Identification and distribution of some developmental isoforms of myosin heavy chains in avian muscle fibres

Summary Two monoclonal antibodies that react with all the slow skeletal myosin heavy chains in the mammalian skeletal muscles appeared to react with only SM1 myosin heavy chain in the post-hatch muscles of chicken. Further studies on the developing chicken showed one of these two antibodies to react with an additional myosin heavy chain in the early embryonic skeletal muscle as well as with the cardiac muscle. It is concluded that this antibody identified a slow muscle-like embryonic isoform of myosin heavy chain during earlier stages of development. While this embryonic isoform was more abundant during early development, the synthesis of SM1 myosin heavy chain was restricted to only presumptive slow muscle cells.     

Changes in the distribution of slow skeletal myosin heavy chain SM1 in developing avian muscle fibres

Summary The use of monoclonal antibodies against fast skeletal and slow skeletal myosin heavy chains (MHC) has shown the presence of significant amounts of slow skeletal type MHC in embryonic skeletal muscles of white leghorn chickens. The presence of this slow skeletal myosin heavy chain (SMHC) was not restricted to presumptive slow muscles only, as it was also observed in presumptive fast skeletal muscles. As was the case for embryonic MHC reactive with the antibody against fast skeletal myosin heavy chain (FMHC), the presence of SMHC could be detected at the earliest stages of myogenesis. It appeared to be present in most muscle cells during early embryonic development. The changes in its cellular distribution during subsequent embryonic and post-hatch period indicated its suppression in a certain proportion of the cells in both presumptive fast and slow skeletal muscles. Its time course of suppression, however, was much prolonged, not synchronized, and varied in fast and slow skeletal muscles during both embryonic and post-hatch development.     

Postnatal expression of myosin heavy chains in muscle spindles of the rat

Summary The immunocytochemical expression of two myosin isoforms in intrafusal muscle fibers was examined in soleus muscles of neonatal (zero to six days postpartum) and adult rats. Monoclonal antibodies specific for myosin heavy chains of the slow-tonic anterior latissimus dorsi (ALD58) and fast-twitch pectoralis (MF30) muscles of the chicken were used. In adults ALD58 bound to the intracapsular regions of bag₁ and bag₂ fibers and MF30 bound to the intracapsular regions of bag₂ and chain fibers. The extracapsular regions of intrafusal fibers and all extrafusal fibers did not react to ALD58 or MF30. Bag₁ and bag₂ fibers of neonatal rats expressed immature myosin patterns but chain fibers did not. The adult pattern of immunoreactivity of intrafusal fibers developed by the fourth postnatal day, when the patterns of motor but not sensory innervation in the spindle are still immature. Data suggest that the expression and maintenance of the specific anti-myosin immunoreactivity of intrafusal fibers during postnatal development of rat spindles is dependent upon sensory but not motor innervation. Moreover, afferents might regulate the gene expression responsible for synthesis of myosins isoforms specific to intrafusal fibers only in those myonuclei located within the capsule, but not in the myonuclei in extracapsular regions of intrafusal fibers.     

Developmental myosin heavy chain progression in avian type IIB muscle fibres

Summary Myosin heavy chain species were investigated during development in avian pectoralis major muscle (type IIB fibres) by high resolution anion-exchange chromatography of the myosin head region, subfragment-1. At 15 daysin ovo four distinct fast-type heavy chain species, I, II, III and IV, in order of elution, were identified. By 19 daysin ovo, form IV had become predominant and remained the major species through 3-days post-hatch. This form has been named theperihatch form. Between 3 and 5 days post-hatch, a second massive change occurred such that by 5 days post-hatch a new species, V, apparent at 19 daysin ovo in small amounts, dominated and at 8 days post-hatch was the only heavy chain species present. Form V, which corresponds to that previously identified as theposthatch form, continued as the major species through 20 days post-hatch and was replaced slowly by the adult form. N-terminal sequencing of CNBr peptides from three subfragment-1 heavy chain species, the peri-hatch (form IV), the post-hatch (form V) and adult, revealed differences in amino acid sequence consistent with the three being products of different genes. These results confirm and extend recent reports of complexity in fast heavy chain expression prior to hatching in the chicken (Hofmannet al., 1988; Van Horn & Crow, 1989).     

