in the cytoplasm of regenerating and partially damaged fibres and was lost in necrotic fibres, although there were differences in the staining reactions of the two antidesmin antibodies used. In fibres which had undergone neurogenic atrophy, cytoplasmic lectin binding was seen only with Griffonia simplicifolia 1 lectin, and desmin was expressed more strongly than in normal fibres. Lectin binding and immunohistochemical staining from desmin can supplement the information obtained from muscle biopsies by conventional histochemical methods and lead to a better understanding of the mechanisms of muscle damage.     

Conspicuous accumulation of a single-stranded DNA binding protein in skeletal muscle fibers in inclusion body myositis.

In muscle biopsies from patients with inclusion body myositis (IBM), multiple sites were found in many muscle fibers that bound single-stranded but not double-stranded DNA without sequence specificity, as exemplified by several different cDNA probes. This activity was attributable to a protein, because it was abolished by proteases but not by RNAse. Most of the sites of binding were myonuclei, whereas some were rimmed vacuoles, which probably result from nuclear breakdown. No comparable binding was seen in 27 control biopsies. A number of human and viral single-stranded DNA binding proteins exist but our data does not identify the protein responsible for DNA binding in IBM. Our findings reinforce the supposition that nuclear damage plays a basic role in the pathogenesis of IBM.     

The pathological diagnosis of specific inflammatory myopathies.

Pathological diagnosis of dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM) should be possible in almost all cases when an appropriately involved muscle is biopsied. DM shows characteristic patterns of muscle fiber damage and capillary damage. Lymphocytes and macrophages are seen in PM and IBM partially invading non-necrotic fibers. IBM is also characterized by rimmed vacuoles with membranous whorls, characteristic masses of filaments in cytoplasm and sometimes in nuclei, and grouped atrophic fibers. Muscle fiber damage in PM is more variable. Inflammatory myopathy can be associated with HTLV-1 and HIV infection. In the latter a strong resemblance to PM is reported. Separate, still less well characterized forms of inflammatory myopathy occur in young children.     

ALS‐Associated Endoplasmic Reticulum Proteins in Denervated Skeletal Muscle: Implications for Motor Neuron Disease Pathology

Alpha‐motoneurons and muscle fibres are structurally and functionally interdependent. Both cell types particularly rely on endoplasmic reticulum (ER/SR) functions. Mutations of the ER proteins VAPB, SigR1 and HSP27 lead to hereditary motor neuron diseases (MNDs). Here, we determined the expression profile and localization of these ER proteins/chaperons by immunohistochemistry and immunoblotting in biopsy and autopsy muscle tissue of patients with amyotrophic lateral sclerosis (ALS) and other neurogenic muscular atrophies (NMAs) and compared these patterns to mouse models of neurogenic muscular atrophy. Postsynaptic neuromuscular junction staining for VAPB was intense in normal human and mouse muscle and decreased in denervated Nmd^2J mouse muscle fibres. In contrast, VAPB levels together with other chaperones and autophagy markers were increased in extrasynaptic regions of denervated muscle fibres of patients with MNDs and other NMAs, especially at sites of focal myofibrillar disintegration (targets). These findings did not differ between NMAs due to ALS and other causes. G93A‐SOD1 mouse muscle fibres showed a similar pattern of protein level increases in denervated muscle fibres. In addition, they showed globular VAPB‐immunoreactive structures together with misfolded SOD1 protein accumulations, suggesting a primary myopathic change. Our findings indicate that altered expression and localization of these ER proteins and autophagy markers are part of the dynamic response of muscle fibres to denervation. The ER is particularly prominent and vulnerable in both muscle fibres and alpha‐motoneurons. Thus, ER pathology could contribute to the selective build‐up of degenerative changes in the neuromuscular axis in MNDs.     

