Ultrastructural changes in dysferlinopathy support defective membrane repair mechanism

BACKGROUND: The dysferlin gene has recently been shown to be involved in limb girdle muscular dystrophy type 2B and its allelic disease, Miyoshi myopathy, both of which are characterised by an active muscle degeneration and regeneration process. Dysferlin is known to play an essential role in skeletal muscle fibre repair, but the process underlying the pathogenetic mechanism of dysferlinopathy is not completely understood. AIMS: To define both specific alterations of muscle fibres and a possible sequential mechanism of myopathy development. METHODS: A histological, immunohistochemical, and ultrastructural analysis of 10 muscle biopsies from patients with molecularly diagnosed dysferlinopathy. RESULTS: An inflammatory response was seen in most of the muscle biopsies. The immunohistochemical pattern demonstrated active regeneration and inflammation. Non-necrotic fibres showed alterations at different submicroscopic levels, namely: the sarcolemma and basal lamina, subsarcolemmal region, and sarcoplasmic compartment. In the subsarcolemmal region there were prominent aggregations of small vesicles, probably derived from the Golgi apparatus, which consisted of empty, swollen cisternae. In the sarcolemma there were many gaps and microvilli-like projections, whereas the basal lamina was multilayered. CONCLUSIONS: The histopathological, immunohistochemical, and ultrastructural data show that dysferlinopathy is characterised by a very active inflammatory/degenerative process, possibly associated with an inefficient repair and regenerative system. The presence of many crowded vesicles just beneath the sarcolemma provides submicroscopical proof of a defective resealing mechanism, which fails to repair the sarcolemma.     

Ischaemia and reperfusion effects on skeletal muscle tissue: morphological and histochemical studies

This was a study on the oxidative stress due to ischaemia (I) and reperfusion (R) in skeletal muscle tissue. Using a tourniquet, groups of rats were submitted to ischaemia for 4 h, followed by different reperfusion periods. The animals were divided in four groups: control; 4 h of ischaemia (IR); 4 h of ischaemia plus 1 h reperfusion (IR-1 h); 4 h of ischaemia plus 24 h reperfusion (IR-24 h); and 4 h of ischaemia plus 72 h reperfusion (IR-72 h). At the end of the procedures, samples of soleus muscle were collected and frozen in n-hexane at -70 degrees C. Cryostat sections were submitted to haematoxylin-eosin, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) stains. An additional muscle sample was processed for electron microscopy. No alterations were found in control animals. IR group showed fibres had normal aspect besides some round, acidophilic and hypertrophic fibres. There were several fibres with angular outlines and smaller diameters in this group compared with control group. NADH-TR/SDH reaction was moderately intense in most fibres. In some fibres, cytoplasm showed areas without activity and other fibres had very intense reactivity. IR-1 h group showed oedema hypercontracted fibres with disorganized myofibrils, mitochondria with focal lesions and dilated sarcoplasmic reticulum. NADH-TR/SDH reaction was moderate to weak. IR-24 h showed intense inflammatory infiltrate in the endomysium and perimysium. NADH-TR/SDH reaction was similar to IR-1 h. IR-72 h showed necrotic fibres, areas with inflammatory infiltrate, reduced muscle fibres at different stages of necrosis and phagocytosis, and many small round and basophilic fibres characterizing a regeneration process. NADH-TR/SDH reaction was weak to negative. Our results suggest that ischaemia and the subsequent 1-, 24- and 72-h reperfusions induced progressive histological damage. Although progressive, it may be reversible because there were ultrastructural signs of recovery after 72-h reperfusion. This recovery could in part be due to the low oxidative stress identified by the morphological and histochemical analysis.     