Expression of slow and fast myosin heavy chains in overloaded muscles of the developing rat

Summary The present study examines the developmental accumulation of slow myosin heavy chain in the extensor digitorum longus, soleus and plantaris muscles of rats after early post-natal imposition of mechanical overload by removal of synergistic muscles. The proportions of slow and fast myosin heavy chain were measured in each muscle by ELISA. Fibres expressing slow myosin were examined immunocytochemically using a monoclonal antibody specific for slow MHC. Between 30 and 60 days of age, MHC increases by 15% (p<0.001) in the soleus and by 27% (p<0.001) in the plantaris of normally developing, unoperated animals. The effect of overload on the soleus and plantaris is to accelerate the rate of increase in slow MHC accumulation so that levels are respectively 16 and 39% higher than controls by 30 days of age (p<0.001). By 60 days, the control soleus and plantaris attain levels of slow MHC roughly equivalent to their overloaded counterparts. In overloaded plantaris, the increase in levels of slow myosin does not occur at the expense of fast myosin expression. In the EDL there is a normal developmentally regulated decrease in slow MHC accumulation, reflected by a 40% decrease in levels of slow MHC (p<0.0001) and a 50% decrease in the number of slow fibres (p<0.001), between 30 days and 20 weeks of age. This elimination of slow myosin accumulation in the EDL is unimpeded by chronic overload. Thus, muscles react to mechanical overload in a tissue specific manner. The pattern of response is conservative and potentiates normal, long term maturational shifts in myosin heavy chain expression characteristic of each muscle.     

Analysis of myosin heavy chains at the protein level in horse skeletal muscle

Combined methodologies of enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting, traditional myofibrillar ATPase (mATPase) histochemistry and immunocytochemistry of whole biopsied samples were used to study myosin heavy chain (MHC) isoforms in the equine gluteus medius muscle. The ELISA technique allowed the quantification of the three MHC isoforms known to be present in different horse muscles: slow (MHC-I) and two fast (termed MHC-IIA and MCH-IIX). The SDS-PAGE method resolved MHCs in three bands: MHC-I, MHC-IIX and MHC-IIA from the fastest to the slowest migrating band and a quantification by densitometry for each MHC isoform was also possible. The identity of these three MHCs was confirmed by immunoblots with specific monoclonal antibodies. Five fibre types were defined immunohistochemically according to their MHC content: I, I + IIA, IIA, the hybrid IIAX and IIX. When quantitative data obtained with the four different methodologies were combined and compared, they were consistent and, when considered together, showed significant correlation. Nevertheless, the percentage of MHC-IIA histochemically derived was underestimated, while that of MHC-IIX was overestimated in comparison with the immunocytochemical determination of these MHC isoforms. The percentage of MHC-I obtained by ELISA technique was underestimated. In short, these integrated methods for the analysis of MHCs at the protein level demonstrate that equine skeletal muscle does not express the MHC-IIB, so type II fibres have been misclassified in numerous previous studies based upon the very traditional mATPase histochemistry. They also offer new prospects for muscle fibre typing in equine experimental studies and veterinary medicine.     

The re-expression of two myosin heavy chains in regenerated rat muscle spindles

The vascular supply and tendons of the extensor digitorum longus (EDL) muscles of two adult rats were unilaterally severed and the muscles were allowed to regenerate for 40 days. Serial frozen sections of muscle grafts were cut and stained for enzymes that delineated fiber type, sensory endings and motor endings. MF30 and ALD58, two antibodies which react only to intrafusal fibers in normal rat muscle, were reacted against sections of nerve-intact muscle grafts. Data were compared to that from muscles of normal rats. Encapsulated fibers devoid of sensory innervation and some extrafusal fibers in muscle grafts had a weak to moderate reaction to MF30, but no reaction to ALD58. Regenerated, encapsulated fibers with sensory innervation bound both MF30 and ALD58. These data indicate that afferents which reinnervate regenerated spindles retain the capacity to induce expression of spindle-specific myosin isoforms in rats.     

