Effect of ageing on the myosin heavy chain composition of the human sternocleidomastoid muscle.

The myosin heavy chain (MyHC) composition of ageing limb muscles is transformed into a slower phenotype and expresses fast-twitch fibre type atrophy, presumably due to age-related motor unit remodelling and a change in the patterns of physical activity. It is not known if ageing affects the sternocleidomastoid muscle (SCM) in a similar way. The goal of the study was to analyze the MyHC composition and the size of muscle fibres in the ageing SCM by immunohistochemical methods and quantitative analysis and stereology using our own software for morphometry. We hypothesize that with ageing the MyHC composition of SCM transforms similarly as in ageing limb muscles, but the size of the muscle fibres is less effected as in limb muscles. The study was performed on the autopsy samples of the SCM in 12 older males. The results were compared with those published in our previous study on 15 young adult males. An ageing SCM transforms into a slower MyHC profile: the percentage of slow-twitch fibres is enhanced (numerical proportion 44.6 vs. 31.5%, P<0.05; area proportion 57.2 vs. 38.4%, P<0.05). The share of hybrid 2a/2x fibres is diminished (numerical proportion 14.1 vs. 26.8%, P<0.05), the area proportion of all fast-twitch fibres expressing MyHC-2a and 2x is smaller (50.6 vs. 63.5%, P<0.05), and the area proportion of fibres expressing the fastest myosin isoform MyHC-2x is smaller too (19.0 vs. 34.5%, P<0.05). The slower phenotype with the preferential reduction of the fibres expressing the fastest MyHC-2x provide circumstantial evidence for: (i) more fast-twitch than slow-twitch motor units being lost; and (ii) reinnervation by the surviving motor units. There appears to be no significant influence on muscle fibre size, which is congruent with relatively unchanged SCM activity during life.

The utility of three-dimensional optical projection tomography in nerve injection injury imaging.

The examination of nerve microarchitecture has hitherto been limited solely to two-dimensional imaging techniques. The objective of this study was to evaluate the ability of optical projection tomography to discern the nerve microarchitecture and injection injury in three dimensions. Five piglets were studied, whose median and lingual nerves were unilaterally injected post mortem with preset volumes of local anaesthetic, excised and subsequently made transparent with benzyl alcohol benzyl benzoate. Images were captured in three dimensions. The same contralateral nerves were used as controls. Using optical projection tomography, we observed differences between the internal organisation of the median and the lingual nerves, which potentially explain the variations in their susceptibility to injury. This was demonstrated in three dimensions as a disruption to the fascicles in the lingual nerve, and their displacement in the median nerve. This new technology offers potential for studying nerve microarchitecture topography and its tolerance to injection injury.

Myosin heavy chain composition of the human sternocleidomastoid muscle.

The sternocleidomastoid (SCM) muscle is one of the neck muscles responsible for head posture and control of head movement. It functions in rotation, inclination, protraction, extension and flexion of the head, whilst chewing and in exerting increased respiratory efforts. This study is the first one describing the myosin heavy chain (MyHC) isoform composition of the SCM muscle of presumably healthy young males for the purpose of better understanding the contractile properties of the muscle as well as to help in evaluation of pathologically altered structure of the muscle. Autopsy samples were processed immunohistochemically to reveal the MyHC isoform composition. The muscle fibres expressed MyHC-1 (31.5%), -2a (29.7%) and -2x (4.3%) or co-expressed MyHC-2a with MyHC-2x (26.8%), MyHC-1 with MyHC-2a (4.1%) and/or MyHC-1, -2a with -2x (1.1%). In addition to the MyHC isoforms, characteristic of adult limb muscles, a very low percentage of muscle fibres (0.2-2.7%) expressed MyHC-neo, which is normally not found in adult limb muscles. Only two samples exhibited MyHC-neo at a rather higher percentage (6.3% and 7.5%) of muscle fibres. The high share of hybrid fibres and the presence of MyHC-neo in the SCM muscle differ from that of adult limb muscles where hybrid fibres are rare and the expression of immature MyHC isoforms occurs only in pathological or experimental conditions. Since the SCM muscle shares the same embryogenic potential as limb muscles, its distinct MyHC expression appears to be associated with twin innervation and with the intrinsic specialisation to perform multiple functions.