Origin and significance of small muscle fibres in neuromuscular disease

Summary Small muscle fibres, defined as those of less than 40 µm diameter in the male and 30 m in the female were encountered in muscle biopsies of patients with spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), polymyositis (PM) and myopathy/dystrophy. Excessive reactivity with NADH-TR in small fibres did not discriminate between neurogenic and myopathic disorders. Quantification of perifascicular atrophic fibres, the number of nuclei in atrophic fibres, or the presence of isolated or grouped small fibres without histochemical kinship to their surrounding fibres did not aid recognition of the disease process in the groups studied. Small fibres which reacted strongly both with NADH-TR and ATPase at pH 9.4 (Type 3 fibres) constituted 38% of small fibres in the biopsies of SMA; 25% in ALS; but only 1% and 2.7% in PM and myopathy/dystrophy respectively. Thus, the presence of small Type 3 fibres in muscle biopsies may be a useful marker for neurogenic disorders in adults.     

Degenerative and regenerative changes in murine skeletal muscle after injection of venom from the snake Bothrops asper: a histochemical and immunocytochemical study.

The degenerative and regenerative changes in murine skeletal muscle after injection of Bothrops asper venom were studied by histological, lectin histochemical and immunocytochemical techniques. According to our observations, the process was divided into four main stages: (a) During the first 3 days prominent degenerative events took place in skeletal muscle fibres, capillaries, arteries, veins and intramuscular nerves. An inflammatory infiltrate was abundant after the first day and removal of necrotic material was well advanced by the third day. (b) Muscle regeneration was evident by the fourth day. From 4 to 6 days there were two populations of regenerating muscle fibres, one of apparently normal fibres located in areas where capillary vessels were abundant, and another population of groups of regenerative fibres showing signs of degeneration. This second type of fibre was predominant in areas where the number of capillaries was greatly reduced. (c) One and 2 weeks after envenomation areas of small regenerative fibres of normal morphology and areas of degenerating regenerative fibres were observed. The latter were abundant in regions of dense fibrotic tissue and scarce capillaries. (d) Finally, at 4 and 8 weeks after envenomation there were both areas of fibrosis and areas where regenerating muscle fibres predominated. However, the diameter of these fibres was abnormally small, an indication that they may have been atrophic fibres. It is suggested that muscle regeneration is partially impaired after myonecrosis induced by Bothrops asper venom, probably due to the damage induced by this venom on muscle microvasculature and nerves.     

Morphological aspects of rabbit masseter muscle after cervical sympathectomy

The objective of this paper was to study the effect of sympathetic innervation on morphological and histochemical aspects of skeletal muscle tissue. Rabbit masseter muscle was studied using histochemical and immunohistochemical methods for periods of up to 18 months post-sympathectomy. The morphological and enzymatic characteristics of control masseter muscles were similar on both the left and right sides. The main features were muscle fibres with a mosaic pattern and a predominance of type IIa fibres, followed by type I. Type IIb fibres showed very low frequency. Sympathectomized animals showed varying degrees of metabolic and morphological alterations, especially 18 months after sympathectomy. The first five groups showed a higher frequency of type I fibres, whilst the oldest group showed a higher frequency of type IIb fibres. In the oldest group, a significant variation in fibre diameter was observed. Many fibres showed small diameter, atrophy, hypertrophy, splitting, and necrosis. Areas with fibrosis were observed. Thus cervical sympathectomy induced morphological alterations in the masseter muscles. These alterations were, in part, similar to both denervation and myopathy. These findings indicate that sympathetic innervation contributes to the maintenance of the morphological and metabolic features of masseter muscle fibres.     

Ultrastructural pathology in skeletal muscle of mice envenomed with Crotalus vegrandis venom

In this ultrastructural study we examined skeletal muscle fibres from mice intraperitoneally inoculated with a sublethal dose of Crotalus vegrandis (rattlesnake) venom. The group of mice inoculated presented neurological symptoms characterised by respiratory failure and hind limbs paralysis. Skeletal muscle fibres showed different degrees of alterations. Most of them presented the characteristic pattern of necrosis in progress. Atrophied and hypercontracted fibres were frequently seen. Some atrophied and necrotic fibres showed several nucleoli-like bodies in the nucleus. In the atrophic and hypercontracted fibres, sarcoplasmic vacuolation and abnormal mitochondria with stacked cristae were observed. Areas of segmental necrosis were also frequently found. In connection with these altered muscle fibres, capillary abnormalities were detected. This study suggests that in envenomed mice respiratory failure symptoms may be related with muscle damage caused by Crotalus vegrandis venom components.     