Myosin isozymes in avian skeletal muscles. I. Sequential expression of myosin isozymes in developing chicken pectoralis muscles

Myosin has been purified from chicken pectoralis muscle at various stages of development, from 10 days' incubation to approximately 10 months after hatching. Embryonic myosin from the earliest stage showed a high level of ATPase activity, similar to that obtained for adult pectoralis myosin. Two-dimensional peptide mapping of partial chymotryptic digests showed, however, that is heavy chain is quite different from that of adult fast myosin. The immunological crossreactivity observed between embryonic myosin and adult fast (pectoralis) myosin is therefore due to shared antigenic determinants rather than the presence of any adult isoforms. In an accompanying paper we will show that embryonic myosin at 10 days' incubation is not a single species, but consists of at least two heavy chain isozymes. The minor fraction binds slow light chains preferentially, and appears to be largely responsible for the observed crossreactivity with slow (ALD) myosin. None of the embryonic myosins is equivalent to the adult forms. Prior to hatching, LC3f is present only in very small amounts (less than 5%), and the adult light chain pattern, containing LC1f and LC3f in equimolar amounts, is not generated until after one week post-hatching. At about that time a new heavy chain population is detected, different from either the embryonic heavy chain or the adult heavy chain. The adult heavy chain peptide pattern appears from about three weeks' post-hatching, but a map indistinguishable from that of adult myosin is not observed until about 26 weeks. None of the observed differences in peptide maps can be related to different strains of chicken; pectoralis myosin from adult White Rock gave an identical map to that from White Leghorn. Unexpectedly, posterior latissimus dorsi (PLD) myosin from White Leghorn appears to be different from pectoralis myosin from the same strain, despite the histochemical and immunocytochemical similarity of the two muscles. We conclude that myosin polymorphism is widespread in muscle tissue, and that the expression of myosin isozymes and their subunits is under developmental regulation.     

Spatial and temporal patterns of myosin heavy chain expression in developing rat extraocular muscle

Summary The present study describes transitions in myosin heavy chain expression in the extraocular muscles of rats between the ages of E17 and adult. The unique phenotype of the extraocular muscle is reflected in its fibre type composition, which is comprised by six distinct profiles, each defined by location (orbital versus global layer) and innervation pattern (single versus multiple terminals). During extraocular muscle myogenesis, developmental myosin heavy chains were expressed in both primary and secondary fibres from embryonic day E17 through the first postnatal week. At this time, the downregulation of developmental myosin heavy chain isoforms began in the global layer in a fibre type-specific manner, reaching completion only after the first postnatal month. By contrast, developmental isoforms were retained in the overwhelming majority of orbital layer fibres into adulthood and expressed differentially along the length of these fibres. Fast myosin heavy chain was detected pre- and postnatally in developing secondary fibres and in all of the singly innervated fibre types and one of the multiply innervated fibre types in the adult. As many as four fast isoforms were detected in maturing extraocular muscle, including the extraocular muscle-specific myosin heavy chain. Slow myosin heavy chain was expressed in primary fibres throughout development and in one of the multiply innervated fibre types in the adult. In contrast, the pure fast-twitch retractor bulbi initially expressed slow myosin heavy chain in fibres destined to switch to the fast myosin heavy chain developmental programme. Based upon spatial and temporal patterns of myosin heavy chain isoform transitions, we suggest that epigenetic influences, rather than purely myogenic stage-specific factors, are critical in determining the unique extraocular muscle phenotype.     