Adult-onset muscular dystrophy in a cat associated with a presumptive alteration in trafficking of caveolin-3

A 10-year-old neutered female domestic longhaired cat was referred for evaluation of forelimb weakness and lameness. There was hypertrophy and firmness of the musculature with no neurological deficits. Moderate increase of creatine kinase activity was present. Muscle biopsy showed rounded atrophic and hypertrophic fibres, an increased number of centrally located myofibre nuclei, scattered rimmed vacuoles and mild perimysial and endomysial fibrosis. Myofibre necrosis with phagocytosis was present in the gluteal muscle. Immunohistochemistry revealed absence of sarcolemmal caveolin-3 in almost all muscle fibres and sarcoplasmic accumulation of the protein in approximately 30% of myofibres. Normal expression of caveolin-3 was detected by immunoblotting, so protein mislocalization in the sarcoplasm due to aberrant trafficking towards the sarcolemma was suspected. This case represents the first example of muscular dystrophy due to a caveolinopathy in animals.     

Localization of O-GlcNAc-modified proteins in neuromuscular diseases

O-linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification of nucleocytoplasmic proteins that induces the attachment of N-acetylglucosamine to serine or threonine residues of a protein. In contrast to other protein glycosylations, this modification is highly reversible and, similar to phosphorylation, it plays important roles in various cell signals. Here, we immunolocalized O-GlcNAc-modified proteins in muscle biopsy specimens from 40 patients with neuromuscular diseases and controls. In normal muscle fibers, O-GlcNAc was found along plasma membranes and in nuclei. Diffuse and increased cytoplasmic staining of O-GlcNAc was detected in (1) regenerating muscle fibers in muscular dystrophy, myositis, and rhabdomyolysis; (2) a proportion of atrophic fibers in myositis, such as those found in perifascicular regions in dermatomyositis; and (3) vacuolated fibers in sporadic inclusion body myositis (s-IBM) and distal myopathy with rimmed vacuoles (DMRV). Target formations in neurogenic muscular atrophy were O-GlcNAc positive. Increase of O-GlcNAc glycosylation could be associated with the stress response, as these lesions have been shown to be positive for several stress markers. Vacuolar rims in s-IBM and DMRV were sometimes sharply lined by O-GlcNAc-positive deposits, which reflects myonuclear breakdown occurring from the disease.     

Myopathy with core-like structures in a dog

Core-like structures were seen histologically in many of the fibres of the triceps and biceps femoris muscles of an 18-months-old male Great Dane with muscle weakness and moderate proximal muscular atrophy. The structures were lightly staining and lacked cross-striations. Some contained vacuoles and nuclei. Scattered necrotic and presumably regenerating fibres were also present. Ultrastructurally, the cores contained disarrayed filament bundles attached to thickened Z-lines which were compatible with the rods of rod myopathies. The condition was not fully characterized, but has certain similarities to a group of rare human congenital muscular disorders which includes central core disease.     

Electron microscopy in neuromuscular disorders

Electron microscopy has a strategic position in the diagnosis of neuromuscular disorders. In muscular fibers, the main abnormalities include vacuoles, inclusion bodies, and myofibrillar disorganization with or without abnormal inclusion material. Vacuolar changes include lipidic and glycogenic storage vacuoles, rimmed vacuoles, and lysosomal and autophagic vacuoles. Accumulation of abnormal inclusion material is found in nemaline myopathy, actinopathies, and hyaline body myopathy. Myofibrillar disorganization involves cores, multiminicores, and myosin chain depletion. Myofibrillar myopathies associate a pathologic pattern of myofibrillar dissolution and ectopic protein expression. They can be divided into two groups: myofibrillar myopathies with multiple expression proteins and myofibrillar myopathies with desmin and alphaB-crystallin expression only. In these two conditions, electron microscopy shows accumulation of a granulofilamentous material immunoreactive for desmin. At least three genes are implicated: desmin, alphaB-crystallin, and myotilin. Lastly, electron microscopy serves to identify changes, pathogenic or not, which are not shown up by light microscopy. Moreover, electron microscopy gives insight on pathophysiological mechanisms and can guide molecular genetics analysis.     