Wearing of complete dentures reduces slow fibre and enhances hybrid fibre fraction in masseter muscle.

Edentulous conditions and use of complete dentures alter the function of jaw muscles, which is presumably reflected in the myosin heavy chain (MyHC) isoform composition. This study is the first dealing with MyHC isoforms expression in edentulous persons with the aim to clarify to which extent the decreased functional load following teeth loss contributes to the changed muscle phenotype during ageing. We analysed MyHC expression in old masseter muscle at decreased and full functional load by comparing age-matched edentulous and dentate subjects. Edentulous subjects had upper and lower complete dentures. Dentate subjects had at least 24 natural teeth in continuous dental arches with two molars present in each quadrant and normal intermaxillary relationship. The adaptive response to the reduced masticatory load was lower numerical and area proportion of MyHC-1 expressing fibres and higher numerical proportion of hybrid fibres in edentulous compared with dentate subjects with no significant difference in the proportion of MyHC-neo-expressing fibres between both groups. We conclude that the observed differences in the proportion of fibre types between denture wearers and dentate subjects cannot be ascribed to degenerative changes intrinsic to the ageing muscle, but to functional differences in muscle activity and to morphological alterations of stomatognathic system accompanying the complete teeth loss.

Correcting the axial shrinkage of skeletal muscle thick sections visualized by confocal microscopy.

Confocal microscopy is a suitable method for measurements and visualization of skeletal muscle fibres and the neighbouring capillaries. When using 3D images of thick sections the tissue deformation effects should be avoided. We studied the deformation in thick sections of the rat skeletal muscle from complete stacks of images captured with confocal microscope. We measured the apparent thickness of the stacks and compared it to the slice thickness deduced from calibrated microtome settings. The ratio of both values yielded the axial scaling factor for every image stack. Careful sample preparation and treatment of the tissue cryosections with cold Ringer solution minimize the tissue deformation. We conclude that rescaling by the inverse of the axial scaling factor of the stack of optical slices in the direction of the microscope optical axis satisfactorily corrects the axial deformation of skeletal muscle samples.

Characterization of the capillary network in skeletal muscles from 3D data.

In this review we present immunohistochemical methods for visualization of capillaries and muscle fibres in thick muscle sections. Special attention is paid to the procedures that preserve good morphology. Applying confocal microscopy and virtual 3D stereological grids, or tracing of capillaries in virtual reality, length of capillaries within a muscle volume or length of capillaries adjacent to a muscle fibre per fibre length, fibre surface area or fibre volume can be evaluated by an unbiased approach. Moreover, 3D models of capillaries and muscle fibres can be produced. Comparison of the developed methods with counting capillary profiles from 2D sections is discussed and the reader is warned that counting capillary profiles from 2D sections can underestimate the capillary length by as much as 75 percent. Application of the described 3D methodology is illustrated by the anatomical remodelling of capillarity during acute denervation and early reinnervation in the rat soleus and extensor digitorum longus muscles.

Effects of divergent selection for 8-week body weight on postnatal enzyme activity pattern of 3 fiber types in fast muscles of male broilers (Gallus gallus domesticus).

