Expression of MyHC-15 and -2x in human muscle spindles: An immunohistochemical study.

To build on the existing data on the pattern of myosin heavy chain (MyHC) isoforms expression in the human muscle spindles, we aimed to verify whether the 'novel' MyHC-15, -2x and -2b isoforms are co-expressed with the other known isoforms in the human intrafusal fibres. Using a set of antibodies, we attempted to demonstrate nine isoforms (15, slow-tonic, 1, α, 2a, 2x, 2b, embryonic, neonatal) in different regions of intrafusal fibres in the biceps brachii and flexor digitorum profundus muscles. The reactivity of some antibodies with the extrafusal fibres was also tested in the masseter and laryngeal cricothyreoid muscles. In both upper limb muscles, the expression of slow-tonic isoform was a reliable marker for differentiating positive bag fibres from negative chain fibres. Generally, bag1 and bag2 fibres were distinguished in isoform 1 expression; the latter consistently expressed this isoform over their entire length. Although isoform 15 was not abundantly expressed in intrafusal fibres, its expression was pronounced in the extracapsular region of bag fibres. Using a 2x isoform-specific antibody, this isoform was demonstrated in the intracapsular regions of some intrafusal fibres, particularly chain fibres. To the best of our knowledge, this study is the first to demonstrate 15 and 2x isoforms in human intrafusal fibres. However, whether the labelling with an antibody specific for rat 2b isoform reflects the expression of this isoform in bag fibres and some extrafusal ones in the specialised cranial muscles requires further evaluation. The revealed pattern of isoform co-expression only partially agrees with the results of previous, more extensive studies. Nevertheless, it may be inferred that MyHC isoform expression in intrafusal fibres varies along their length, across different muscle spindles and muscles. Furthermore, the estimation of expression may also depend on the antibodies utilised, which may also react differently with intrafusal and extrafusal fibres.

Effect of altered innervation and thyroid hormones on myosin heavy chain expression and fiber type transitions: a mini-review.

In this mini-review, we briefly present the data regarding the effect of extrinsic factors, i.e., innervation and thyroid hormones (TH) on myosin heavy chain genes and isoforms expression and consequently on muscle fiber type transitions. It has been well known that reduced neuromuscular activity, hyperthyroidism or mechanical unloading stimulate slow-to-fast fiber type transitions, while increased neuromuscular activity, hypothyroidism and higher mechanical loading result in fast to slow fiber type transitions. As there is a plethora of results on these topics, we focus mostly on data relevant to our experimental model of slow-to-fast muscle transformation following heterochronous intramuscular isotransplantation in rats with altered TH status.

Slow to fast muscle transformation following heterochronous isotransplantation is influenced by host thyroid hormone status.

We studied the effect of regeneration, altered innervation and thyroid hormone (TH) levels on fiber type transitions in slow soleus (SOL) muscles grafted (GRAFT) into host extensor digitorum longus (EDLh) muscles of euthyroid (EU), hyperthyroid (HT) and hypothyroid (HY) Lewis strain rats. SOL muscles were excised from 3-week to 4-week-old inbred Lewis rats and intramuscularly transplanted into EDLh muscles of 2-month-old female rats of the same strain. The proportions of type 1, 2A, 2X and 2B fibers of GRAFT were determined by immunohistochemistry and compared with those of EDLh muscle and EDL and SOL muscles of the unoperated contralateral hind limb. After an average regeneration period of 6-7 months and after being reinnervated by the "fast" peroneal nerve of EDLh muscle, GRAFT was transformed into a fast muscle. However, the extent of GRAFT transformation varied with different TH states. In the EU rats, GRAFT contained about 95 % of fast fibers, among which type 2X and 2B fibers predominated (about 75 %). The transition toward fast muscle phenotype was more pronounced in HT status, where the fastest type 2B fibers predominated. On the contrary, in HY status, the slow to fast transformation was less pronounced, as GRAFT contained less type 2B and 2X but more type 2A and 1 fibers. We conclude that the type of innervation is the crucial factor for the slow to fast fiber type transitions in GRAFT, but the extent of muscle transformation is further modulated by altered TH status.

Myosin heavy chain-2b transcripts and isoform are expressed in human laryngeal muscles.

