Congenital muscle fibre type disproportion: clinical, morphological and biochemical findings in children

Muscle biopsies from quadriceps femoris muscle of normal subjects and subjects with symptoms of congenital myopathy, aged 1-3 years, were examined for morphological and biochemical differences. Four patients showed clinical signs of Congenital Fibre Type Disproportion (CFTD) as described originally by Brooke (1973), while the other cases showed only signs of hypotonia and diffuse weakness as described by Clancy et al. (1980). No morphological differences between patients with different clinical signs were found in any biopsy sample from the quadriceps femoris muscle, as regards fibre size; type I fibres were always smaller than type II fibres. The difference in fibre size was more than 15% in all cases, and the variability coefficient of the larger type II fibres was less than 250. Nevertheless, as regards fibre occurrence, two patients showed more type I fibres than type II fibres, four patients showed more type II fibres than type I fibres and one patient had a marked type II fibre predominance. Enzyme activities assayed in the crude mitochondrial fraction showed no abnormalities between normal subjects and patients. An increase in the activity of lactate dehydrogenase in the crude extract was found in all cases.     

Fibre types in the locomotory muscles of an antarctic teleost,Notothenia rossii

Summary The metabolic and structural differentiation of locomotory muscles of Notothenia rossii has been investigated. In this species sustained locomotion is achieved by sculling with enlarged pectoral fins (labriform locomotion), whilst the segmental myotomal muscle is reserved for burst activity. Red, white and subepidermal fibres can be distinguished in the trunk by histochemical and ultrastructural criteria. The main pectoral muscle (m. adductor profundus) consists entirely of red fibres. These three main fibres types show differences in histochemical staining profiles, capillarization, myofibril shape and packing, and lipid and mitochondrial content. The fractional volume of mitochondria amounts to 38% for pectoral, 30% for red myotomal and 1.9% for white myotomal fibres. Enzyme activities of red pectoral muscle are consistent with a higher potential for aerobic glucose and fatty acid oxidation than for the red myotomal fibres. Mg²⁺ Ca²⁺ -myofibrillar ATPase activities are similar for red pectoral and myotomal muscles and approximately half of those white fibres. Specialisations of N. rossii muscles associated with labriform swimming and locomotion at Antarctic temperatures are discussed.     

ENU Mutagenesis Reveals a Novel Phenotype of Reduced Limb Strength in Mice Lacking Fibrillin 2

Background

Fibrillins 1 (FBN1) and 2 (FBN2) are components of microfibrils, microfilaments that are present in many connective tissues, either alone or in association with elastin. Marfan''s syndrome and congenital contractural arachnodactyly (CCA) result from dominant mutations in the genes FBN1 and FBN2 respectively. Patients with both conditions often present with specific muscle atrophy or weakness, yet this has not been reported in the mouse models. In the case of Fbn1, this is due to perinatal lethality of the homozygous null mice making measurements of strength difficult. In the case of Fbn2, four different mutant alleles have been described in the mouse and in all cases syndactyly was reported as the defining phenotypic feature of homozygotes.

Methodology/Principal Findings

As part of a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified a mouse mutant, Mariusz, which exhibited muscle weakness along with hindlimb syndactyly. We identified an amber nonsense mutation in Fbn2 in this mouse mutant. Examination of a previously characterised Fbn2-null mutant, Fbn2^fp, identified a similar muscle weakness phenotype. The two Fbn2 mutant alleles complement each other confirming that the weakness is the result of a lack of Fbn2 activity. Skeletal muscle from mutants proved to be abnormal with higher than average numbers of fibres with centrally placed nuclei, an indicator that there are some regenerating muscle fibres. Physiological tests indicated that the mutant muscle produces significantly less maximal force, possibly as a result of the muscles being relatively smaller in Mariusz mice.

Conclusions

These findings indicate that Fbn2 is involved in integrity of structures required for strength in limb movement. As human patients with mutations in the fibrillin genes FBN1 and FBN2 often present with muscle weakness and atrophy as a symptom, Fbn2-null mice will be a useful model for examining this aspect of the disease process further.     

Acute lindane poisoning with development of muscle necrosis.