Fibre type specific expression of Leu19-antigen and N-CAM in skeletal muscle in various stages after experimental denervation

Leu-19 antigen, which seems to be identical with neural cell adhesion molecule (N-CAM), plays a major role in the innervation of muscle cells, and in adult muscle appears after denervation and during regeneration of muscle fibres, where it acts as part of a signalling system increasing the probability of re-innervation. This combined enzyme-histochemical and immunohistochemical study examined whether this signalling process was regulated in a uniform or differential pattern for type 1 and type 2 muscle fibres. The subscapular nerve of 18 rabbits was transsected with subsequent complete denervation of the supraspinatus muscle. Leu19 and N-CAM immunohistochemistry was performed 2 to 64 days after surgery. Whereas in normal muscle there are virtually no Leu-19/N-CAM positive muscle fibres; from day 2 after denervation an increasing proportion of fibres expressed Leu-19/N-CAM, prior to any neurogenic atrophy. In the early stage of denervation Leul9/N-CAM expression was confined to type 1 fibres. After 11 days nearly all fibres were Leul9/N-CAM positive irrespective of their fibre type. Sixty-four days after denervation type 1 fibres became Leu19/N-CAM negative, while atrophic type 2 fibres showed intensive staining. Thus, expression of Leu-19 antigenity is differently regulated in both fibre types.     

Fatal lipid storage myopathy with deficiency of cytochrome-c-oxidase and carnitine

Summary Two newborn female siblings fell ill with apathy, failure of suckling and a generalized progressive muscular hypotonia. Death occured at the age of 7 weeks, obviously caused by impairment of respiratory musculature. Biochemical studies in one child revealed carnitine deficiency especially in skeletal muscle; hepatic encephalopathy was absent. Both children had a generalized hyperaminoaciduria, an unusual finding in primary carnitine deficiency.Besides fatty metamorphosis of the liver, bilateral hydroureters and tubular calcifications of both kidneys, morphological studies showed a generalized lipid storage myopathy which predominated in Type-I-fibres and was accentuated in the muscles of the neck. Enzymehistochemical electron microscopy in longterm frozen muscle demonstrated that cytochrome-c-oxidase activity was absent not only in myopathic but also in most of the morphological unchanged muscle fibres. Only some fibres and endothelial cells displayed normal activity of mitochondria. Biochemically no cytochrome aa₃ (cytochrome-c-oxidase) could be found in skeletal muscle; cytochrome b was almost undetectable. - In newborns with fatal lipid storage myopathy and carnitine deficiency it seems necessary to look for additional defects in the respiratory chain. Enzyme histochemical electron microscopy is a sensitive method in identifying cytochrome-c-oxidase even after a 12 months period of storage.     

Morphological alterations in skeletal muscle of spontaneously hypertensive rats

The Extensor digitorum longus (EDL) and the soleus muscles of spontaneously hypertensive rats (SHR) were studied in comparison with those of their normal counterparts, the Wistar Kyoto (WKY) rats. Quantitative assessment of capillaries and muscle fibre typing was done with optical microscopy, while the study of capillary abnormalities was performed by ultrastructural observation. There were no differences in fibre type proportion or in capillarity indexes between the SHR and the control rats. A reduction in the area of IIB fibres was found in the EDL muscle of the hypertensive animals. The ultrastructural study showed abnormalities in the capillaries of both muscles in SHR, the cross section of the endothelial cells was enlarged; there was irregular distribution of caveolae and pinocytic vesicles, the capillary basement membrane showed irregular width, with parts engrossed and reduplicated. Some pericytes were prominent. There were macrophages present in the interstitial space. In some muscle fibres there was disorganization of the sarcomere structure, swelling of the sarcotubular system, abundant autophagic vacuoles, and proliferative satellite cells. There were abundant collagen fibrils. The presence of cellular rests, autophagic vacuoles and loss of sarcolemma indicated necrosis. It can be concluded, that in SHR, muscle capillaries showed alterations that may be the substrate of functional rarefaction, although anatomical rarefaction (number reduction) could not be demonstrated. In EDL and soleus muscles of SHR, signs of a mild myopathy with focal fibrosis were present.     