Three fast myosin heavy chain (MyHC) isoforms, i.e. MyHC-2a, -2x and -2b, are expressed in skeletal muscles of smaller mammals. In contrast, only MyHC-2a and -2x have been revealed in humans so far. The expression of MyHC isoforms is known to be wider in the functionally more specialized laryngeal muscles. Though mRNA transcripts of the MyHC-2b gene were found to be expressed in certain human skeletal and laryngeal muscles, the corresponding isoform has not been demonstrated in these muscles. To our knowledge, we are the first to demonstrate not only the expression of MyHC-2b transcripts using an in situ hybridization technique but also the corresponding protein, i.e. the MyHC-2b isoform, in some human laryngeal muscles by immunohistochemistry but not by polyacrylamide gel electrophoresis. Using a set of antibodies specific to MyHC isoforms, we demonstrated that MyHC-2b was always co-expressed with the major MyHC isoforms, not only with the fast ones (MyHC-2a and -2x) but with the slow isoform (MyHC-1) as well.

Histochemical and immunohistochemical profile of human and rat ocular medial rectus muscles.

PURPOSE: To compare the organization of human and rat ocular medial recti muscles (MR). METHODS: The cryosections of human and rat MR were processed for myofibrillar ATPase (mATPase), succinate dehydrogenase and glycerol-3-phosphate dehydrogenase. To reveal myosin heavy chain (MyHC) isoforms, specific monoclonal antibodies against MyHC-1/beta- slow, alpha-cardiac (-alpha), -2a, -2x, -2b, -extraocular (eom), -embryonic (-emb) and -neonatal (-neo) were applied. The MyHC gene expression was studied by in situ hybridization in human muscle. RESULTS: The muscle fibers were arranged in two distinct layers in both species. In the orbital layer most fibers were highly oxidative and expressed fast MyHC isoforms, whereas slow and oxidative fibers expressed MyHC-1 and -alpha, some of them also MyHC-2a, -2x, -eom, very rarely -emb, and -neo. In the global layer, slow fibers with very low oxidative and glycolytic activity and three types of fast fibers, glycolytic, oxidative and oxidative-glycolytic, could be distinguished. The slow medium-sized fibers with mATPase activity stable at pH 4.4 expressed mostly MyHC-1 and -alpha in rat, while in humans they co-expressed MyHC-1 with -2b, -2x, -eom, and -neo. In both species, the fast fibers showed variable mATPase activity after preincubation at pH 9.4, and co-expressed various combinations of MyHC-2b, -2x, -2a and -eom but not -emb and -neo. MyHC-2b expressing fibers were larger and glycolytic, while MyHC-2a expressing fibers were smaller and highly oxidative in both species. To our knowledge, the present study is the first that demonstrated the expression of MyHC-2b in any of human skeletal muscles. Though the expression of MyHC genes did not correlate with the immunohistochemical profile of fibers in human MR, the expression of MyHC-2b gene was undoubtedly confirmed. CONCLUSIONS: Rat MR represent a good model that can be applied to study human MR in experiment or disease, however certain differences are to be expected due to specific oculomotor demands in humans.

Demonstration of myosin heavy chain isoforms in rat and humans: the specificity of seven available monoclonal antibodies used in immunohistochemical and immunoblotting methods.

The aim of this paper was to present our experience with seven monoclonal antibodies, six of them were applied in immunohistochemistry and immunoblotting of MyHC isoforms in rats and humans, one of them, 6H1 (Lucas et al., 2000), was tested in human muscle sections only. The four antibodies specific to rat MyHC isoforms, BA-D5,SC-71, BF-35,BF-F3 (Schiaffino et al.,1989) reacted as declared both on muscle sections and immunoblots of rat except SC-71 antibody, which stained MyHC-2a and -2x bands in blots. One of the two commercially available antibodies, A4.74 antibody,reliably marked type 2a fibres of rat,but in blots it weakly stained MyHC-2a and -2x isoforms when used undiluted. The other one, F113.15F4, stained type 2a and 2x fibres and corresponding MyHC bands in blots. Therefore, using this antibody rat MyHC-2x can be additionally confirmed, which can be otherwise demonstrated only on the principle of exclusion with BF-35.Using the same set of antibodies human fast MyHC isoforms can be revealed less clearly. Namely, SC-71 and A4.74 antibodies intensively stained histochemical type 2a,predominantly expressing 2a MyHC transcripts and moderately type 2x fibres, expressing mostly 2x MyHC transcripts, in blots the antibodies recognized both fast isoforms. The 6H1antibody was the only one that selectively labelled type 2x fibres, whereas BF-35 left unstained only a variable proportion of histochemical type 2x fibres and MyHC-2x in blots. F113.15F4 did not distinguish between human fast fibre types and corresponding MyHC isoforms in blots. The negativeresults obtained with BF-F3 in muscle sections and in blots are in agreement with the absence of MyHC-2b in human skeletal muscles. Our results imply that the reactivity of antibodies specific to distinct MyHC isoforms should be carefully evaluated not only among various species but with the two different techniques used as well.

