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.     

The expression of NFATc1 in adult rat skeletal muscle fibres

Although numerous studies have recently implicated the calcineurin-nuclear factor of activated T-cells (Cn-NFAT) signalling pathway in the regulation of activity-dependent fibre type switching in adult mammalian skeletal muscles, little is known about the endogenous expression of NFAT proteins in the various fibre types present in these muscles. In this study, the immunolocalization of NFATc1 (also known as NFATc or NFAT2) in the extensor digitorum longus (EDL; a mainly fast-twitch muscle) and the soleus (a predominantly slow-twitch muscle) muscles of adult ( approximately 90-day-old) Wistar rats was investigated. The results show that NFATc1 is expressed only in oxidative fibres (i.e. type I and type IIA fibres) that stain intensely for succinate dehydrogenase activity irrespective of whether they are from the fast- or slow-twitch muscle. Thus, 99 +/- 4% (n = 7 rats) of the muscle fibres in the soleus and 42 +/- 2% (n = 7 rats) of those in the EDL expressed NFATc1. In the soleus muscle fibres, NFATc1 was localized mainly in the fibre nuclei, whereas in the EDL fibres it was localized in both the cytoplasm and the nuclei. However, no difference in its localization was observed between type I and type IIA fibres in both muscles. Western blot experiments showed that the soleus expressed more NFATc1 proteins than the EDL. From these results, we suggest that NFATc1 controls the number and distribution of both type I and type IIA fibres, as well as the oxidative capacity of adult mammalian skeletal muscles.     

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.     

Hindlimb suspension induces the expression of multiple myosin heavy chain isoforms in single fibres of the rat soleus muscle

The aim of our experiment was to test the hypothesis that the performance of maximal isometric exercise every 20 s would reduce the intermediate frequency force, i.e. the force that appears while stimulating the muscle at 15 and 20 Hz, and would produce less decrease the force at 10 and 50 Hz, while Pt would increase. Such changes in stimulated force should demonstrate the coexistence of potentiation, low frequency fatigue (LFF) and `post-contractile depression' (PCD). The quadriceps muscle of 14 healthy men (aged 19–37 years) was studied. The results have shown, that during isometric exercise of maximal intensity there was significant (P < 0.05) decrease in P15 and P20, increase in Pt, however, MVC and P10 and P50 was unchanged (P > 0.05). LFF manifested itself most significantly which is evident from decrease in P20/P50. During recovery after work there was significant increase in LFF and decrease in P50 which is indicative of the manifestation of PCD. Besides, there was significant (P < 0.05) decrease immediately after exercise in RTP20 and RTP50, while no changes in T50 and RT. There were no significant changes (P > 0.05) however, either in RTP20 and RTP50 or in T50 and RT 20 min after exercise if compared to the initial and immediately post-exercise values.     

The contractile properties of slow muscle fibres in sheep extraocular muscle.

1. The diameters of nerve fibres in the sheep trochlear nerve were measured and they fell into two distinct groups. 2. Selective stimulation of the small diameter group of nerve fibres gave rise to a slow contraction of the superior oblique muscle which was attributed to multiply innervated muscle fibres. 3. The slow fibre contraction following a single stimulus to the small diameter nerves had a rise time of 20-50 msec and was 150-980 mg in size. On repetitive stimulation, a maximum slow fibre contraction was developed at 120-140 Hz, and maximum velocity of tension development at 170-200 Hz. 4. The maximum slow fibre tetanic tension was 7-0-12-3 g, which amounted to 5-3% of the whole muscle tetanic tension, while the maximum contracture following an injection of suxamethonium was 7% of the whole muscle tetanic tension. 5. The slow fibres were very resistant to fatigue and their contraction increased the resistance of the muscle to stretching. 6. Discrepancies from earlier work and the possible significance of the slow fibres are discussed.     

Fibre types in extraocular muscles: a new myosin isoform in the fast fibres

Summary We report on the existence of a myosin heavy chain (MHC) isoform with unique structural properties in extraocular (EO) muscles. Differences in MHC composition are apparent using a polyclonal antibody prepared against myosin isolated from bovine EO muscle myosin. In enzyme immunoassays and western blotting experiments, this anti-EO myosin antibody reacted specifically with the heavy chains of EO muscle myosin and not with the heavy chains of other myosins. The distribution of this new MHC isoform in the globe rotating muscles from different mammalian species was analysed using a panel of specific anti-myosin antibodies and comparing the histochemical myosin ATPase profile of muscle fibres with their isomyosin content. Most fibres which display a type 2 ATPase reaction pattern were selectively labelled by anti-EO antibodies. A few type 2 fibres were found to react with both anti-EO and anti-2A myosin antibodies and others, located almost exclusively in the orbital layers, reacted with anti-foetals as well as anti-EO antibodies.The presence of a distinct form of myosin in EO muscle fibres is probably related to the particular functional characteristics of these muscles, which are known to be exceptionally fast-contracting but display a very low tension output.     