Ultrastructural correlates of ischaemic contracture during global subtotal ischaemia in the rat heart.

The development of left ventricular ischaemic contracture and its correlation with ultrastructural and sarcolemmal permeability defects were studied in isolated rat hearts during global subtotal ischaemia. With acetate as substrate the hearts exhibited a rise in diastolic tension after 8-10 min at which time small foci of contracted myocytes were scattered throughout the myocardium. In hearts with 5% of the maximum diastolic tension (termed 5% contracture), the foci were situated predominantly in the subendocardium and papillary muscle. Contracted myocytes in these foci were capable of excluding ionic lanthanum thus demonstrating retention of normal sarcolemmal permeability properties. With 30% contracture ultrastructural damage had spread to the subepicardium and with further contracture there was an associated increase in the number and size of foci in all regions. In these foci, swelling of the tubular sarcolemmal system and occasionally of the sarcoplasmic reticulum appeared to precede myofibrillar contraction. At 50% contracture lanthanum influx into contracted cells became more frequent. Hearts developed full contracture by 15-18 min at which time most myocytes were contracted and retained lanthanum intracellularly. The heterogeneity of the response at a cellular level may offer a possible explanation for the lack of correlation between contracture and tissue ATP. A possible sequence of structural injury leading to impaired calcium homeostasis is also suggested.     

Evaluation of muscle capillary basement membrane in inflammatory myopathy

The capillary basement membranes from 16 skeletal muscle biopsies from patients with a clinical and histological diagnosis of inflammatory myopathy and from six controls were analysed ultrastructurally and morphometrically. Resin sections from 244 endomysial capillaries were examined by light microscope, and the results were correlated with findings seen in electron micrographs of these capillaries. The ultrastructural morphometric measurements and the statistical analysis showed that the capillary basement membrane was thick and multilaminated in 87% specimens affected by inflammatory myopathy. No thick or multilaminated basement membrane was observed in controls. In inflammatory myopathy the endomysial space next to the capillaries contained an increased amount of collagen fibrils and showed signs of a chronic reparative process. It is suggested that the thick multilaminated basement membrane in inflammatory myopathy represents an advanced stage of vascular regeneration.     

Myocardial fibre calcification.

Three cases of myocardial fibre calcification found at post-mortem examination are described. In one case there was antemortem hypercalcaemia and hyperphosphataemia and the case was clearly an example of metastatic calcification. In the other two cases there was ischaemic myocardial necrosis and calcification was seen in fibres which were not overtly necrotic, but which were both in proximity to (the majority) and remote from the necrotic zones. Since renal failure with hyperphosphataemia was present in both cases, these were considered to be examples of augmented (by the hyperphosphataemia) dystrophic calcification. The histological, histochemical and ultrastructural features were identical in the three cases. Hydroxyapatite formation was observed initially in mitochondria, followed by spillage of crystals into the cytosol and ultimately into the interstitium. It is suggested that the fundamental lesion is a dysfunction of the fibre membrane; the similarity of this reaction with the calcification seen in skeletal muscle fibres in various myopathies is noted and a unifying hypothesis of the mechanism of skeletal and cardiac muscle fibre calcification is thereby suggested.     

Reaction of skeletal muscle to small implants of titanium or stainless steel: a quantitative histological and autoradiographic study.

Small wire implants of titanium or stainless steel were inserted into mouse leg muscles to test the reaction of regenerating skeletal muscle. Muscle fibres regenerated rapidly, starting at 3 d; by 2 wk all implants were encapsulated in thin (10 microns) fibrous tissue capsule surrounded by myotubes for 15-20 microns. Quantitatively there were no detectable differences in muscle regeneration between the two metals. The initiation of myoblast precursor cell replication was determined in regenerating muscle next to the implants. Tritiated thymidine was injected 18-156 h after implant insertion and labelled myotube nuclei in the regenerated muscle indicated that their precursors had started DNA synthesis 24 h after implant insertion. This is similar to myogenesis in many other muscle lesions and indicated that neither titanium nor stainless steel retarded muscle regeneration.     