A 35-year-old man ingested food contaminated with lindane, an insecticide containing almost pure gamma hexachlorocyclohexane. Grand mal seizures and severe acidemia developed rapidly. The seizures recurred for nearly 2 hours, then ceased. In addition, the patient had muscle weakness and pain, headaches, episodic hypertension, myoglobinuria, acute renal failure and anemia. Pancreatitis developed 13 days after the ingestion of lindane. A muscle biopsy on the 15th day of illness demonstrated widespread necrosis and regeneration of muscle fibres. The patient''s condition improved and he was discharged 24 days after the onset of his illness. During the year following the poisoning the patient noted difficulty with recent memory, loss of libido and easy fatigability. One year after lindane ingestion the results of physical examination, including those for muscle power and bulk, were normal.     

Effects of training on axial muscle of two cyprinid species: Chondrostoma nasus (L.) and Leuciscus cephalus (L.)

The effect of an endurance training program lasting 17 weeks was studied in two cyprinid species, Chondrostoma nasus (L.) and Leuciscus cephalus (L.). Red, intermediate and white axial muscle were investigated. Morphometrical analysis revealed that training induced, in both species, increased red and intermediate muscle mass, fibre diameter and capillarization. Differences between species in the response to training were observed for volume densities of mitochondria and lipid. In contrast to C. nasus, L. cephalus show higher values for these compartments in red and intermediate fibres. The results are considered adaptational changes which increase the aerobic capacity of red and intermediate muscle fibres to meet higher sustained swimming activities.     

A study of the peripheral innervation and muscle fibre types of Ictalurus nebulosus (Lesueur) and Ictalurus punctatus (Rafinesque)

The innervation pattern and fibre types of the axial musculature of two closely related catfish species with differing lifestyles, Ictalurus nebulosus (Lesueur) and I. punctatus (Rafinesque) were investigated. Both fish displayed the multiple innervation pattern in the red muscle. However, the white muscle of I. nebulosus demonstrated terminal innervation while I. puncrurus displayed multiply innervated white muscle fibres. Fibre typing utilizing histochemical techniques for glyco-gen, lipid, succinic dehydrogenase and glucose-6-phosphate dehydrogenase revealed the typical teleostean distribution of red, intermediate and white muscle fibres in both fish. Staining was greatest in the red muscle fibres and least in the white muscle fibres. The white muscle fibres of I. punctatus stained slightly more for lipid than the white fibres of I. nebulosus which may be correlated with a greater aerobic capacity related to lifestyle and possibly innervation.     

Muscle fibre types and metabolism in post-larval and adult stages of notothenioid fish

Summary A histochemical study was carried out on muscle fibre types in the myotomes of post-larval and adult stages of seven species of notothenioid fish. There was little interspecific variation in the distribution of muscle fibre types in post-larvae. Slow fibres (diameter range 15–60 m) which stained darkly for succinic dehydrogenase activity (SDHase) formed a superficial layer 1–2 fibres thick around the entire lateral surface of the trunk. In all species a narrow band of very small diameter fibres (diameter range 5–62 m), with only weak staining activity, occurred between the skin and slow fibre layer. These have the characteristics of tonic fibres found in other teleosts. The remainder of the myotome was composed of fast muscle fibres (diameter range 9–75 m), which stain weakly for SDHase, -glycerophosphate dehydrogenase, glycogen and lipid. Slow muscle fibres were only a minor component of the trunk muscles of adult stages of the pelagic species Champsocephalus gunnari and Pseudochaenichthys georgianus, consistent with a reliance on pectoral fin swimming during sustained activity. Of the other species examined only Psilodraco breviceps and Notothenia gibberifrons had more than a few percent of slow muscle in the trunk (20%–30% in posterior myotomes), suggesting a greater involvement of sub-carangiform swimming at cruising speeds. The ultrastructure of slow fibres from the pectoral fin adductor and myotomal muscles of a haemoglobinless (P. georgianus) and red-blooded species (P. breviceps), both active swimmers, were compared. Fibres contained loosely packed, and regularly shaped myofibrils numerous mitochondria, glycogen granules and occasional lipid droplets. Mitochondria occupied >50% of fibre volume in the haemoglobinless species P. georgianus, each myofibril was surrounded by one or more mitochondria with densely packed cristae. No significant differences, however, were found in mean diameter between fibres from red-blooded and haemoglobinless species. The activities of key enzymes of energy metabolism were determined in the slow (pectoral) and fast (myotomal) muscles of N. gibberifrons. In contrast to other demersal Antarctic fish examined, much higher glycolytic activities were found in fast muscle fibres, probably reflecting greater endurance during burst swimming.     

