Myosin heavy chains in extraocular muscle fibres: Distribution, regulation and function.

This review examines kinetic properties and distribution of the 11 isoforms of myosin heavy chain (MyHC) expressed in extraocular muscle (EOM) fibre types and the regulation and function of these MyHCs. Although recruitment and discharge characteristics of ocular motoneurons during fixation and eye movements are well documented, work directly linking these properties with motor unit contractile speed and MyHC composition is lacking. Recruitment of motor units according to Henneman's size principle has some support in EOMs but needs consolidation. Both neurogenic and myogenic mechanisms regulate MyHC expression as in other muscle allotypes. Developmentally, multiply-innervated (MIFs) and singly-innervated fibres (SIFs) are derived presumably from distinct myoblast lineages, ending up expressing MyHCs in the slow and fast ends of the kinetic spectrum respectively. They modulate the synaptic inputs of their motoneurons through different retrogradely transported neurotrophins, thereby specifying their tonic and phasic impulse patterns. Immunohistochemical analyses of EOMs regenerating in situ and in limb muscle beds suggest that the very impulse patterns driving various ocular movements equip effectors with appropriate MyHC compositions and speeds to accomplish their tasks. These experiments also suggest that satellite cells of SIFs and MIFs are distinct lineages expressing different MyHCs during regeneration. MyHC compositions and functional characteristics of orbital fibres show longitudinal variations that facilitate linear ocular rotation during saccades. Palisade endings on global MIFs are postulated to respond to active and passive tensions by triggering axon reflexes that play important roles during fixation, saccades and vergence. How EOMs implement Listings law during ocular rotation is discussed.

Changes in Myosin Heavy Chain Isoforms Along the Length of Orbital Fibers in Rabbit Extraocular Muscle.

Purpose: Extraocular muscles express 10 myosin heavy chain (MyHC) isoforms that cater for a wide range of contractile speeds. We aim to characterize the variations in MyHC expression along the length of singly (SIFs) and multiply innervated fibers (MIFs) in the orbital layer of rabbit superior rectus muscle. Methods: Monospecific antibodies to nine MyHCs, including an anti-slow-tonic antibody characterized here were used to immunohistochemically map variations in MyHC distribution in serial sections along the muscle's full length. Results: The fastest MyHC, EO, is expressed at the endplate zone (EPZ) of SIFs, flanked proximally and distally by segments expressing the slower 2A, with or without embryonic MyHC. MIFs with constant diameter express α-cardiac MyHC at the EPZ, flanked by segments co-expressing α-cardiac/embryonic and possibly slow-tonic MyHCs. MIFs with varying diameter also express α-cardiac MyHC at the EPZ in their thin, central region, flanked by thin segments co-expressing α-cardiac/embryonic MyHCs, with long proximal and distal extensions of larger diameter that co-express embryonic/slow-tonic and α-cardiac or ß/slow MyHCs. Conclusions: Orbital fiber types express multiple MyHCs, with faster ones in SIFs, slower ones in MIFs, but all have fast EPZs and slower end segments. We hypothesize that these unique MyHC distributions enable these fibers to relax in two kinetically distinct phases while acting in an antagonistic manner during a saccade: the fast phases facilitate acceleration of eyeball rotation during agonist contraction, while the slow phases help its deceleration toward the visual target, thereby linearizing the saccade. These properties also facilitate pulley movements to implement Listing's law.

X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers.

The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments.

Chronic low-frequency stimulation transforms cat masticatory muscle fibers into jaw-slow fibers.