Expression of extraocular myosin heavy chain in rabbit laryngeal muscle

Summary The intrinsic laryngeal muscles of mammals are functionally heterogeneous, some of these muscles (e.g. the thyroarytenoid) contract extremely rapidly, like extraocular muscle, whilst others (e.g. the cricothyroid) contract as fast as limb fast muscle. The extraordinarily rapid contraction speed of extraocular muscles is associated with a fast myosin not found in limb muscles. In this work we explored the possibility that the thyroarytenoid muscle may also express this extraocular-specific fast myosin by raising a monoclonal antibody (mab 4A6) against its heavy chain. Electrophoretic separation of native isomyosins revealed that both the extraocular and the thyroarytenoid have two similar bands migrating ahead of bands found in limb fast or cricothyroid myosins. These two bands bound mab 4A6. The thyroarytenoid muscle can be divided into two divisions, a vocalis division which is important in phonation and an external division which functions in closing the glottis. Fibres in the vocalis are heterogeneous, some stain with mab 4A6, whilst others stain with mabs against limb myosin heavy chains. Fibres in the external division stain almost homogeneous with mab 4A6. The immunohistochemical staining pattern in the cricothyroid muscle resembled that of fast limb muscle: no fibres stained with mab 4A6. Thus, the high speed of contraction of the thyroarytenoid is associated with the same myosin heavy chain found in extraocular muscles, this characteristic is presumably an evolutionary adaptation for rapid closure of the glottis to enhance airway defense mechanisms.     

Force and myosin content variation in isolated intact single muscle fibres from Rana temporaria

 We studied the relation between force normalized by dry mass per unit length and the myosin fraction of muscle dry mass. The two tibialis anterior muscles were dissected from 12 frogs (Rana temporaria). Then, from one muscle, two single fast-twitch fibres were isolated. Each fibre was mounted isometrically in Ringer’s solution, and electrically stimulated using a standardized protocol. Peak force production, normalized by the fibre’s dry mass per unit length, varied by a factor of 1.4. Little variation in normalized force was measured between fibres from the same animal, whereas between animals a significant difference was found (P<0.05). The contralateral muscle was used to determine the myosin fraction of the dry mass. The relationship between the fraction myosin of the dry mass and force normalized by dry mass per unit length showed a high correlation (r = 0.81; n = 12). From this we conclude that variation in normalized tetanic force is determined greatly (65%) by variations in myosin content. Received: 23 January 1997 / Received after revision: 14 March 1997 / Accepted: 26 March 1997     

Clenbuterol induces expression of multiple myosin heavy chain isoforms in rat soleus fibres

Clenbuterol, a beta2-agonist, administration results in hypertrophy of fast fibres and an increase in the fast myosin heavy chain (MHC) composition of both fast and slow muscles. The present study was designed to determine the phenotypic response at the single fibre level. Clenbuterol was added to the drinking water (30 mg L(-1)) of adult male Wistar rats for 4 weeks. Single fibres from the soleus muscle of control (10 rats; 555 fibres) and clenbuterol-treated (10 rats; 577 fibres) were dissected and their MHC isoform composition was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. Body, heart, and soleus weights were 9, 24, and 27% higher in clenbuterol-treated than control rats. The mean cross-sectional areas of fast and slow/fast hybrid fibres were approximately 64 and approximately 74% larger in the clenbuterol-treated than control rats, whereas the size of the slow fibres were similar in the two groups. Fibres from control soleus showed three MHC patterns: pure type I (84%), pure type IIa (4%), and type I + IIa (12%) MHC. Some fibres from clenbuterol-treated soleus showed a de novo expression of type IIx MHC resulting in the following fibre type proportions: pure type I (62%), pure type IIa (2%), type I + IIa (26%), type I + IIa + IIx (6%), and type IIa + IIx (1%). In those fibres containing multiple MHCs, there was a shift towards the faster MHC isoforms after clenbuterol treatment. These data indicate that clenbuterol results in muscle fibre hypertrophy, stimulates a de novo expression of type IIx MHC and increases the percentage of fibres containing multiple MHC isoforms in the rat soleus muscle. These phenotypic changes at the single fibre level are consistent with a clenbuterol-related shift in the functional properties of the soleus towards those observed in a faster muscle.     