Localisation and quantification of dehydrogenase activities in single muscle fibres of mdx gastrocnemius

The kinetics of succinate (SDH) and lactate (LDH) dehydrogenases were determined in single muscle fibres in unfixed sections of the gastrocnemius of dystrophic mdx mice (with an X-linked genetic disorder lacking a cytoskeletal protein, dystrophin) and age-matched C57BL/10 control mice. Quantitative gel substrate-film techniques and a real-time image analysis system were used. Three main fibre types were observed in regenerated mdx gastrocnemius and in corresponding controls: small fibres (S) with high SDH and LDH initial reaction velocities and activities, large fibres (L) with low activities of these dehydrogenases and intermediate-sized fibres (I) with intermediate enzyme activities. The small and intermediate fibres in both mdx and control muscles exhibited respectively high and moderate subsarcolemmal SDH and LDH activities attributable to accumulated mitochondria. The ratios of the initial velocities of the intrinsic enzyme reactions in the sarcoplasm, excluding the subsarcolemmal regions, of mdx muscle fibres compared to those in control fibres were 0.958 (S), 1.09 (I) and 0.959 (L) for SDH, and 1.03 (S), 1.06 (I) and 1.07 (L) for LDH. A parameter a, a measure of the diffusion of LDH out of muscle sections during incubation on gel substrate films, was found to be 0.981 and 1.00 in mdx and control muscles, respectively. Thus there are no significant differences in the activities and microenvironments of the enzymes between regenerated mdx muscle fibres and normal control muscle fibres. These data suggest that dystrophin deficiency in mdx muscles has no effects on the interactions of LDH with cytoskeletal proteins or on SDH activities in mitochondria whose number and morphology differ in mdx muscle fibres compared to those in normal controls. SDH and LDH activities were also found in the mitochondria clustered on two longitudinally directed poles of each central nucleus in regenerated mdx muscle fibres. They were proportional to the activities in the sarcoplasm excluding the subsarcolemmal regions. Accepted: 12 October 1999     

Bronchitis in Bristol

Eleven autopsied cases of bronchiolar emphysema are reported. In all, both lungs were involved. Their pleural surfaces were finely bosselated, presenting an appearance resembling that of the liver in Laennec''s cirrhosis. The lungs were firm, they cut with increased fibrous resistance, and the cut surfaces were honeycombed with cysts.Microscopically, the cysts originated in terminal bronchioles. Their walls were thickened with fibrous tissue, elastic fibres and prominent smooth muscle. Areas of lung parenchyma were replaced by fibrous tissue rich in elastic fibres.The etiology of this disease is unknown. Inherent weakness of the myoelastic wall of the respiratory bronchiole, hypoplasia of the distal segment of the respiratory unit, and superimposed recurrent respiratory infections probably are essential in its pathogenesis. The pulmonary changes cause interference with hemorespiratory gaseous exchange.Death was due to respiratory failure in seven cases, to cardiac failure in three and to superimposed staphylococcal pneumonia in one.     

Quantitative histochemistry of three mouse hind-limb muscles: the relationship between calcium-stimulated myofibrillar ATPase and succinate dehydrogenase activities

Summary A quantitative modification of Meijer's calcium-lead capture method, for the demonstration of calcium-stimulated myofibrillar ATPase activity at physiological pH, is described. A range of myofibrillar ATPase activities has been found among fast muscle fibres in two mouse hind-limb muscles. The myofibrillar ATPase activity of fast muscle fibres is 1.5–3 times higher than the myofibrillar ATPase activity of slow muscle fibres.Myofibrillar ATPase activities and succinate dehydrogenase activities of individual muscle fibres have been determined in serial sections. Activities of the two enzymes are correlated positively in soleus (fast and slow fibres), and negatively in plantaris (almost all fast) and extensor digitorum longus muscle (all fast). However, this correlation is not significant among the oxidative fibres in the extensor digitorum longus. The fibres of the latter muscle cannot be classified satisfactorily into two sub-types.     