Intact myelinated fibres in biopsies of ventral spinal roots after preganglionic traction injury to the brachial plexus. A proof that Sherrington's ‘wrong way afferents’ exist in man?

Bell-Magendie's law of separation of spinal function states that afferent and efferent fibres join the spinal cord separately in ventral and dorsal spinal nerve roots. For over 100 years there have been reports that challenge the exclusiveness of this law in mammals; very few studies have referred to man. We conducted a prospective morphological study in patients with preganglionic traction injuries of the brachial plexus to address this question. Avulsed ventral and dorsal roots were examined after variable intervals from the injury for histological and ultrastructural evidence for myelinated afferent fibres entering the cord via the ventral roots. Intact myelinated fibres were found in all ventral root specimens, but the majority of fibres in later biopsies are regenerative. A small number of fibres could be demonstrated that are likely to be 'wrong way ventral afferents'. Their number is falsely low due to wallerian degeneration of dorsal and ventral afferents following the mechanical and ischaemic effects of traction injury. Our findings are the first morphological evidence in human material that Bell-Magendie's law might not entirely be correct and they underline the difficulties in comparing traumatic with experimental rhizotomy.     

Experimental myopathy induced by amphiphilic cationic compounds including several psychotropic drugs.

Several amphiphilic cationic compounds with different therapeutic actions, among them several psychotropic drugs, which have previously been described as inducing generalized lipidosis, were examined for their myotoxic effects in rats. Chronic administration of high oral doses of chlorphentermine, chlorcyclizine, l-chloro-amitriptyline, triparanol, quinacrine (mepacrine), iprindole or imipramine caused a myopathy closely resembling the chloroquine-induced myopathy described earlier in humans and in animals. The essential features were, in decreasing order of frequency of observation: (a) longitudinal branching of muscle fibres giving rise to clusters of subfibres with greatly varying diameters; (b) cytoplasmic vacuoles of varying sizes: and (c) necrosis of muscle fibres. As shown by sequential investigations, fibre branching resulted from incomplete regeneration subsequent to fibre necrosis. Upon withdrawal of the drugs, the myopathy was slowly reversible.Cytoplasmic vacuolation and necrosis of muscle fibres appear to be, at least primarily, independent events. Necrosis is probably not causally related to the drug-induced generalized lipidosis nor to the drug-induced alterations previously described in motor nerve endings. Development of necrosis is tentatively suggested as being triggered by a direct effect of the cationic amphiphilic compounds upon the plasma membrane. Both side effects of the present drugs, myopathy and generalized lipidosis, are thought to be directly related to the physicochemical characteristics of the compounds.     

Expression of developmental myosin and morphological characteristics in adult rat skeletal muscle following exercise-induced injury

The extent and stability of the expression of developmental isoforms of myosin heavy chain (MHCd), and their association with cellular morphology, were determined in adult rat skeletal muscle fibres following injury induced by eccentrically-biased exercise. Adult female Wistar rats [274 (10)?g] were either assigned as non-exercised controls or subjected to 30?min of treadmill exercise (grade, ?16°; speed, 15?m?·?min^?1), and then sacrificed following 1, 2, 4, 7, or 12 days of recovery (n?=?5–6 per group). Histologically and immunohistologically stained serial, transverse cryosections of the soleus (S), vastus intermedius (VI), and tibialis anterior (TA) muscles were examined using light microscopy and digital imaging. Fibres staining positively for MHCd (MHCd⁺) were seldom detected in the TA. In the VI and S, higher proportions of MHCd⁺ fibres (0.8% and 2.5%, respectively) were observed in rats at 4 and 7 days post-exercise, in comparison to all other groups combined (0.2%, 1.2%; P?≤?0.01). In S, MHCd⁺ fibres were observed less frequently by 12 days (0.7%) than at 7 days (2.6%) following exercise. The majority (85.1%) of the MHCd⁺ fibres had morphological characteristics indicative of either damage, degeneration, repair or regeneration. Most of the MHCd⁺ fibres also expressed adult slow, and/or fast myosin heavy chain. Quantitatively, the MHCd⁺ fibres were smaller (<2500?μm²) and more angular than fibres not expressing MHCd. Thus, there was a transient increase in a small, but distinct population of MHCd⁺ fibres following unaccustomed, functional exercise in adult rat S and VI muscles. The observed close coupling of MHCd expression with morphological changes within muscle fibres suggests that these characteristics have a common, initial exercise-induced injury-related stimulus.     