Bilateral asymmetric deficiency of the pectoralis major muscle

We observed a rare, bilateral congenital deficiency of the pectoralis major muscle in a 72-year-old female cadaver in our gross anatomy dissection laboratory. The outward appearance of the anterior thoracic wall, which included well-developed breasts, revealed no obvious abnormalities. Upon dissection, the following features were observed: 1) on the left side, the sternal portion of the sternocostal head of the pectoralis major muscle was absent, the costal portion of the sternocostal head and the clavicular head were both well developed, a normal pectoralis minor was present, and the deltoid and subclavius muscles were not hypertrophied as is often the case when the pectoralis major muscle is deficient; 2) on the right side, the entire pectoralis major muscle was absent and the pectoralis minor, deltoid, and coracobrachialis muscles were infiltrated with connective tissue and fat; and 3) on both sides, the lateral pectoral nerves were absent and the medial pectoral nerves were present. The absence of the lateral pectoral nerves suggests that the deficiencies in the pectoralis major muscles are congenital malformations resulting from a developmental failure of the embryonic muscles rather than a sequel to polio or Poland's syndrome.     

Aplasia of pectoralis major muscle and renal anomalies

We describe two patients with aplasia of the pectoralis major muscle and renal anomalies. We think such an association, which has been reported previously, represents a new congenital malformation, probably not a syndrome but an acro-pectoro-renal field defect. We recommend renal ultrasonography be done on all children with aplasia of the pectoralis major.     

Identification of alpha-cardiac myosin heavy chain mRNA and protein in extraocular muscle of the adult rabbit

Summary Extraocular muscles contain both fast-twitch and multiply-innervated, tonic-contracting fibres. In rat, these fibres collectively express numerous myosin heavy chain isoforms including fast-type embryonic and neonatal, adult slow twitch type I and fast twitch type II, and a fast isoform unique to extraocular muscle. Immunocytochemical and Western blotting results are presented which suggest that, in rabbit, an additional species, the -cardiac myosin heavy chain, is present. The immunoreactive species is found in all rabbit extraocular muscles and in the rotatory extraocular muscles is expressed in almost all fibres which do not contain a fast myosin heavy chain. Positive identification of this isoform as the -cardiac myosin heavy chain was obtained by sequencing a cloned PCR product derived from extraocular muscle mRNA unique to the 3-end of rabbit -cardiac myosin heavy chain mRNA. This is the first unequivocal demonstration of -cardiac myosin heavy chain expression in extraocular muscle.     

Anterior intercostobrachial nerve penetrating the pectoralis minor or major muscle

Two previously unknown anomalies of the anterior intercostobrachial nerve were described. In one case, the anterior intercostobrachial nerve penetrated the pectoralis minor muscle. In the other case, it penetrated the pectoralis major muscle. In both cases, the anomalous nerve supplied the skin of the upper arm.     

Myosin isozymes in avian skeletal muscles. II. Fractionation of myosin isozymes from adult and embryonic chicken pectoralis muscle by immuno-affinity chromatography

Chicken pectoralis consists primarily of large white fibres, which react exclusively with antibodies prepared against adult fast myosin. There is, however, a small region of uniformly red fibres which responds to antibodies against adult slow myosin as well as adult fast myosin. The myosin extracted from this red region is also heterogeneous as shown by the presence of both slow and fast light chains. By means of immunoadsorbents, it has been possible to separate the 'red myosin' into a 'fast' component and a 'slow' component. These two fractions have been characterized with respect to their light and heavy chain content by one-dimensional and two-dimensional gel electrophoresis. The myosin heavy chain was reduced to the smaller fragments required for electrophoresis by proteolytic degradation. We conclude from the electrophoretic patterns that the 'fast' and 'slow' myosin components from the pectoralis red region closely resemble the myosin from the white region of the pectoralis and the myosin from the slow anterior latissimus dorsi (ALD) muscle. The demonstration of a 'slow myosin' in adult pectoralis muscle raises the possibility that the crossreactivity of embryonic pectoralis myosin with anti-slow (ALD) myosin antibodies might be due to the presence of such slow components in embryonic chicken muscle. Direct isolation of a slow component from embryonic pectoralis was achieved by immunoadsorption, as described for adult mixed muscle myosin. Analysis of the subunit composition by gel electrophoresis shows an enrichment in adult-type slow light chains, but the heavy chain pattern is quite distinct from that of adult slow heavy chain. These studies suggest that several myosin isozymes exist in embryonic chicken pectoralis, but that none is identical to those myosins found in the different fibres of the adult pectoralis muscle.