A divergent selection experiment was conducted for 8-wk BW in chickens. At 3, 6, 9, and 12 wk of age, samples of pectoralis profundus (PP) and biceps femoris (BF) muscles from fast-growing and slow-growing lines were used to estimate the enzyme activities and muscle fiber diameter. Microphotometric measurements made in situ of succinate dehydrogenase (SDH, EC 1.3.99.1) and glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.99.5) were completed on serial sections of PP and BF muscles from male chickens, in order to examine the ratio of SDH:GPDH activity in single fibers. On the basis of the SDH:GPDH activity ratios, muscle fibers were divided using cluster analysis into 3 populations of different fiber types (O = oxidative, OG = oxidative-glycolytic, and G = glycolytic). Cockerels of the SGL attained an 8.1-fold increase and those of the FGL a 6.8-fold increase in BW at 12 wk compared with that at 3 wk of age. The O, OG, and G type fibers of the BF muscles of the SGL had significantly (P ≤ 0.001) lower SDH:GPDH activity ratios than those of the FGL. A step decrease in the SDH:GPDH activity of O, OG, and G fibers in the PP of both lines occurred, and this differed significantly between SGL and FGL (P ≤ 0.001). Age and line effects influenced the diameter of the 3 fiber types in the BF muscle only. In contrast to this response, all 3 fiber types of the PP muscles reached similar diameters in both lines during the growth process from wk 3 to 12. From the results of this study, we concluded that the activities of metabolic enzymes in skeletal muscle fibers are under the influence of muscle type, age, and selection pressure. Microphotometry is a suitable method for the evaluation of enzyme activity measured in a single muscle fiber. The method enables precise estimation of enzyme activities, especially in muscles composed of populations of different metabolic fiber types.

Software for muscle fibre type classification and analysis.

Fibre type determination requires a large series of differently stained muscle sections. The manual identification of individual fibres through the series is tedious and time consuming. This paper presents a software that enables (i) adjusting the position of individual fibres through a series of differently stained sections (image registration) and identification of individual fibres through the series as well as (ii) muscle fibre classification and (iii) quantitative analysis. The data output of the system is the following: numerical and areal proportions of fibre types, fibre type size and optical density (grey level) of the final reaction product in every fibre. The muscle fibre type can be determined stepwise, based on one set of stained sections while further, newly stained sections can be added to the already defined muscle fibre profile. Several advantages of the presented software application in skeletal muscle research are presented. The system is semiquantitative, flexible, and user friendly.

Software for muscle fibre type classification and analysis.

Fibre type determination requires a large series of differently stained muscle sections. The manual identification of individual fibres through the series is tedious and time consuming. This paper presents a software that enables (i) adjusting the position of individual fibres through a series of differently stained sections (image registration) and identification of individual fibres through the series as well as (ii) muscle fibre classification and (iii) quantitative analysis. The data output of the system is the following: numerical and areal proportions of fibre types, fibre type size and optical density (grey level) of the final reaction product in every fibre. The muscle fibre type can be determined stepwise, based on one set of stained sections while further, newly stained sections can be added to the already defined muscle fibre profile. Several advantages of the presented software application in skeletal muscle research are presented. The system is semiquantitative, flexible, and user friendly.

A novel staining method for quantification and 3D visualisation of capillaries and muscle fibres.

The aim of this study was to introduce a combined fluorescent staining that clearly demonstrates capillaries and distinguishes them from the basal lamina of muscle fibres in skeletal muscle tissue. The triple staining with CD31, Griffonia (Bandeira) simplicifolia lectin (GSL I) and laminin efficiently distinguishes vascular endothelium from the basal lamina of skeletal muscle fibres in physiological and pathological conditions. The presented triple staining method has several advantages, which facilitate quantitative analysis of the capillary network, and its relation to individual muscle fibres.

Long-term changes in myosin heavy chain composition after botulinum toxin a injection into rat medial rectus muscle.

