Evidence for low muscle capillary supply as a pathogenic factor in chronic compartment syndrome.

There is a paucity of data regarding the pathogenesis of chronic exertional compartment syndrome (CECS), its consequences for the muscles and the effects of treatment with fasciotomy. We analyzed biopsies from the tibialis anterior muscle, from nine patients, obtained during a decompressing fasciotomy and during follow-up 1 year later. Control biopsies were obtained from nine normal subjects. Muscle capillarity, fiber-type composition and fiber area were analyzed with enzyme- and immunohistochemistry and morphometry. At baseline, CECS patients had lower capillary density (273 vs 378 capillaries/mm(2) , P=0.008), lower number of capillaries around muscle fibers (4.5 vs 5.7, P=0.004) and lower number of capillaries in relation to the muscle fiber area (1.1 vs 1.5, P=0.01) compared with normal controls. The fiber-type composition and fiber area did not differ, but focal signs of neuromuscular damage were observed in the CECS samples. At 1-year follow-up after fasciotomy, the fiber area and the number of fibers containing developmental myosin heavy chains were increased, but no enhancement of the capillary network was detected. Thus, morphologically, patients with CECS seemed to have reduced microcirculation capacity. Fasciotomy appeared to trigger a regenerative response in the muscle, however, without any increase in the capillary bed.

Satellite cell dysfunction contributes to the progressive muscle atrophy in myotonic dystrophy type 1.

AIMS: Myotonic dystrophy type 1 (DM1), one of the most common forms of inherited neuromuscular disorders in the adult, is characterized by progressive muscle weakness and wasting leading to distal muscle atrophy whereas proximal muscles of the same patients are spared during the early phase of the disease. In this report, the role of satellite cell dysfunction in the progressive muscular atrophy has been investigated. METHODS: Biopsies were obtained from distal and proximal muscles of the same DM1 patients. Histological and immunohistological analyses were carried out and the past regenerative history of the muscle was evaluated. Satellite cell number was quantified in vivo and proliferative capacity was determined in vitro. RESULTS: The size of the CTG expansion was positively correlated with the severity of the symptoms and the degree of muscle histopathology. Marked atrophy associated with typical DM1 features was observed in distal muscles of severely affected patients whereas proximal muscles were relatively spared. The number of satellite cells was significantly increased (twofold) in the distal muscles whereas very little regeneration was observed as confirmed by telomere analyses and developmental MyHC staining (0.3-3%). The satellite cells isolated from the DM1 distal muscles had a reduced proliferative capacity (36%) and stopped growing prematurely with telomeres longer than control cells (8.4 vs. 7.1 kb), indicating that the behaviour of these precursor cells was modified. CONCLUSIONS: Our results indicate that alterations in the basic functions of the satellite cells progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy.

Unique basement membrane structure of human pancreatic islets: implications for beta-cell growth and differentiation.

Basement membranes (BMs) are an important part of the physiological microenvironment of pancreatic islet cells. In mouse islets, beta-cells interact directly with BMs of capillary endothelial cells. We have shown that in the human islets, the capillaries are surrounded by a double BM both in foetal and adult tissues. The endocrine islet cells are facing a BM that is separate from the endothelia. Laminins are the functionally most important component of BMs. The only laminin isoform present in the human endocrine islet BM is laminin-511 (previously known as laminin 10). The islet cells facing this BM have a strong and polarized expression of Lutheran glycoprotein, which is a well-known receptor for the laminin alpha 5 chain. Dispersed human islet cells adhere to purified human laminin-511 and the binding is equally effectively blocked by a soluble form of Lutheran as by antibody against integrin beta1. Our results reveal unique features of the BM structure of human islets, different from rodents. This information has potentially important implications for the generation of an optimal microenvironment for beta-cell function, proliferation and differentiation.

Blood vessels of human islets of Langerhans are surrounded by a double basement membrane.