Cat masticatory muscle during regeneration expresses masticatory-specific myofibrillar proteins upon innervation by a fast muscle nerve but acquires the jaw-slow phenotype when innervated by a slow muscle nerve. Here, we test the hypothesis that chronic low-frequency stimulation simulating impulses from the slow nerve can result in masticatory-to-slow fiber-type transformation. In six cats, the temporalis muscle was continuously stimulated directly at 10 Hz for up to 12 weeks using a stimulator affixed to the skull. Stimulated muscles were analyzed by immunohistochemistry using, among others, monoclonal antibodies against masticatory-specific myosin heavy chain (MyHC), myosin binding protein-C, and tropomyosins. Under the electrodes, stimulation induced muscle regeneration, which generated slow fibers. Deep to the electrodes, at two to three weeks, two distinct populations of masticatory fibers began to express slow MyHC: 1) evenly distributed fibers that completely suppressed masticatory-specific proteins but transiently co-expressed fetal MyHCs, and 2) incompletely transformed fibers that express slow and masticatory but not fetal MyHCs. SDS-PAGE confirmed de novo expression of slow MyHC and ß-tropomyosin in the stimulated muscles. We conclude that chronic low-frequency stimulation induces masticatory-to-slow fiber-type conversion. The two populations of transforming masticatory fibers may differ in their mode of activation or lineage of their myogenic cells.

Regulation of jaw-specific isoforms of myosin-binding protein-C and tropomyosin in regenerating cat temporalis muscle innervated by limb fast and slow motor nerves.

Cat jaw-closing muscles are a distinct muscle allotype characterized by the expression of masticatory-specific myofibrillar proteins. Transplantation studies showed that expression of masticatory myosin heavy chain (m-MyHC) is promoted by fast motor nerves, but suppressed by slow motor nerves. We investigated whether masticatory myosin-binding protein-C (m-MBP-C) and masticatory tropomyosin (m-Tm) are similarly regulated. Temporalis muscle strips were transplanted into limb muscle beds to allow innervation by fast or slow muscle nerve during regeneration. Regenerated muscles were examined postoperatively up to 168 days by peroxidase IHC using monoclonal antibodies to m-MyHC, m-MBP-C, and m-Tm. Regenerates in both muscle beds expressed fetal and slow MyHCs, m-MyHC, m-MBP-C, and m-Tm during the first 4 weeks. Longer-term regenerates innervated by fast nerve suppressed fetal and slow MyHCs, retaining m-MyHC, m-MBP-C, and m-Tm, whereas fibers innervated by slow nerve suppressed fetal MyHCs and the three masticatory-specific proteins, induced slow MyHC, and showed immunohistochemical characteristics of jaw-slow fibers. We concluded that expression of m-MBP-C and m-Tm is coregulated by m-MyHC and that neural impulses to limb slow muscle are capable of suppressing masticatory-specific proteins and to channel gene expression along the jaw-slow phenotype unique to jaw-closing muscle.

Immunohistochemical analysis of the effects of cross-innervation of murine thyroarytenoid and sternohyoid muscles.

This work uses cross-innervation of respiratory muscles of different developmental origins to probe myogenic and neurogenic mechanisms regulating their fiber types. The thyroarytenoid (TA) originates from the sixth branchial arch, whereas the sternohyoid (SH) is derived from somitic mesoderm. Immunohistochemical analysis using highly specific monoclonal antibodies to myosin heavy chain (MyHC) isoforms reveals that normal rat SH comprises slow, 2a, 2x, and 2b fibers, as in limb fast muscles, whereas the external division of the TA has only 2b/eo fibers coexpressing 2B and extraocular (EO) MyHCs. Twelve weeks after cross-innervation with the recurrent laryngeal nerve, the SH retained slow and 2a fibers, greatly increased the proportion of 2x fibers, and their 2b fibers failed to express EO MyHC. In the cross-innervated TA, the SH nerve failed to induce slow and 2A MyHC expression and failed to suppress EO MyHC expression in 2b/eo fibers. However, 2x fibers amounting to 4.2% appeared de novo in the external division of the TA. We conclude that although MyHC gene expression in these muscles can be modulated by neural activity, the patterns of response to altered innervation are largely myogenically determined, thus supporting the idea that SH and TA differ in muscle allotype.

Expression of masticatory-specific isoforms of myosin heavy-chain, myosin-binding protein-C and tropomyosin in muscle fibers and satellite cell cultures of cat masticatory muscle.