Effect of high-fat mixed lipid diet and swimming on fibre types in skeletal muscles of rats with colon tumours.

Skeletal muscle fibre types, whose characteristics are determined by myosin heavy chain (MyHC) isoforms, can adapt to changed physiological demands with changed MyHC isoform expression resulting in the fibre type transitions. The endurance training is known to induce fast-to-slow transitions and has beneficial effect in carcinogenesis, whereas the effect of an excessive fat intake and its interaction with the effect of swimming are less conclusive. Therefore, we studied the effect of high-fat mixed lipid (HFML) diet and long-term (21-week) swimming on fibre type transitions and their average diameters by immunohistochemical demonstration of MyHC isoforms in slow soleus (SOL), fast extensor digitorum longus (EDL), and mixed gastrocnemius medialis and lateralis (GM, GL) muscles, divided to deep and superficial portions (GMd, GMs, GLd, GLs), of sedentary and swimming Wistar rats with experimentally (dimethylhydrazine) induced colon tumours and fed either with HFML or low-fat corn oil (LFCO) diet. HFML diet induced only a trend for fast-to-slow transitions in SOL and in the opposite direction in GMd. Swimming triggered significant transitions in unexpected slow-to-fast direction in SOL, whereas in GMs the transitions had tendency to proceed in the expected fast-to-slow direction. The average diameters of fibre types were mostly unaffected. Hence, it can be concluded that if present, the effects of HFML diet and swimming on fibre type transitions were counteractive and muscle-specific implying that each muscle possesses its own adaptive range of response to changed physiological conditions.

Adaptive potential of human biceps femoris muscle demonstrated by histochemical, immunohistochemical and mechanomyographical methods.

The goal of this study was to estimate the ability of biceps femoris (BF) muscle, a hamstring muscle crucial for biarticulate movement, to adapt to changed functional demands. For this purpose and due to ethical reasons, in a group of healthy sedentary men and of 15 sprinters, a non-invasive mechanomyography (MMG) method was used to measure the muscle twitch contraction times (Tc). These correlate with the proportions of slow and fast fibres in the muscle. To further elucidate the data obtained by MMG method and to obtain reference data for the muscle, the fiber type proportions in autoptic samples of BF in sedentary young men were determined according to histochemical reaction for myofibrillar adenosine triphosphatase (mATPase). In one BF sample also myosin heavy chain (MyHC) isoform expression was demonstrated immunohistochemically. With MMG we indirectly demonstrated that biceps femoris muscle has a strong potential to transform into faster contracting muscle after sprint training, since the average Tc in sprinters was much lower (19.5 +/- 2.3 ms) than in the sedentary group (30.25 +/- 3.5 ms). The results of the histochemical and immunohistochemical analysis of BF muscle also imply a high adapting potential of this muscle. Beside type 1, 2a and 2x (2b) fibres a relatively high proportion of intermediate type 2c fibres (5.7% +/- 0.7), which co-expressed MyHC-1 and -2a, was found. Therefore, type 2c might represent a potential pool of fibres, capable of transformation either to slow type 1 or to fast type 2a in order to tune the functional response of BF muscle according to the actual functional demands of the organism.

Developmental myosins: expression patterns and functional significance.