Increased expression of the normal cellular isoform of prion protein in inclusion-body myositis, inflammatory myopathies and denervation atrophy

The cellular isoform of the prion protein (PrPc) is a glycosylphosphatidylinositol-anchored glycoprotein, normally expressed in neural and non-neural tissues, including skeletal muscle. In transmissible spongiform encephalopathies, or prion diseases, PrPc, which is soluble in nondenaturing detergent and sensitive to proteinase K (PK)-treatment, represents the molecular substrate for the production of a detergent-insoluble and PK-resistant isoform, termed PrP(Sc). In human prion diseases, PrP(Sc) accumulation occurs only in brain tissues, with the exception of new variant Creutzfeldt-Jakob disease, where PrP(Sc) is also detected in lymphoid tissues. Increased amounts of prion protein expression and deposition have been described in pathological muscle fibers of two human muscle disorders, called sporadic inclusion-body myositis (s-IBM) and hereditary inclusion-body myopathy, but it is unknown whether accumulated prion protein reflects normal PrPc or PrP(Sc). We investigated the biochemical characteristics of prion protein in normal human muscle, s-IBM, other inflammatory myopathies and denervation atrophy. We report that 1) both the glycoform profile and size of the normal muscle PrPc are different from those of human brain PrPc; 2) in addition to s-IBM, increased PrPc expression is seen in polymyositis, dermatomyositis and neurogenic muscle atrophy, but PrPc glycoforms are unchanged; 3) only the normal PrPc isoform, and not PrP(Sc), is detected in s-IBM. The present results exclude that s-IBM is a prion disease.     

Mitochondrial DNA variants in inclusion body myositis characterized by deep sequencing

Muscle pathology in inclusion body myositis (IBM) typically includes inflammatory cell infiltration, muscle fibers with rimmed vacuoles and cytochrome c oxidase (COX)‐deficient fibers. Previous studies have revealed clonal expansion of large mitochondrial DNA (mtDNA) deletions in the COX‐deficient muscle fibers. Technical limitations have prevented complete investigations of the mtDNA deletions and other mtDNA variants. Detailed characterization by deep sequencing of mtDNA in muscle samples from 21 IBM patients and 10 age‐matched controls was performed after whole genome sequencing with a mean depth of mtDNA coverage of 46,000x. Multiple large mtDNA deletions and duplications were identified in all IBM and control muscle samples. In general, the IBM muscles demonstrated a larger number of deletions and duplications with a mean heteroplasmy level of 10% (range 1%‐35%) compared to controls (1%, range 0.2%‐3%). There was also a small increase in the number of somatic single nucleotide variants in IBM muscle. More than 200 rearrangements were recurrent in at least two or more IBM muscles while 26 were found in both IBM and control muscles. The deletions and duplications, with a high recurrence rate, were mainly observed in three mtDNA regions, m.534‐4429, m.6330‐13993, and m.8636‐16072, where some were flanked by repetitive sequences. The mtDNA copy number in IBM muscle was reduced to 42% of controls. Immunohistochemical and western blot analyses of IBM muscle revealed combined complex I and complex IV deficiency affecting the COX‐deficient fibers. In conclusion, deep sequencing and quantitation of mtDNA variants revealed that IBM muscles had markedly increased levels of large deletions and duplications, and there were also indications of increased somatic single nucleotide variants and reduced mtDNA copy numbers compared to age‐matched controls. The distribution and type of variants were similar in IBM muscle and controls indicating an accelerated aging process in IBM muscle, possibly associated with chronic inflammation.     

The roles of the synaptic basal lamina and of innervation in directing the accumulation of a synaptic molecule, mAb 3B6 antigen, in regenerating skeletal muscles