We test the hypothesis that cat jaw satellite cells belong to a distinct lineage preprogrammed to express masticatory-specific isoforms of myosin heavy-chain (m-MyHC), myosin-binding protein-C (m-MBP-C), and tropomyosin (m-Tm) during myogenesis in vitro. A monoclonal antibody (MAb) against m-MyHC and MAbs raised here against cat m-MBP-C and m-Tm were used to stain cryostat sections of cat masseter muscle and cultured myotubes derived from satellite cells of cat temporalis and limb muscles, using peroxidase immunohistochemistry. MAbs against m-MBP-C bound purified m-MBP-C in Western blots. MAbs against m-Tm failed to react with m-Tm in Western blots, but reacted with native m-Tm in gel electrophoresis-derived ELISA. In cat masseter sections, MAbs against m-MyHC, m-MBP-C, and m-Tm stained all masticatory fibers, but not the jaw-slow fibers. Cat jaw and limb muscle cultures mature significantly more slowly relative to rodent cultures. However, at 3 weeks, all three MAbs extensively stained temporalis myotubes, whereas they apparently stained isolated myotubes weakly in cat limb and rat jaw cultures. We conclude that satellite cells of masticatory fibers are preprogrammed to express these isoforms during myogenesis in vitro. These results consolidate the notion that masticatory and limb muscle allotypes are distinct.

Effects of hypothyroidism on myosin heavy chain composition and fibre types of fast skeletal muscles in a small marsupial, Antechinus flavipes.

Effects of drug-induced hypothyroidism on myosin heavy chain (MyHC) content and fibre types of fast skeletal muscles were studied in a small marsupial, Antechinus flavipes. SDS-PAGE of MyHCs from the tibialis anterior and gastrocnemius revealed four isoforms, 2B, 2X, 2A and slow, in that order of decreasing abundance. After 5 weeks treatment with methimazole, the functionally fastest 2B MyHC significantly decreased, while 2X, 2A and slow MyHCs increased. Immunohistochemistry using monospecific antibodies to each of the four MyHCs revealed decreased 2b and 2x fibres, and increased 2a and hybrid fibres co-expressing two or three MyHCs. In the normally homogeneously fast superficial regions of these muscles, evenly distributed slow-staining fibres appeared, resembling the distribution of slow primary myotubes in fast muscles during development. Hybrid fibres containing 2A and slow MyHCs were virtually absent. These results are more detailed but broadly similar to the earlier studies on eutherians. We hypothesize that hypothyroidism essentially reverses the effects of thyroid hormone on MyHC gene expression of muscle fibres during myogenesis, which differ according to the developmental origin of the fibre: it induces slow MyHC expression in 2b fibres derived from fast primary myotubes, and shifts fast MyHC expression in fibres of secondary origin towards 2A, but not slow, MyHC.

Protruding masticatory (superfast) myosin heads from staggered thick filaments of dog jaw muscle revealed by X-ray diffraction.

To characterize the structure of jaw muscle fibres expressing masticatory (superfast) myosin, X-ray diffraction patterns of glycerinated fibres of dog masseter were compared with those of dog tibialis anterior in the relaxed state. Meridional reflections of masseter fibres were laterally broad, indicating that myosin filaments are staggered along the filament axis. Compared with tibialis anterior fibres, the peak of the first myosin layer line of masseter fibres was lower in intensity and shifted towards the meridian, while lattice spacings were larger at a similar sarcomere length. These suggest that the myosin heads of masticatory fibres are mobile, and tend to protrude from the filament shaft towards actin filaments. Lowering temperature or treating with N-phenylmaleimide shifted the peak of the first myosin layer line of tibialis anterior fibres towards the meridian and the resulting profile resembled that of masseter fibres. This suggests that the protruding mobile heads in the non-treated masticatory fibres are in the ATP-bound state. The increased population of weakly binding cross-bridges may contribute towards the high specific force of masticatory fibres during contraction. Electron micrographs confirmed the staggered alignment of thick filaments along the filament axis within sarcomeres of masticatory fibres, a feature that may confer efficient force development over a wide range of the sarcomere lengths.