AIMS/HYPOTHESIS: Based on mouse study findings, pancreatic islet cells are supposed to lack basement membrane (BM) and interact directly with vascular endothelial BM. Until now, the BM composition of human islets has remained elusive. METHODS: Immunohistochemistry with specific monoclonal and polyclonal antibodies as well as electron microscopy were used to study BM organisation and composition in human adult islets. Isolated islet cells and function-blocking monoclonal antibodies and recombinant soluble Lutheran peptide were further used to study islet cell adhesion to laminin (Lm)-511. Short-term cultures of islets were used to study Lutheran and integrin distribution. RESULTS: Immunohistochemistry revealed a unique organisation for human Lm-511/521 as a peri-islet BM, which co-invaginated into islets with vessels, forming an outer endocrine BM of the intra-islet vascular channels, and was distinct from the vascular BM that additionally contained Lm-411/421. These findings were verified by electron microscopy. Lutheran glycoprotein, a receptor for the Lm alpha5 chain, was found prominently on endocrine cells, as identified by immunohistochemistry and RT-PCR, whereas alpha(3) and beta(1) integrins were more diffusely distributed. High Lutheran content was also found on endocrine cell membranes in short-term culture of human islets. The adhesion of dispersed beta cells to Lm-511 was inhibited equally effectively by antibodies to integrin and alpha(3) and beta(1) subunits, and by soluble Lutheran peptide. CONCLUSIONS/INTERPRETATION: The present results disclose a hitherto unrecognised BM organisation and adhesion mechanisms in human pancreatic islets as distinct from mouse islets.

Fibre composition of human intrinsic tongue muscles.

The muscle fibre composition of three human intrinsic tongue muscles, the longitudinalis, verticalis and transversus, was investigated in four anterior to posterior regions of the tongue using morphological and enzyme- and immunohistochemical techniques. All three muscles typically contained type I, IIA and IM/IIC fibres. Type I fibres expressed slow myosin heavy chain (MyHC), type II fibres fast MyHC, mainly fast A MyHC, whereas type IM/IIC coexpressed slow and fast MyHCs. Type II fibres were in the majority (60%), but regional differences in proportion and diameter of fibre types were obvious. The anterior region of the tongue contained a predominance of relatively small type II fibres (71%), in contrast to the posterior region which instead showed a majority of larger type I and type IM/IIC fibres (66%). In general, the fibre diameter was larger in the posterior region. This muscle fibre composition of the tongue differs from those of limb, orofacial and masticatory muscles, probably reflecting genotypic as well as phenotypic functional specialization in oral function. The predominance of type II fibres and the regional differences in fibre composition, together with intricate muscle structure, suggest generally fast and flexible actions in positioning and shaping the tongue, during vital tasks such as mastication, swallowing, respiration and speech.

Satellite cells and training in the elderly.

In the present review, we describe the effects of ageing on human muscle fibres, underlining that each human muscle is unique, meaning that the phenotype becomes specifically changed upon ageing in different muscles, and that the satellite cells are key cells in the regeneration and growth of muscle fibres. Satellite cells are closely associated with muscle fibres, located outside the muscle fibre sarcolemma but beneath the basement lamina. They are quiescent cells, which become activated by stimulation, like muscle fibre injury or increased muscle tension, start replicating and are responsible for the repair of injured muscle fibres and the growth of muscle fibres. The degree of replication is governed by the telomeric clock, which is affected upon excessive bouts of degeneration and regeneration as in muscular dystrophies. The telomeric clock, as in dystrophies, does not seem to be a limiting factor in ageing of human muscle. The number of satellite cells, although reduced in number in aged human muscles, has enough number of cell divisions left to ensure repair throughout the human life span. We propose that an active life, with sufficient general muscular activity, should be recommended to reduce the impairment of skeletal muscle function upon ageing.

Intrafusal fiber type composition of muscle spindles in the first human lumbrical muscle.