PURPOSE: To study long-term changes of extraocular muscles after botulinum toxin (Botx) A-induced paralysis, with special emphasis on myosin heavy chain (MyHC) isoform pattern in muscle fibers. METHODS: Botx A (5 IU) was injected into the ocular medial rectus (MR) muscles of adult rats. After 1, 5, and 8 months muscle cross sections were examined immunohistochemically, histochemically, and morphometrically. MyHC content was analyzed by gel electrophoresis. RESULTS: Paralyzed MR muscles displayed mildly atrophic and hypertrophic muscle fibers and decreased oxidative metabolism, due to decreased succinate dehydrogenase activity. However, muscle morphology was not grossly disturbed. MyHC profile was shifted toward slower isoforms. Electrophoretic analysis showed that the share of MyHCI, and especially of MyHCIIa and MyHCIIx/d, increased several fold, whereas the share of MyHCIIb decreased heavily during the first 5 months. Immunohistochemical analysis generally mirrored the results obtained by electrophoresis. Moreover, specific extraocular MyHC isoform MyHCeom disappeared and could not be detected during the whole experimental period. The portion of MyHCIIb relatively increased 8 months after Botx A injection, although the MyHC profile was still far from normal. CONCLUSIONS: These long-lasting changes in Botx A-paralyzed ocular MR muscles most probably reflect their inability to regain their unique functional characteristics after new motor end plate formation and recovery of muscle contraction.

Effect of chronic uncontrollable stress on myosin heavy chain expression in rat masseter muscle.

This study has evaluated the influence of chronic uncontrollable painless stress on the expression of myosin heavy chain (MyHC) isoforms in deep and superficial parts of the rat masseter muscle. Muscle cross sections of three month old Wistar rats were processed for immunohistochemical staining. In control muscle the three adult fast MyHC isoforms (-2a, -2x/d, -2b), were nonhomogenously distributed: in the smaller anterior part of the muscle only fast MyHC isoforms were expressed. The major superficial and posterior part of the muscle fibres also contained MyHC-1 (10%) and only 7% expressed of MyHC-2b. Psychological stress significantly modified the expression of particular MyHC isoforms in both parts of the muscle. In the anterior part of the experimental masseter the proportion of MyHC-2b expressing fibres decreased and 10% of the muscle fibres expressed MyHC-1. The superficial and posterior part of the experimental muscle contained significantly more muscle fibres expressing MyHC-2b isoforms. Regarding whole cross-sectional area transition towards faster phenotype was evident due to a higher percentage of either pure MyHC-2b containing fibres or hybrid MyHC-2x/d and -2b expressing fibres. The results of this study demonstrate that psychological stress is accompanied by a modification of muscle phenotype which is region specific. Opposite changes in deep and superficial part of the muscle could be ascribed to differences in genetic programme, function or susceptibility to hormones in both parts of masseter.

Muscle activity-resistant acetylcholine receptor accumulation is induced in places of former motor endplates in ectopically innervated regenerating rat muscles.

Expression of acetylcholine receptors (AChRs) in the extrajunctional muscle regions, but not in the neuromuscular junctions, is repressed by propagated electric activity in muscle fibers. During regeneration, subsynaptic-like specializations accumulating AChRs are induced in new myotubes by agrin attached to the synaptic basal lamina at the places of former motor endplates even in the absence of innervation. We examined whether AChRs still accumulated at these places when the regenerating muscles were ectopically innervated and the former synaptic places became extrajunctional. Rat soleus muscles were injured by bupivacaine and ischemia to produce complete myofiber degeneration. The soleus muscle nerve was permanently severed and the muscle was ectopically innervated by the peroneal nerve a few millimeters away from the former junctional region. After 4 weeks of regeneration, the muscles contracted upon nerve stimulation, showed little atrophy and the cross-section areas of their fibers were completely above the range in non-innervated regenerating muscles, indicating successful innervation. Subsynaptic-like specializations in the former junctional region still accumulated AChRs (and acetylcholinesterase) although no motor nerve endings were observed in their vicinity and the cross-section area of their fibers clearly demonstrated that they were ectopically innervated. We conclude that the expression of AChRs at the places of the former neuromuscular junctions in the ectopically innervated regenerated soleus muscles is activity-independent.

Dynamic nature of fibre-type specific expression of myosin heavy chain transcripts in 14 different human skeletal muscles.