Clenbuterol induces expression of multiple myosin heavy chain isoforms in rat soleus fibres

Uterine secretory cells receive a sympathetic cholinergic secremotor innervation. Nitric oxide (NO) has been suggested to be a second messenger of neurogenic modulated glandular secretion of the seminal vesicle. Thus a similar pattern for nervous induced carbohydrate secretion of the endometrium was assumed. The nitric oxide synthase (NOS) activity was estimated via formation of L ‐citrulline from L ‐arginine and histochemically with the nicotinamide‐adenine dinucleotide phosphate diaphorase (NADPH‐d) nitro blue technique. The carbohydrate secretion from everted uterine horns placed in organ baths was estimated. A calcium dependent formation of citrulline was found in the uterine horn suggesting an NOS activity. Strong NADPH staining cells were found in the glandular ducts of the endometrium and in the epithelial linings of the oviduct. Carbachol induced carbohydrate secretion of the endometrium while N‐nitro L ‐arginin (L ‐NNA) and N‐nitro L ‐arginin methyl ester (L ‐NAME) inhibited the carbachol induced secretion. The isomer D ‐NAME had no effect on carbachol induced secretion. When L ‐arginine was administered together with L ‐NNA no inhibitory effect on carbachol induced secretion was seen. L ‐arginine only had no effect on carbohydrate secretion. The NO donor glyceryl tritrate increased carbohydrate secretion but no synergistic effect was seen in combination with carbachol. The results suggest that glandular NO production is a prerequisite for muscarinic carbohydrate secretion of the endometrium.     

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.     

Properties of isolated adult rat muscle fibres maintained in tissue culture.

1. The permeability of the isolated skin of a neotenous urodele amphibian, the mudpuppy Necturus maculosus, to Na, Cl, urea and water was measured. 2. Unidirectional transcutaneous flux measurements and the action of ouabain and amiloride, showed that there was normally no active Cl or Na transport, nor a Cl/Cl exchange diffusion process. 3. Amphotericin B initiated a transcutaneous potential difference and short-circuit current, which could be inhibited by ouabain. 4. The short-circuit current was nearly equivalent to the net Na Transport and this was also inhibited by ouabain. 5. A transcutaneous active Na transport mechanism thus appears to be incipient in the mudpuppy but is limited by a low permeability of the outer barrier of the cells. 6. Vasotocin increased the skin's diffusion permeability for water but had no effect on the influx of Na or urea. 7. The function of Necturus skin is in several respects unique compared to that of other amphibians.     

Activation of two types of fibres in rat extraocular muscles.

1. The contractile responses of the inferior rectus, one of the extraocular muscles of the rat, to a depolarization induced by an elevation of the potassium concentration in the external medium ([K]O) have been studied 'in vitro'. 2. The elevation of [K]O to 20 and 30 mM-K produced contractures that consisted of a sustained or tonic tension. When [K]O was increased to 50 mM or more a well-defined transient or phasic tension appeared before the tonic response. The increment of [K]O above 50 mM enhanced the phasic component and depressed the tonic tension. The maximal tonic tension, usually evoked by 50 mM-K, is about 50% of the tetanic tension, shows a gradual decline with time and lasts for hours. Control experiments performed in diaphragm showed that this muscle only responds with phasic tensions. 3. The difference in the repriming of the phasic and tonic responses when tensions were induced with salines containing low or normal [Cl] suggests that the muscle fibres responsible for the tonic tension are poorly permeable to Cl-. 4. The amplitude of the tonic tension was reduced by Ca deprivation and by an elevation of [Ca] in the saline to 10 mM. 5. It is concluded that in rat extraocular muscles, an increase in [K]O activates two types of muscle fibres: singly and multiply innervated. These appear to be functionally equivalent to the twitch and slow fibres of amphibian and avian muscle and would give rise to the phasic and tonic components of the contracture, respectively.     

Effects of age and gender on shortening velocity and myosin isoforms in single rat muscle fibres

The maximum velocity of unloaded shortening (V₀) and the myosin heavy chain (MyHC) and light chain (MyLC) isoform composition were determined in single fibres from soleus and extensor digitorum longus (EDL) muscles of male and female rats 3–6 and 22–24 months old. In the soleus muscle, the β/slow (type I MyHC) isoform predominated in both young and old animals, irrespective of gender. In the EDL, fibres expressing type IIX MyHC or a combination of IIX and IIB (IIXB) MyHC isoforms were predominant in old rats, while type IIB MyHC fibres predominated in young individuals of both genders. The V₀ of soleus fibres expressing the type I MyHC isoform decreased (P < 0.001) by 40% with age in spite of an unchanged MyLC composition. In the EDL, the V₀ of fibres expressing IIX, IIXB and IIB MyHC isoforms did not change with age or differ between males and females. In conclusion, similar age-related changes in V₀ and MyHC composition were observed in single muscle cells from both male and female rats. The present results demonstrate that the relationship between V₀ and MyHC isoform composition at the single fibre level is similar in male and female rats, and that similar qualitative changes take place during ageing in both genders.     