We studied muscle spindles in the first lumbrical muscle of adult humans using myofibrillar ATPase (mATPase) activity. We found that muscle spindles exhibited a marked variability with respect to the number, position, length and detailed histochemical features of nuclear bag1, nuclear bag2 and nuclear chain fibers. Regarding mATPase activity, the nuclear bag2 fibers displayed lower alkali-stable mATPase activity along their length and many nuclear bag1 fibers tended to have lower acid-stable activity in the outer B region, whereas nuclear chain fibers exhibited medium acid-stable mATPase activity at pH 4.6. Almost 10% of spindle fibers displayed atypical features, as they were either located only at one spindle pole or exhibited mixed characteristics at either pole. The number of intrafusal fibers per spindle varied between 8 and 24. Strikingly, only 2 pairs from 22 muscle spindles had identical allotments of their intrafusal fibers. Muscle spindles in the first human lumbrical muscle contained more intrafusal fibers (12.3 +/- 4 per spindle on average) and especially relatively more nuclear bag fibers compared to other human skeletal muscles. Since each spindle apparently represents a unique morphological and physiological entity, the observed variability in the number and characteristics of intrafusal fibers in the first human lumbrical muscle likely reflects a wide range of finely tuned muscle spindle responses.

Myosin heavy chain IIa gene mutation E706K is pathogenic and its expression increases with age.

BACKGROUND: The authors recently described a new autosomal dominant myopathy (OMIM 605637 inclusion body myopathy 3) associated with a missense mutation in the myosin heavy chain (MyHC) IIa gene (MyHC IIa, Human Gene Map [HGM] locus MYH2). Young patients showed minor changes in their muscle biopsies, although dystrophic alterations and rimmed vacuoles with 15- to 20-nm tubulofilaments identical to those in sporadic inclusion body myositis (s-IBM) were observed in some of the adult (especially older) patients. The current study was undertaken to investigate the relation between expression of the mutant MyHC IIa and pathologic changes in muscle. METHODS: The expression of MyHC IIa in nine muscle specimens from six individuals carrying the mutation was analyzed by immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a new reverse transcriptase--PCR method to measure the relative abundance of the various MyHC transcripts. RESULTS: Young patients with muscle weakness and minor pathologic changes in muscle expressed MyHC IIa at undetectable levels. MyHC IIa was expressed at high levels in adults with a progressive clinical course and dystrophic muscle changes. In these cases, a large number of muscle fibers were hybrids with expression of more than one MyHC isoform. Both MyHC IIa alleles were equally expressed. The relative level of MyHC IIa transcripts exceeded that of the corresponding protein, indicating an increased turnover of mutated protein. MyHC IIa expression was a consistent finding in muscle fibers with rimmed vacuoles. CONCLUSIONS: The clear correlation between pathologic changes and expression of MyHC IIa indicates that defects in MyHC may lead not only to muscle weakness but also to muscle degeneration. The consistent expression of MyHC IIa in muscle fibers with rimmed vacuoles indicates that the breakdown of sarcomeric proteins is a key element in the pathogenesis of rimmed vacuoles of s-IBM type.

Human single masseter muscle fibers contain unique combinations of myosin and myosin binding protein C isoforms.

Striated craniofacial and limb muscles differ in their embryological origin, regulatory program during myogenesis, and innervation. In an attempt to explore the effects of these differences on the striated muscle phenotype in humans, the expression of myosin and myosin-associated thick filament proteins were studied at the single fiber level both in the human jaw-closing masseter muscle and in two limb muscles (biceps brachii and quadriceps femoris muscles). In the masseter, unique combinations of myosin heavy chain (MyHC) and myosin binding protein C (MyBP-C) isoforms were observed at the single fiber level. Compared to the limb muscles, the MyHC isoform expression was more complex in the masseter while the opposite was observed for MyBP-C. In limb muscles, a coordinated expression of three MyHC and three MyBP-C isoforms were observed, i.e., single fibers contained one or two MyHC isoforms, and up to three MyBP-C isoforms. Also, the relative content of the different MyBP-C isoforms correlated with the MyHC isoform expression. In the masseter, on the other hand, up to five different MyHC isoforms could be observed in the same fiber, but only one MyBP-C isoform was identified irrespective MyHC isoform expression. This MyBP-C isoform had a migration rate similar to the slow MyBP-C isoform in limb muscle fibers. In conclusion, a unique myofibrillar protein isoform expression was observed in the human masseter muscle fibers, suggesting significant differences in structural and functional properties between muscle fibers from human masseter and limb muscles.

Effects on physical performance and pain from three dynamic training programs for women with work-related trapezius myalgia.