The main goal of this study was to find out, whether the appearance of fibres without evident myosin heavy chain (MyHC) transcript expression (negative fibres) implies the existence of additional MyHC transcripts in human muscle fibres. Fourteen different skeletal muscles were analysed also to verify how MyHC transcript expression matches histochemical phenotypes of fibres. For this purpose, the expression of beta-slow, 2a and 2x MyHC transcripts, demonstrated by in situ hybridisation technique, was analysed within type I, IIC, IIA, IIAX and IIX fibres, determined according to the activity of myofibrillar ATPase. Additionally, MyHC isoform expression was immunohistochemically demonstrated and metabolic profiles of negative fibres were estimated. From a total of 4444 muscle fibres analysed, only 0.8% of fibres were negative, among them type I prevailed, the remainder were type IIA and IIX fibres. The majority of fibres expressed only beta, 2a and 2x MyHC transcripts and they mostly matched type I, IIA and IIX fibres respectively, but two minor hybrid fibre groups (beta/2a and 2ax) exhibited variable histochemical phenotype. The infrequency, the prevailing oxidative-glycolytic metabolic profile of negative type I fibres and frequent co-appearance with transitional type IIC fibres imply that the negative fibres rather result from fibre type transition than express an additional slow or even 2b MyHC transcripts. The appearance of hybrid and mismatched fibres additionally indicates that fibre type transition occurs also in presumably normal skeletal muscles, what enables the muscles to tune even with minimal changes in mechanical demands.

Three-dimensional study of the capillary supply of skeletal muscle fibres using confocal microscopy.

Three-dimensional (3D) study of capillary network of individual muscle fibres in rat extensor digitorum longus (EDL) and soleus (SOL) muscles is presented. Stereology and 3D reconstruction techniques were applied to stacks of serial optical sections recorded by a confocal microscope from thick muscle slices. The results suggest that SOL muscle fibres have a larger surface area and volume as well as a larger length of capillaries per fibre length than EDL. On the other hand, these two muscles have a similar ratio of capillary length to fibre surface area. The 3D approach to evaluation of muscle fibre capillarization brings many advantages over traditional measurements made on single muscle sections and could also be applied to the study of angiogenesis in other tissues.

Changes of enzyme activities in the myocardium and skeletal muscle fibres of cardiomyopathic hamsters. A cytophotometrical study.

Cytophotometrical measurements of enzyme activities were performed in the myocardium and skeletal muscle fibres from normal and cardiomyopathic hamsters (BIO 8262) during ageing from 12-14 to 120-190 days. Myocardium as well as vastus lateralis muscles of cardiomyopathic hamsters showed changes in enzyme activities. The skeletal muscle fibres were typed into slow-oxidative, fast-oxidative glycolytic and fast-glycolytic to investigate fibre type-related changes in muscles of cardiomyopathic hamsters. The following myopathic changes were mainly found: Myofibrillic ATPase was depressed in the myocardium of both ventricles in all investigated age stages. The ATPase activity of the right ventricle was more decreased than that of the left one. Additionally, a metabolic shift was observed in myocardium and slow-oxidative muscle fibres at the onset of clinical symptoms, which appeared from day 150 to day 190. During the period from 42 up to 190 days of life an increase of oxidative (succinate dehydrogenase) activity was measured in the myocardium of both ventricles and in slow oxidative fibres of vastus lateralis muscle as a proximal muscle. At earlier ages, the fast fibres of myopathic vastus lateralis muscle showed higher glycolytic (glycerol-3-phosphate dehydrogenase) activity than those of normal muscles. However, at the age of 120-190 days the metabolic profile of fast fibres was normalized. In gastrocnemius muscle as a distal muscle no changes of enzyme activities were measured, suggesting the investigated hereditary myopathy effected proximal, but not distal muscles.

Fiber types in the mouse levator auris longus muscle: a convenient preparation to study muscle and nerve plasticity.