Developing skeletal muscles express unique myosin isoforms, including embryonic and neonatal myosin heavy chains, coded by the myosin heavy chain 3 (MYH3) and MYH8 genes, respectively, and myosin light chain 1 embryonic/atrial, encoded by the myosin light chain 4 (MYL4) gene. These myosin isoforms are transiently expressed during embryonic and fetal development and disappear shortly after birth when adult fast and slow myosins become prevalent. However, developmental myosins persist throughout adult stages in specialized muscles, such as the extraocular and jaw-closing muscles, and in the intrafusal fibers of the muscle spindles. These myosins are re-expressed during muscle regeneration and provide a specific marker of regenerating fibers in the pathologic skeletal muscle. Mutations in MYH3 or MYH8 are responsible for distal arthrogryposis syndromes, characterized by congenital joint contractures and orofacial dysmorphisms, supporting the importance of muscle contractile activity and body movements in joint development and in shaping the form of the face during fetal development. The biochemical and biophysical properties of developmental myosins have only partially been defined, and their functional significance is not yet clear. One possibility is that these myosins are specialized in contracting against low loads, and thus, they may be adapted to the prenatal environment, when fetal muscles contract against a very low load compared to postnatal muscles.

Fibre type composition of soleus and extensor digitorum longus muscles in normal female inbred Lewis rats.

We have analysed the fibre type composition of soleus and extensor digitorum longus (EDL) muscles of normal female 4-6-month-old inbred Lewis rats. This rat strain is used in our ongoing study of the effects of thyroid hormone on myosin heavy chain (MyHC) isoform expression. On the basis of the mATPase reaction, soleus muscles contained 96.1 +/- 2.9% of type 1 fibres supplemented by 2A fibres. EDL muscles contained type 1 (5.5 +/- 1.0%), type 2A (18.8 +/- 1.7%) and type 2B (75.7 +/- 2.2%) fibres. Immunohistochemical analysis and SDS gel electrophoresis confirmed that most fibres in the soleus muscle expressed the type 1 (slow) MyHC isoform and that only a small proportion of fibres expressed the fast 2a MyHC isoform. Immunohistochemical analysis and SDS gel electrophoresis demonstrated that almost half of the 2B fibres of EDL muscles expressed the 2x/d MyHC isoform. In both muscle types, many fibres expressed more than one MyHC isoform. The content of slow fibres in the soleus muscle of female inbred Lewis rats was slightly higher than that reported for Wistar rats, but was considerably higher than that of Sprague-Dawley rats, whereas substantial differences were not found in the proportion of slow and fast fibre types in EDL muscles in these strains.

Enzyme- and immunohistochemical aspects of skeletal muscle fibers in brown bear (Ursus arctos).

To further elucidate the pattern of MHC isoform expression in skeletal muscles of large mammals, in this study the skeletal muscles of brown bear, one of the largest mammalian predators with an extraordinary locomotor capacity, were analyzed. Fiber types in longissimus dorsi, triceps brachii caput longum, and rectus femoris muscles were determined according to the myofibrillar ATPase (mATPase) histochemistry and MHC isoform expression, revealed by a set of antibodies specific to MHC isoforms. The oxidative (SDH) and glycolytic enzyme (alpha-GPDH) capacity of fibers was demonstrated as well. By mATPase histochemistry five fiber types, i.e., I, IIC, IIA, IIAX, IIX were distinguished. Analyzing the MHC isoform expression, we assume that MHC-I, -IIa, and -IIx are expressed in the muscles of adolescent bears. MHC-I isoform was expressed in Type-I fibers and coexpressed with presumably -IIa isoform, in Type-IIC fibers. Surprisingly, two antibodies specific to rat MHC-IIa stained those fast fibers, that were histochemically and immunohistochemically classified as Type IIX. This assumption was additionally confirmed by complete absence of fiber staining with antibody specific to rat MHC-IIb and all fast fiber staining with antibody that according to our experience recognizes MHC-IIa and -IIx of rat. Furthermore, quite high-oxidative capacity of all fast fiber types and their weak glycolytic capacity also imply for MHC-IIa and -IIx isoform expression in fast fibers of bear. However, in adult, full-grown animal, only MHC-I and MHC-IIa isoforms were expressed. The expression of only two fast isoforms in bear, like in many other large mammals (humans, cat, dog, goat, cattle, and horse) obviously meets the weight-bearing and locomotor demands of these mammals.

Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth.

To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (alpha-GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I. The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4-6 weeks of age, which coincides with the increased physical activity of puppies.