To compare training programs for women with trapezius myalgia regarding physical performance and pain, 102 women were randomized to strength, endurance, co-ordination and non-training groups. Before and after the intervention, static strength and dynamic muscular endurance in shoulder muscles were measured on a Cybex II dynamometer. Muscle activity in shoulder muscles was monitored via surface EMG. The signal amplitude ratio between the active and passive phase of repeated contractions indicated the ability to relax. Pain at present, pain in general and pain at worst were measured on visual analogue scales. After training, within group comparisons showed that the training groups rated less pain, and in the strength training group ratings of pain at worst differed from the non-training group. Using the non-training group as a reference, static strength increased in the strength and endurance training groups and muscular endurance in all training groups. The study indicates that regular exercises with strength, endurance or co-ordination training of neck/shoulder muscles might alleviate pain for women with work-related trapezius myalgia.

Desmin-related myopathies in mice and man.

Desmin, the main intermediate filament (IF) protein in skeletal and heart muscle cells, is of great importance as a part of the cytoskeleton. The IFs surround and interlink myofibrils, and connect the peripheral myofibrils with the sarcolemma. In myotendinous junctions and neuromuscular junctions of skeletal muscle fibres, desmin is enriched. In the heart, desmin is increased at intercalated discs, the attachment between cardiomyocytes, and it is the main component in Purkinje fibres of the conduction system. Desmin is the first muscle-specific protein to appear during myogenesis. Nevertheless, lack of desmin, as shown from experiments with desmin knockout (K/O) mice, does not influence myogenesis or myofibrillogenesis. However, the desmin knock-out mice postnatally develop a cardiomyopathy and a muscle dystrophy in highly used skeletal muscles. In other skeletal muscles the organization of myofibrils is remarkably unaffected. Thus, the main consequence of the lack of desmin is that the muscle fibres become more susceptible to damage. The loss of membrane integrity leads to a dystrophic process, with degeneration and fibrosis. In the heart cardiac failure develops, whereas in affected skeletal muscles regenerative attempts are seen. In humans, accumulations of desmin have been a hallmark for presumptive desmin myopathies. Recent investigations have shown that some families with such a myopathy have a defect in the gene coding for alphaB-crystallin, whereas others have mutations in the desmin gene. Typical features of these patients are cardiac affections and muscle weakness. Thus, mutations in the desmin gene is pathogenic for a distinct type of muscle disorder.

Myosin heavy chain composition of the human lateral pterygoid and digastric muscles in young adults and elderly.

The myosin heavy chain (MyHC) content in different parts of, two jaw opening muscle, the human lateral pterygoid and the digastric muscles of five young adult and five elderly subjects (mean age 22 and 73 years, respectively) was determined, using gel electrophoresis and immunohistochemical methods. The lateral pterygoid of both young and elderly contained predominantly slow MyHC, and fast A MyHC was the major fast isoform. In contrast, the digastric was composed of slow, fast A and fast X MyHCs in about equal proportions in both age groups. About half of the lateral pterygoid fibres contained mixtures of slow and fast MyHCs, often together with alpha-cardiac MyHC. In the digastric, co-existence of slow and fast MyHCs was rare, and alpha-cardiac MyHC was lacking. On the other hand, co-expression of fast A and fast X MyHCs was found more often in the digastric than in the lateral pterygoid. In both age groups about half of the digastric IIB fibres contained solely fast X MyHC. In the lateral pterygoid, type IIB fibres with pure fast X MyHC was found in only one subject. The lateral pterygoid in elderly showed a significant amount of fibres with solely fast A MyHC, which were occasionally found in young adults. In the digastric, no significant differences were found between young and elderly, although the muscles of elderly contained lower mean value of slow MyHC, as compared to that of young muscles. It is concluded that the lateral pterygoid and the digastric muscles differ not only in the MyHC composition but also in modifications of the MyHC phenotypes during aging, suggesting that they have separate roles in jaw opening function.

The effects of different training programs on the trapezius muscle of women with work-related neck and shoulder myalgia.