Immunohistochemical analysis of laryngeal muscles in normal horses and horses with subclinical recurrent laryngeal neuropathy.

We used immunohistochemistry to examine myosin heavy-chain (MyHC)-based fiber-type profiles of the right and left cricoarytenoideus dorsalis (CAD) and arytenoideus transversus (TrA) muscles of six horses without laryngoscopic evidence of recurrent laryngeal neuropathy (RLN). Results showed that CAD and TrA muscles have the same slow, 2a, and 2x fibers as equine limb muscles, but not the faster contracting fibers expressing extraocular and 2B MyHCs found in laryngeal muscles of small mammals. Muscles from three horses showed fiber-type grouping bilaterally in the TrA muscles, but only in the left CAD. Fiber-type grouping suggests that denervation and reinnervation of fibers had occurred, and that these horses had subclinical RLN. There was a virtual elimination of 2x fibers in these muscles, accompanied by a significant increase in the percentage of 2a and slow fibers, and hypertrophy of these fiber types. The results suggest that multiple pathophysiological mechanisms are at work in early RLN, including selective denervation and reinnervation of 2x muscle fibers, corruption of neural impulse traffic that regulates 2x and slow muscle fiber types, and compensatory hypertrophy of remaining fibers. We conclude that horses afflicted with mild RLN are able to remain subclinical by compensatory hypertrophy of surviving muscle fibers.

Immunohistochemical analysis of myosin heavy chain expression in laryngeal muscles of the rabbit, cat, and baboon.

We studied myosin heavy chain (MyHC) expression and fiber type distribution in laryngeal muscles in the rabbit, cat, and baboon using immunohistochemistry with highly MyHC-specific antibodies. Two types of variation in MyHC expression were found: between muscles of different function within species and within specific muscles between species. Within species, thyroarytenoid (Ta), an adductor, had faster MyHCs and fiber type profiles than the abductor, posterior cricoarytenoid (PCA), which expressed faster MyHCs than the vocal fold tensor, cricothyroid (CT). Between species, laryngeal muscles generally expressed faster MyHCs in small animals than in larger ones: extraocular (EO) MyHC was expressed in the Ta and PCA of the rabbit but not in the cat and baboon, whereas 2B MyHC was expressed in these muscles of the cat but not of the baboon. The CT expressed only MyHC isoforms and fiber types found in the limb muscles of the same species. These results are discussed in light of the hypothesis that the between-species variations in laryngeal muscle fiber types are evolutionary adaptations in response to changes in body mass and respiratory frequency. Within-species variations in fiber types ensure that protective closure of the glottis is always faster than movements regulating airflow during respiration.

Myosin isoforms and fibre types in limb muscles of Australian marsupials: adaptations to hopping and non-hopping locomotion.

Using immunohistochemistry and SDS-PAGE, we studied the myosin heavy chain (MyHC) composition and fibre type distribution of hindlimb muscles of hopping and non-hopping Australian marsupials. We showed that hindlimb muscles of a bandicoot (Isoodon obesulus, order Peramelomorphia) and a small macropodoid, the brushtail bettong (Bettongia penicillata) expressed four MyHCs, slow, 2a, 2x and 2b, and had the corresponding fibre types as other macropods reported earlier. The fastest and most powerful 2b fibres predominated in most bettong hindlimb muscles, but were absent in the gastrocnemius and the flexor digitorum profundus, which are involved in elastic strain energy saving during hopping. The gastrocnemius of four large macropodids also showed little or no 2b MyHC, whereas this isoform was abundant in their tibialis anterior, which is not involved in elastic energy saving. In contrast, 2b MyHC predominated in the gastrocnemius of four non-hopping marsupials. These results suggest that absence of 2b fibres may be a general feature of macropodoid muscles involved in elastic energy saving. Large eutherians except llamas and pigs also have no 2b fibres. We hypothesize that 2x and 2a fibres perform better than 2b fibres in the storage and recovery of kinetic energy during locomotion in both marsupials and eutherians.