The histochemical composition of the levator auris longus (LAL) muscle has been investigated in adult NMRi mice. Histochemical reaction for myofibrillar adenosine triphosphatase (ATPase) after preincubation in alkaline and acidic media, nicotine amideadenine-dinucleotide dehidrogenase (NADH-dehydrogenase), and alpha-glycerophosphate dehydrogenase were performed on cryosections of LAL muscle. Expression of myosin heavy chain (MyHC) isoforms was detected with the immunoperoxidase method applying monoclonal antibodies against MyHC isoforms -1, -2a, -2x/d, and -2b, as well as by sodium dodecylsulfate (SDS) glycerol gel electrophoresis. The muscle was proven to be a pure fast-twitch muscle. The most numerous fibers in LAL muscles contained MyHC-2b and some MyHC-2a. Histochemically, pure IIA fibers with oxidative metabolism and pure IIB fibers with glycolytic metabolism were detected. In contrast to the majority of mature control muscles, numerous hybrid fibers coexpressing MyHC-2x/d with MyHC-2a or MyHC-2b were present. Both hybrids were oxidative-glycolytic; additionally, some hybrids containing MyHC-2a were oxidative. In one out of six muscles, traces of MyHC-1 were detected both with immunoperoxidase staining and with SDS glycerol gel electrophoresis. Rare fibers that exceptionally expressed small amounts of MyHC-1 always coexpressed MyHC-2a, which is an additional proof that pure type I fibers do not exist in LAL. Due to these histochemical characteristics and to its previously described morphological features, the use of the LAL muscle as a model for various studies, particularly muscle and nerve interactions, is emphasized.

Fibre types and myosin heavy chain expression in the ocular medial rectus muscle of the adult rat.

Myosin heavy chain (MHC) expression was determined immunohistochemically in individual muscle fibre types characterised by activities of ATPase and the key oxidative and glycolytic enzymes in rat ocular medial rectus (MR) muscles. In the global layer (GL), glycolytic activity of muscle fibres was higher and oxidative activity lower, than in the orbital layer (OL). Muscle fibres in the former displayed rosette-like organisation with a slow fibre surrounded by several fast fibres, which expressed either MHCIIa or MHCIIb, but many co-expressed both isoforms. In the OL some slow fibres co-expressed MHCIIa. Extraocular MHC isoform (MHCeom) could not be determined immunohistochemically and no pure MHCIIx/d containing fibres were found, suggesting that these isoforms, demonstrated electrophoretically, are co-expressed with others. Slow muscle fibres in both layers co-expressed MHCbeta slow, MHCalpha cardiac and MHC-slow tonic. Neonatal isoform (MHCneo) was co-expressed in several fast and slow muscle fibres in the orbital, but not global layer. Slow fibres in the GL displayed very low oxidative activity. Electrophoretic analysis of ocular MR muscle homogenates revealed that about 50% of total MHC was MHCIIb, MHCeom was quite prominent (25%), and MHCIIa, MHCIIx/d and MHCI contributed each about 8%. MHCneo, MHCslow tonic and MHCalpha cardiac could not be identified as separate bands.

Myosin heavy chain profiles in regenerated fast and slow muscles innervated by the same motor nerve become nearly identical.

Plasticity of mature muscles exposed to different activation patterns is limited, probably due to restricted adaptive range of their muscle fibres. In this study, we tested whether satellite cells derived from slow muscles can give rise to a normal fast muscle, if transplanted to the fast muscle bed. Marcaine-treated rat soleus and extensor digitorum longus (EDL) muscles were transplanted to the EDL muscle bed and innervated by the 'EDL' nerve. Six months later expression of myosin heavy chain isoforms was analysed by areal densities of fibres, binding specific monoclonal antibodies, and by SDS gel electrophoresis. Both regenerated muscles closely resembled each other. Their myosin heavy chain profiles were similar to those in fast muscles although they were not identical to that in the control EDL muscle. Since not even regenerated EDL was able to reach the myosin heavy chain isoform profile of mature EDL muscle, our experimental model did not permit studying the adaptive capacity of satellite cells in different muscles in its whole extent. However, the results favour the multipotential myoblast stem cell population in rat muscles and underline the importance of the extrinsic regulation of muscle phenotype.