The aim of this study was to examine the effects of training on the structural characteristics of the trapezius muscle in women with work-related trapezius myalgia. Muscle biopsies were taken before and after 10 weeks of three different training programs (strength, endurance and coordination). Enzyme-immunohistochemical analysis was performed to assess muscle fibre types, fibre area, capillary supply and cytochrome c oxidase (COX) activity. There was an increase in the proportion of type IIA fibres in strength trained group (P < 0.05). Strength training elicited a preferential increase in the area of type II fibres (P < 0.05); both strength and endurance programs induced an increase in the number of capillaries around type I and IIA muscle fibres. Finally, all training programs induced a decrease in the proportion of COX-negative fibres. In conclusion, the trapezius muscle of women with neck and shoulder myalgia is characterised by a great potential of adaptation to physical exercise over a period of 10 weeks. The significant changes in the number of capillaries and the specific changes induced by training at the level of muscle fibres might well explain the improvement of muscle function.

Differences in the distribution of synemin, paranemin, and plectin in skeletal muscles of wild-type and desmin knock-out mice.

Mice lacking the gene encoding for the intermediate filament protein desmin have a surprisingly normal myofibrillar organization in skeletal muscle fibers, although myopathy develops in highly used muscles. In the present study we examined how synemin, paranemin, and plectin, three key cytoskeletal proteins related to desmin, are organized in normal and desmin knock-out (K/O) mice. We show that in wild-type mice, synemin, paranemin, and plectin were colocalized with desmin in Z-disc-associated striations and at the sarcolemma. All three proteins were also present at the myotendinous junctions and in the postsynaptic area of motor endplates. In the desmin K/O mice the distribution of plectin was unaffected, whereas synemin and paranemin were partly affected. The Z-disc-associated striations were in general no longer present in between the myofibrils. In contrast, at the myotendinous and neuromuscular junctions synemin and paranemin were still present. Our study shows that plectin differs from synemin and paranemin in its binding properties to the myofibrillar Z-discs and that the cytoskeleton in junctional areas is particularly complex in its organization.

Human extraocular muscles: unique pattern of myosin heavy chain expression during myotube formation.

PURPOSE: To study the myosin heavy chain composition of the human extraocular muscles (EOMs) during development. METHODS: EOMs from human fetuses of 8 to 22 weeks of gestation were studied with immunocytochemistry and gel electrophoresis. Antibodies specific against nine isoforms of myosin heavy chain (MyHC) were used in serial frozen sections. RESULTS: The developing EOMs had a delayed time course of myotube formation and a unique composition and distribution of MyHCs compared with human limb skeletal muscle. The primary myotubes coexpressed two developmental isoforms of MyHCI from the earliest stages. The third developmental MyHCI delineated the future orbital layer at 10 to 12 weeks of gestation. MyHC-slow tonic also appeared early, whereas MyHC alpha-cardiac and MyHC-extraocular, important components of adult EOM, were never detected at the gestational ages studied. CONCLUSIONS: The developmental features of the EOMs differed significantly from those reported for limb muscles of the corresponding ages. It is clear that the knowledge of limb muscle development does not fully apply to more specialized muscles, such as the eye muscles. The extreme complexity displayed by the EOMs probably reflects their distinct embryonic origin, innervation, and regulatory program of myogenesis.

Concomitant increases in myonuclear and satellite cell content in female trapezius muscle following strength training.

A skeletal muscle fibre maintains its cytoplasmic volume by means of hundreds of myonuclei distributed along its entire length. Therefore it is hypothesised that changes in fibre size would involve modifications in myonuclear number. In this study, we have examined whether 10 weeks of strength training can induce changes in the number of myonuclei and satellite cells in female trapezius muscles. Biopsies were taken pre- and posttraining from the upper part of the descending trapezius muscle of nine subjects. Muscle samples were analysed for fibre area and myonuclear and satellite cell number using immunohistochemistry. There was a 36% increase in the cross-sectional area of muscle fibres. The hypertrophy of muscle fibres was accompanied by an approximately 70% increase in myonuclear number and a 46% increase in the number of satellite cells. Myonuclei number was positively correlated to satellite cell number indicating that a muscle with an increased concentration of myonuclei will contain a correspondingly higher number of satellite cells. The acquisition of additional myonuclei appears to be required to support the enlargement of multinucleated muscle cells following 10 weeks of strength training. Increased satellite cell content suggests that mitotic divisions of satellite cells produced daughter cells that became satellite cells.