Heterogeneity of alpha-cardiac myosin heavy chains in a small marsupial, Antechinus flavipes, and the effect of hypothyroidism on its ventricular myosins.

The effect of drug-induced hypothyroidism on ventricular myosin gene expression was explored in a small marsupial, Antechinus flavipes. Pyrophosphate gel electrophoresis, SDS-PAGE and western blotting were used to analyse changes in native myosin isoforms and myosin heavy chains (MyHCs) in response to hypothyroidism. In some animals, five instead of the normal three native myosin components were found: V(1a), V(1b),V(1c), V(2) and V(3), in order of decreasing mobility. In western blots, V(1a), V(1b), and V(1c) reacted with anti-alpha-MyHC antibody, but not with anti-beta-MyHC, whereas V(2) and V(3) reacted with anti-beta-MyHC antibody. SDS-PAGE of the unusual ventricular myosins revealed three MyHC isoforms, two of which bound anti-alpha-MyHC antibody while the third bound anti-beta-MyHC antibody. We conclude that V(1a), V(1b), V(1c) are triplets arising from the dimerization of two distinct alpha-MyHC isoforms. Hypothyroidism, verified by metabolic studies, decreased alpha-MyHC content significantly (t-test, P < 0.001) from 91.6 +/- 5.9% (SEM, n = 4) in control animals to 67.2 +/- 5.7% (SEM, n = 4) in hypothyroid animals, with a concomitant increase in beta-MyHC content. We conclude that in adult marsupials, ventricular myosins are also responsive to changes in the thyroid state as found in eutherians, and suggest that evolution of the molecular mechanisms underlying this thyroid responsiveness predate the divergence of marsupials and eutherians.

Developmental changes in ventricular myosin isoenzymes of the tammar wallaby.

Ventricular myosin in eutherian mammals undergoes a perinatal change in response to a sharp rise in thyroid hormone levels during development. In this investigation, changes in ventricular myosin heavy chains (MyHCs) of the tammar wallaby (Macropus eugenii) from early pouch life to adulthood were analysed using native gel electrophoresis, SDS-PAGE and western blotting. Adult wallaby ventricle showed three myosin isoenzymes, V(1), V(2) and V(3); western blots using specific anti-alpha-MyHC and anti-beta-MyHC antibodies showed their MyHC compositions to be alphaalpha, alphabeta and betabeta, respectively. Ventricular muscle in early pouch joeys expressed predominantly beta-MyHC. Up to 200 days, the time of initial pouch exit, alpha-MyHC content was around 5%. Thereafter, there was a sharp increase of alpha-MyHC expression to 35% by 242 days of age, eventually falling back to 23% in the adult. These changes correlate with known surges in plasma levels of thyroid hormones around pouch exit. The results suggest that ventricular myosins in a marsupial mammal also undergo a developmental change, and that marsupial ventricular myosins are thyroid responsive as in eutherians. The increased alpha-MyHC expression empowers the heart to meet the enhanced cardiovascular demands of out-of-pouch activity and the thermogenic action of thyroid hormones.

Marsupial cardiac myosins are similar to those of eutherians in subunit composition and in the correlation of their expression with body size.