Neuromuscular organization of avian flight muscle: architecture of single muscle fibres in muscle units of the pectoralis (pars thoracicus) of pigeon (Columba livia)

The M. pectoralis (pars thoracicus) of pigeons (Columba livia) is comprised of short muscle fibres that do not extend from muscle origin to insertion but overlap ''in-series''. Individual pectoralis motor units are limited in territory to a portion of muscle length and are comprised of either fast twitch, oxidative and glycolytic fibres (FOG) or fast twitch and glycolytic fibres (FG). FOG fibres make up 88 to 90% of the total muscle population and have a mean diameter one-half of that of the relatively large FG fibres. Here we report on the organization of individual fibres identified in six muscle units depleted of glycogen, three comprised of FOG fibres and three comprised of FG fibres. For each motor unit, fibre counts revealed unequal numbers of depleted fibres in different unit cross-sections. We traced individual fibres in one unit comprised of FOG fibres and a second comprised of FG fibres. Six fibres from a FOG unit (total length 15.45 mm) ranged from 10.11 to 11.82 mm in length and averaged (± s.d.) 10.74 ± 0.79 mm. All originated bluntly (en mass) from a fascicle near the proximal end of the muscle unit and all terminated intramuscularly. Five of these ended in a taper and one ended bluntly. Fibres coursed on average for 70% of the muscle unit length. Six fibres from a FG unit (total length 34.76 mm) ranged from 8.97 to 18.38 mm in length and averaged 15.32 ± 3.75 mm. All originated bluntly and terminated intramuscularly; one of these ended in a taper and five ended bluntly. Fibres coursed on average for 44% of the muscle unit length. Because fibres of individual muscle units do not extend the whole muscle unit territory, the effective cross-sectional area changes along the motor unit length. These non-uniformities in the distribution of fibres within a muscle unit emphasize that the functional interactions within and between motor units are complex.     

Histochemical characterization of myotomal muscle in the bluntnose minnow, Pimephales notatus Rafinesque

A histochemical study of the axial muscles in the bluntnose minnow, Pimephales notatus Rafinesque, revealed three main types of fibres-red, white and pink. These were distin- guished on the basis of glycogen or lipid contents, or of NADH diaphorase and actomyosin ATPase activities. White muscle formed the largest fraction of the total, whereas red ranged from slightly > 1% of the total cross-sectional area in the immediately post-pectoral fin region to slightly <4% in the post-anal region. Pink muscle formed only a few layers of fibres between red and white. Results are discussed in relation to possible division of labour among the different types of fibres. However, it is emphasized that fibre types may be more functionally versatile in fish muscles than has frequently been supposed.     

Activity patterns of phosphofructokinase,glyceraldehydephosphate dehydrogenase,lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle

Summary Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.     

Activity patterns of phosphofructokinase, glyceraldehydephosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle.

Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.     

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

Summary This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.     

An ultrastructural and histochemical study of the axial musculature in the African lungfish

Summary Red, intermediate, and white axial muscle fibres of African lungfish were studied using histochemical techniques and electron microscopy. Gross dissection revealed the presence of a small wedge of red coloured muscle along the lateral line. This wedge was shown by histochemical demonstrations of lactate and succinate dehydrogenases, of adenosine triphosphatases, and of lipid to be composed of a mosaic of red and intermediate fibres measuring 23.63 and 34.30 m in average diameter, respectively. The bulk of the myotome was composed of white fibres having an average diameter of 67.35 m. Mitochondrial density, capillarity and lipid content were very low for all fibres. These data suggest that the axial musculature is geared primarily for anaerobic function. The mosaic arrangement of fibres, and the lack of a subsarcolemmal band of mitochondria suggests that the lungfish have a muscle organisation that is transitional between lower vertebrates and amphibians.     