The expression of androgen receptors in human neck and limb muscles: effects of training and self-administration of androgenic-anabolic steroids.

The purpose of this study was to investigate the immunohistochemical expression of androgen receptors (AR) in human vastus lateralis and trapezius muscles and to determine whether long-term strength training and self-administration of androgenic-anabolic steroids are accompanied by changes in AR content. Biopsy samples were taken from eight high-level power-lifters (P), nine high-level power-lifters who used anabolic steroids (PAS) and six untrained subjects (U). Myonuclei and AR were visualised in cross-sections stained with the monoclonal antibody against AR and 4',6-diamidino-2-phenylindole. The proportion of AR-containing myonuclei per fibre cross-section was higher in the trapezius than in the vastus lateralis (P<0.05). In the trapezius, the proportion of AR-containing myonuclei was higher in P compared to U and in PAS compared to both P and U (P<0. 05). On the contrary, in the vastus lateralis, there were no differences in AR content between the three groups. Myonuclear number in both muscles was higher in P compared to U and in PAS compared to both P and U (P<0.05). In conclusion, AR content differs greatly between human neck and limb muscles. Moreover, the regulation of AR-containing myonuclei following training and self-administration of androgenic-anabolic steroids is muscle dependent.

Laminin chains in developing and adult human myotendinous junctions.

In addition to being the specialized site for transmission of force from the muscle to the tendon, the myotendinous junction (MTJ) also plays an important role in muscle splitting during morphogenesis. An early event in the formation of the MTJ is a regional deposition of basement membranes. We used immunocytochemistry to investigate the distribution of laminin chains during the development of MTJs in human limb muscle at 8-22 weeks of gestation (wg) and in adult MTJs. We used polyclonal antibodies and a new monoclonal antibody (MAb) against the human laminin alpha1 G4/G5 domains. At 8-10 wg, laminin alpha1 and laminin alpha5 chains were specifically localized to the MTJ. Laminin alpha1 chain remained restricted to the MTJ at 22 wg as the laminin beta2 chain had appeared, whereas the laminin alpha5 chain became deposited along the entire length of the myotubes from 12 wg. In the adult MTJ, only vestigial amounts of laminin alpha1 and laminin alpha5 chains could be detected. On the basis of co-distribution data, we speculate that laminin alpha1 chain in the forming MTJ undergoes an isoform switch from laminin 1 to laminin 3. Our data indicate a potentially important role for laminin alpha1 chain in skeletal muscle formation. (J Histochem Cytochem 48:201-209, 2000)

Effects of anabolic steroids on the muscle cells of strength-trained athletes.

PURPOSE: Athletes who use anabolic steroids get larger and stronger muscles. How this is reflected at the level of the muscle fibers has not yet been established and was the topic of this investigation. METHODS: Muscle biopsies were obtained from the trapezius muscles of high-level power lifters who have reported the use of anabolic steroids in high doses for several years and from high-level power lifters who have never used these drugs. Enzyme-immunohistochemical investigation was performed to assess muscle fiber types, fiber area, myonuclear number, frequency of satellite cells, and fibers expressing developmental protein isoforms. RESULTS: The overall muscle fiber composition was the same in both groups. The mean area for each fiber type in the reported steroid users was larger than that in the nonsteroid users (P < 0.05). The number of myonuclei and the proportion of central nuclei were also significantly higher in the reported steroid users (P < 0.05). Likewise, the frequency of fibers expressing developmental protein isoforms was significantly higher in the reported steroid users group (P < 0.05). CONCLUSION: Intake of anabolic steroids and strength-training induce an increase in muscle size by both hypertrophy and the formation of new muscle fibers. We propose that activation of satellite cells is a key process and is enhanced by the steroid use. The incorporation of the satellite cells into preexisting fibers to maintain a constant nuclear to cytoplasmic ratio seems to be a fundamental mechanism for muscle fiber growth. Although all the subjects in this study have the same level of performance, the possibility of genetic differences between the two groups cannot be completely excluded.