Cardiac myosins and their subunit compositions were studied in ten species of marsupial mammals. Using native gel electrophoresis, ventricular myosin in macropodoids showed three isoforms, V(1), V(2) and V(3), and western blots using specific anti-alpha- and anti-beta-cardiac myosin heavy chain (MyHC) antibodies showed their MyHC compositions to be alphaalpha, alphabeta and betabeta, respectively. Atrial myosin showed alphaalpha MyHC composition but differed from V(1) in light chain composition. Small marsupials (Sminthopsis crassicaudata, Antechinus stuartii, Antechinus flavipes) showed virtually pure V(1), while the larger (1-3 kg) Pseudocheirus peregrinus and Trichosurus vulpecula showed virtually pure V(3). The five macropodoids (Bettongia penicillata, Macropus eugenii, Wallabia bicolour, M. rufus and M. giganteus), ranging in body mass from 2 to 66 kg, expressed considerably more alpha-MyHC (22.8%) than expected for their body size. These results show that cardiac myosins in marsupial mammals are substantially the same as their eutherian counterparts in subunit composition and in the correlation of their expression with body size, the latter feature underlies the scaling of resting heart rate and cardiac cross-bridge kinetics with specific metabolic rate. The data from macropodoids further suggest that expression of cardiac myosins in mammals may also be influenced by their metabolic scope.

Cross-bridge kinetics of fast and slow fibres of cat jaw and limb muscles: correlations with myosin subunit composition.

Mechanical properties of the jaw-closing muscles of the cat are poorly understood. These muscles are known to differ in myosin and fibre type compositions from limb muscles. This work aims to correlate mechanical properties of single fibres in cat jaw and limb muscles with their myosin subunit compositions. The stiffness minimum frequency, f(min), which reflects isometric cross-bridge kinetics, was measured in Ca(2+)-activated glycerinated fast and slow fibres from cat jaw and limb muscles for temperatures ranging between 15 and 30 degrees C by mechanical perturbation analysis. At 15 degrees C, f(min) was 0.5 Hz for limb-slow fibres, 4-6 Hz for jaw-slow fibres, and 10-13 Hz for limb-fast and jaw-fast fibres. The activation energy for f(min) obtained from the slope of the Arrhenius plot for limb-slow fibres was 30-40% higher than values for the other three types of fibres. SDS-PAGE and western blotting using highly specific antibodies verified that limb-fast fibres contained IIA or IIX myosin heavy chain (MyHC). Jaw-fast fibres expressed masticatory MyHC while both jaw-fast and jaw-slow fibres expressed masticatory myosin light chains (MLCs). The nucleotide sequences of the 3' ends of the slow MyHC cDNAs isolated from cat masseter and soleus cDNA libraries showed identical coding and 3'-untranslated regions, suggesting that jaw-slow and limb-slow fibres express the same slow MyHC gene. We conclude that the isometric cross-bridge cycling kinetics of jaw-fast and limb-fast fibres detected by f(min) are indistinguishable in spite of differences in MyHC and light chain compositions. However, jaw-slow fibres, in which the same slow MyHCs are found in combination with MLCs of the jaw type, show enhanced cross-bridge cycling kinetics and reduced activation energy for cross-bridge detachment.

Myosin isoforms and fibre types in jaw-closing muscles of Australian marsupials.

Myosin heavy chains (MyHCs) and fibre types in the masseter muscle of seven species of Australian marsupials (brushtail and ringtail possums, bettong, bandicoot, dunnart, two species of antechinuses) spanning three orders were studied by native myosin electrophoresis, SDS-PAGE, immunoblotting and immunohistochemistry. We found only two fibre types in the masseter muscles of these animals: (1) masticatory fibres expressing masticatory MyHC, and (2) hybrid alpha/beta fibres that co-express alpha-cardiac and beta-cardiac MyHCs. Masticatory fibres predominate in most species, being appropriate for predation or for chewing tough vegetable matter. The relative abundance of alpha/beta fibres decreased from 60% to 0 in the order: ringtail possum > brushtail possum > bettong > bandicoot > dunnart/antechinus. These variations in masseter fibre type are correlated with decreasing amounts of vegetable matter in the diets of these animals. The results are in contrast to earlier work on masseter fibres of macropodids that expressed alpha-cardiac MyHC almost homogeneously. The fact that the bettong (Family: Potoroidae), which belong to the same marsupial superfamily (Macropodoidea) as kangaroos and wallabies (Family: Macropodidae), has not specialized in the exclusive expression of alpha-cardiac MyHC as members of the latter family suggests that this specialization was of recent phylogenetic origin (30 million years before present).