The fine structure of muscle fibres of roach, Rutilus rutilus (L.), and chub, Leuciscus cephalus (L.), Cyprinidae, Teleostei: interspecific differences and effects of habitat and season

Red and white axial muscle fibres from roach and chub were investigated by electron microscopy. Fish from three different localities were compared. Qualitative and quantitative analysis of myofibrils, mitochondria, lipid and subsarcolemmal cytoplasm with regard to muscle fibre type, species, season and habitat were made. Muscle fibre types differ significantly with the exception of the subsarcolemmal cytoplasm in roach. Within-species lipid content of red fibres differs between seasons. However, the most marked effect on red muscle fibres within species and season as regards volume density of lipid and mitochondria can be attributed to the different localities. The results are discussed in relation to mode of life and differences in habitat.     

Abnormal actin binding of aberrant β-tropomyosins is a molecular cause of muscle weakness in TPM2-related nemaline and cap myopathy

NM (nemaline myopathy) is a rare genetic muscle disorder defined on the basis of muscle weakness and the presence of structural abnormalities in the muscle fibres, i.e. nemaline bodies. The related disorder cap myopathy is defined by cap-like structures located peripherally in the muscle fibres. Both disorders may be caused by mutations in the TPM2 gene encoding β-Tm (tropomyosin). Tm controls muscle contraction by inhibiting actin-myosin interaction in a calcium-sensitive manner. In the present study, we have investigated the pathogenetic mechanisms underlying five disease-causing mutations in Tm. We show that four of the mutations cause changes in affinity for actin, which may cause muscle weakness in these patients, whereas two show defective Ca2+ activation of contractility. We have also mapped the amino acids altered by the mutation to regions important for actin binding and note that two of the mutations cause altered protein conformation, which could account for impaired actin affinity.     

Biochemical verification of quantitative histochemical analysis of succinate dehydrogenase activity in skeletal muscle fibres

Summary The purpose of this investigation was to examine critically the validity of a computerized quantitative microphotometric histochemical technique for the determination of succinate dehydrogenase (SDH) activity in skeletal muscle fibres. Sections from the anterior costal diaphragm were removed from Fischer-344 rats (n = 12) and assayed histochemically to determine SDH activity. The SDH activity in individual muscle fibres was computed using a computerized microphotometric histochemical technique which involves measurement of the optical density of deposited diformazan derived from nitroblue tetrazolium within the fibres. To validate the histochemical technique, whole muscle SDH activities were calculated from the histochemical procedure and were compared to SDH activities determined from whole muscle homogenates via a standard quantitative biochemical assay. The mean within-day variability of the computerized microphotometric histochemical technique of determining SDH activity was 6% (range = 0.5–10.9%) for an area containing ~50 fibres and 6.1% (range = 1.05–14.9%) for an individual muscle fibre. Similarly, the mean between-day variability of the microphotometric histochemical technique of determining SDH activity was 5.9% (range = 2.6–13.9%) for an area containing ~50 fibres and 6.6% (range = 2.2–13.9%) for an individual muscle fibre. The inter-class correlation coefficient between biochemically determined SDH activity and histochemically determined SDH activity was r = 0.83 (p < 0.05). Collectively, these data demonstrate that the quantitative histochemical technique of Blanco et al. (1988) is both valid and reliable in the determination of SDH activity in skeletal muscle fibres.     

The characterization of Tasmanian devil Sarcophilius harrisii pelage fibres and their associated lipids

The Tasmanian devil (Sarcophilius harrisii) is the largest living marsupial carnivore left on Earth. In this paper we report the results of the first thorough characterization of the keratin fibres comprising the Tasmanian devil pelage. The fibre's morphology, structure, composition and surface have been investigated. The results have been compared with those of a number of other mammalian species including carnivores and herbivores. The fibres structure was found to be consistent with that expected for a keratin fibre. From the results of the bound lipid analysis it can be concluded that the Tasmanian devil is a typical mammal in which the 21‐carbon atom anteiso branched fatty acid is the predominant bound fatty acid. This is consistent with the Tasmanian devil's position in the mammalian phylogenetic tree. The amino acid analysis places the devil in line with other carnivores. The high cystine and proline content may correlate with the Tasmanian devil's diet which is rich in muscle and collagen proteins.