Summary We have recently described a novel nonhomogeneous distribution of a muscle synaptic molecule following denervation. Monoclonal antibody (mAb) 3B6 antigen, a molecule concentrated at endplate/junctional regions and myotendinous junctions in innervated muscles, appears in denervated muscles in restricted perijunctional regions that are continuous with and centered on endplates. In the present study we examine the roles of the synaptic basal lamina and of innervation in directing the accumulation of the molecule in newly formed regenerating muscle fibres. In denervated regenerating muscle fibres, mAb 3B6 antigen was associated with the plasma membrane and localized at former junctional and perijunctional regions. In those muscle fibres which displayed the perijunctional distribution, the molecule was preferentially colocalized with and centered on former endplate areas, Altogether, a preference for the localization of mAb 3B6 at former endplate regions was observed in 86–90% of denervated regenerating myofibres. A similar preference was observed in 97–99% of innervated regenerating muscle fibres. However, whereas 85.9% of denervated regenerating muscle fibres displayed a perijunctional distribution of the molecule, only 50.5% of innervated regenerating myofibres exhibited a perijunctional distribution. In addition, mAb 3B6 antigen was detected in the cytoplasm of most of the denervated regenerating myofibres but in none of the innervated ones. These results indicate that the basal lamina directs the preferential accumulation of mAb 3B6 antigen at original synaptic sites. Innervation, which is not a prerequisite for the expression of the molecule by regenerating muscle, down-regulates its overall production and presence in perijunctional regions.     

Inclusion body myositis : clinical and histopathological features of 36 patients

Summary Thirty-six patients (15 females, 21 males) with inclusion body myositis (IBM) were studied. The diagnosis was established according to clinical and histopathological criteria. Clinical features were insidious onset of slowly progressive muscle weakness and wasting with depressed or absent tendon reflexes especially in the lower limbs. The pattern of muscle weakness was variable. The majority of patients (58%) showed proximal and symmetrical weakness usually most prominent in the legs. Isolated distal (6%) and asymmetrical weakness (19%) was less frequently observed. Myalgia occurred in 42% of the patients. The age at onset of symptoms ranged from 20 to 73 years (mean 47 years). Serum creatine kinase levels were normal (11%) or mildly elevated (89%). Needle electromyography revealed myopathic features in about 80% of the patients, and results of nerve conduction studies were normal in most of the cases. The predominant histopathological findings were numerous muscle fibers with rimmed vacuoles (100% of the patients), groups of atrophic fibers (92%), and inflammatory infiltrates (89%). The inflammatory infiltrates were located predominantly at endomysial sites and were composed mainly of T8 cells. Electron microscopy showed characteristic intracytoplasmic filamentous inclusions in all 36 cases. Immunosuppressive treatment in 16 patients failed to prevent disease progression in all but one patient with an associated Sjögren's syndrome. It is concluded that the consistent combination of typical histopathological findings and characteristic clinical features offers a firm basis for the diagnosis of IBM. IBM should be suspected in any adult patient presenting with clinical signs of a chronic polymyositis unresponsive to immunosuppressive therapy. The etiology and pathogenesis of IBM remain to be established.Abbreviations IBM inclusion body myositis - CK creatine kinase - MHC major histocompatibility complex     

Histopathological changes in skeletal muscle associated with chronic ischaemia

Muscle biopsy is an essential part in the diagnostic workup in patients with suspected neuromuscular disorders. It is therefore important to be aware of morphological alterations that can be caused by systemic factors or natural ageing. Chronic limb ischaemia is frequent in elderly individuals. This study was performed to examine histopathological and mitochondrial changes in muscle in patients with chronic critical limb ischaemia. Muscle biopsy of skeletal muscle of the lower limb of patients with chronic ischaemia leading to amputation was performed and compared with muscle biopsies of healthy, age‐matched controls. The histopathological abnormalities included fibrosis, necrosis, atrophy, glycogen depletion, internal nuclei, rimmed vacuoles, fibre type grouping, cytochrome c oxidase deficient fibres, MHC‐I upregulation, and signs of microangiopathy. The only alteration found in age‐matched controls was a few cytochrome c oxidase deficient fibres. There were also increased levels of multiple mitochondrial DNA deletions in ischaemic muscles compared with controls. Critical limb ischaemia is associated with significant histopathological changes in muscle tissue and also increased levels of mitochondrial DNA deletions. Since the alterations mimic different primary myopathic changes, chronic ischaemia is important to consider as a differential diagnosis in elderly individuals, investigated with muscle biopsy for muscle disease.