Insulin-like growth factor I slows the rate of denervation induced skeletal muscle atrophy.

Loss of the nerve supply to skeletal muscle results in a relentless loss of muscle mass (atrophy) over time. The ability of insulin-like growth factor-1 to reduce atrophy resulting from denervation was examined after transection of the sciatic nerve in transgenic MLC/mIGF-1 mice that over-express mIGF-1 specifically in differentiated myofibres. The cross sectional area (CSA) of all types of myofibres and specifically type IIB myofibres was measured in tibialis anterior muscles from transgenic and wild-type mice at 28 days after denervation. There was a marked myofibre atrophy ( approximately 60%) in the muscles of wild-type mice over this time with increased numbers of myofibres with small CSA. In the muscles of MLC/mIGF-1 mice, over-expression of mIGF-1 reduced the rate of denervation induced myofibre atrophy by approximately 30% and preserved myofibres with larger CSA, compared to wild-type muscles. It is proposed that the protective effect of mIGF-1 on denervated myofibres might be due to reduced protein breakdown.

Targeted expression of insulin-like growth factor-I reduces early myofiber necrosis in dystrophic mdx mice.

Necrosis of dystrophic myofibers in Duchenne muscular dystrophy and mdx mice results from defects in the subsarcolemmal protein dystrophin that cause membrane fragility and tears in the sarcolemma, and these lead to the destruction of the myofibers. The present study specifically tests whether overexpression of mIGF-1 in mdx/mIGF-1 transgenic mice reduces myofiber breakdown during the acute onset phase of dystrophy (at 21 days). The extent of muscle damage and Evans blue dye (EBD) staining of myofibers was quantitated histologically for mdx/mIGF-1 and their mdx littermates from 15 to 30 days of age. Overexpression of mIGF-1 strikingly reduced the extent of myofiber damage (histology and EBD staining) by up to 97% in tibialis anterior and quadriceps muscles at 21-22 days after birth. In the mdx diaphragm, the onset of muscle breakdown was earlier (by 15 days after birth) but no significant protective effect of IGF-1 was apparent within the first month of age in mdx/IGF-1 mice. These novel observations show that increased mIGF-1 within mdx myofibers specifically reduces the breakdown of dystrophic muscle during the acute onset of muscle degeneration. This mechanism of action can account for the long-term reduced severity of the dystropathology in mdx mice that overexpress mIGF-1 and provides promising opportunities for therapeutic strategies.

The development of longitudinal variation of Myosin isoforms in the orbital fibers of extraocular muscles of rats.

PURPOSE: To examine the appearance of longitudinal variation of extraocular and embryonic myosin heavy chain (MyHC) isoforms during the development of orbital singly innervated fibers of rat extraocular muscles (EOMs). METHODS: EOMs were dissected from rat pups of various ages and stained with isoform-specific monoclonal antibodies to the embryonic and extraocular MyHC isoforms and to neurofilaments, as well as with labeled alpha-bungarotoxin. The orbital layers of whole muscles were examined by confocal microscopy. RNase protection assays for the embryonic (Myh3) and extraocular (Myh13) MyHC isoform mRNAs were also performed. RESULTS: At 10 days postpartum, the EOM MyHC RNA was first detected by RNase protection assay. At 11 days postpartum, the extraocular isoform was detected in the orbital fibers as two thin stripes just proximal and distal to the neuromuscular junction (NMJ). Over the next few weeks, the area occupied by the extraocular isoform increased to include the entire central region of the orbital fibers at and surrounding the NMJ. At the same time, the embryonic isoform became excluded from the region of the NMJ. CONCLUSIONS: The orbital layer fibers of rat EOMs contain a longitudinal variation in MyHC isoforms not seen in other skeletal muscles. Development of this longitudinal variation begins as a late event postpartum; and the first appearance of it may be closely linked to neural contact. This targeting of MyHC isoforms to distinct domains is unique to EOMs.