Cardiac ankyrin repeat protein is preferentially induced in atrophic myofibers of congenital myopathy and spinal muscular atrophy

Cardiac ankyrin repeat protein (CARP), which is structurally characterized by the presence of four ankyrin repeat motifs in its central region, is believed to be localized in the nucleus and to participate in the regulation of cardiac-specific gene expression in cardiomyocytes. However, we recently found that CARP was induced in skeletal muscle by denervation, leading us to speculate that CARP may be induced under some pathological conditions. In the present study, we immunohistochemically analyzed the expression of CARP in 11 cases of spinal muscular atrophy (SMA) and 14 cases of congenital myopathy. In SMA, CARP was expressed selectively in severely atrophic myofibers, suggesting that CARP expression may reflect the status of muscle atrophy. Furthermore, in the congenital myopathies, the expression patterns of CARP were distinct among the subtypes, which included nemaline myopathy, myotubular myopathy, central core disease, and congenital fiber type disproportion. Although CARP was preferentially expressed in severely damaged myofibers in nemaline myopathy, it was not detected in central core disease. These findings suggest that immunohistochemical evaluation of CARP may be helpful in the diagnosis of SMA and the congenital myopathies.     

Dystrophy-like myopathy in the cat

Two cases of feline myopathy are described which were associated with moderate locomotor disturbances. At necropsy, a pale coloured skeletal musculature was found with severe hypertrophy of diaphragmatic musculature. Histologically, the myopathy was characterized by varying fibre diameter, internal nuclei, moderate degeneration and necrosis of solitary muscle fibres and slight to moderate endomysial and perimysial fibrosis. Only very few regenerating muscle fibres were present. The histological findings are compatible with those found in human muscular dystrophies and so this feline myopathy may be considered as a dystrophy-like myopathy.     

X-linked myotubular myopathy: clinical and pathological findings in a family

A five-generation family with recessively inherited X-linked myotubular myopathy was investigated. Two of the affected boys, who were siblings and were verified by muscle biopsy to have the disease, died 3 days and 3 months, respectively, after birth. They showed marked hypotonus from birth, general muscle weakness and asphyxia. Three other boys, who were probably affected by the disease, had severe asphyxia and died shortly after birth. In three of the five cases there was polyhydramnios. The muscle biopsies of the two siblings revealed predominance of small fibres with central nuclei and accumulation of mitochondria in the central parts of the fibres. In one of the boys mainly the type 1 fibres were hypotrophic. The postmortem examination revealed variation in the involvement of different muscles, the anterior tibial muscle being the most severely affected. Intrafusal muscle fibres and myocardium were apparently unaffected. There was no involvement of the spinal cord. The clinical examination of two obligate carriers in the family revealed no muscle weakness but the muscle biopsy showed pathological changes including greatly increased variability of fibre size, and many fibres with central nuclei. The findings indicate that muscle biopsy is of value in genetic counselling to detect carriers although the observed changes were unspecific.     

Muscle degeneration in chicks caused by Senna occidentalis seeds.

Acute intoxication with Senna occidentalis seeds was studied in chicks. Seven-day-old chicks were fed ground dried seeds of this plant mixed with regular chicken ration at a concentration of 4% by weight for 15 days. Feed intake and body weight were markedly affected and a high level of lethality was observed. Necropsy examination of chicks from the experimental group revealed paleness and atrophy of thoracic muscles. Degenerative and necrotic fibres were observed in skeletal muscle by histological examination. Muscle histochemistry showed accumulation of lipids and numerous acid phosphatase-positive muscle fibres. Electron microscopy revealed atrophic muscle fibres, lipid storage, dilatation of the sarcoplasmic reticulum and abnormal mitochondria.