Physiological properties of dissociated muscle fibres obtained from innervated and denervated adult rat muscle.

1. Adult rat flexor digitorum brevis muscles were dissociated by treatment with collagenase and trituration. Several hundred isolated fibres were obtained from each muscle. 2. Most isolated fibres appeared to be intact as judged by some morphological and physiological criteria, although resting membrane potentials were about -60 mV, which is somewhat lower than normal. 3. A small percentage of the muscle fibres were branched. 4. Acetylcholine sensitivity was measured iontophoretically. The sensitivity fell abruptly outside the margin of the end-plate. Extrajunctional sensitivty was detected on all fibres, and declined smoothly away from the end-plate to an undetectable level over a distance of about 200 micron. On a few fibres, ACh sensitivity was mapped circumferentially from the end-plate. It appeared to decline with distance in a manner similar to the longitudinal sensitivity gradient. 5. Fibres dissociated from muscles denervated a week earlier were sensitive to ACh everywhere on their surfaces.     

Stretch activation and isoforms of myosin heavy chain and troponin-T of rat skeletal muscle fibres

Recent studies on single mammalian skeletal muscle fibres revealed a correlation between the kinetics of stretch-induced delayed force increase (stretch activation) and the isoforms of the myosin heavy chain. This observation suggests a causal relation between stretch activation and myosin heavy chain. However, the assumption is weakened by the fact that isoforms of other myofibrillar proteins tend to be coexpressed with myosin heavy chain isoforms. The relation between the isoforms of the tropomyosin-binding troponin subunit and myosin heavy chain is unknown. For a variety of reasons, tropomyosin-binding troponin subunit is a possible candidate for being involved in stretch activation. Therefore, we measured stretch activation of single, maximally Ca2+-activated skinned rat skeletal muscle fibres and characterized them by their myosin heavy chain composition, as well as by the isoform species of tropomyosin-binding troponin subunit. Four myosin heavy chain isoforms (I, IIa, IId or IIx and IIb) and six tropomyosin-binding troponin subunit isoforms (TnT1s, TnT2s, TnT1f, TnT2f, TnT3f, TnT4f) were distinguis hed. The following preferential coexpression patterns of the myosin heavy chain and tropomyosin-binding troponin subunit isoforms were observed: MHCI-TnT1s, MHCIIa-TnT3f, MHCIId-TnT1f, and MHCIIb-TnT4f. Stretch activation kinetics was found to be correlated with the myosin heavy chain isoform complement also in fibres not displaying one of the preferential MHC-TnTf isoform coexpression patterns. This corroborates the assumption of a causal relation between myosin heavy chain and stretch activation This revised version was published online in July 2006 with corrections to the Cover Date.     

Dependence of cross-bridge kinetics on myosin light chain isoforms in rabbit and rat skeletal muscle fibres

Cross-bridge kinetics underlying stretch-induced force transients was studied in fibres with different myosin light chain (MLC) isoforms from skeletal muscles of rabbit and rat. The force transients were induced by stepwise stretches (< 0.3% of fibre length) applied on maximally Ca²⁺-activated skinned fibres. Fast fibre types IIB, IID (or IIX) and IIA and the slow fibre type I containing the myosin heavy chain isoforms MHC-IIb, MHC-IId (or MHC-IIx), MHC-IIa and MHC-I, respectively, were investigated. The MLC isoform content varied within fibre types. Fast fibre types contained the fast regulatory MLC isoform MLC2f and different proportions of the fast alkali MLC isoforms MLC1f and MLC3f. Type I fibres contained the slow regulatory MLC isoform MLC2s and the slow alkali MLC isoform MLC1s. Slow MLC isoforms were also present in several type IIA fibres. The kinetics of force transients differed by a factor of about 30 between fibre types (order from fastest to slowest kinetics: IIB > IID > IIA ≫ I). The kinetics of the force transients was not dependent on the relative content of MLC1f and MLC3f. Type IIA fibres containing fast and slow MLC isoforms were about 1.2 times slower than type IIA fibres containing only fast MLC isoforms. We conclude that while the cross-bridge kinetics is mainly determined by the MHC isoforms present, it is affected by fast and slow MLC isoforms but not by the relative content of MLC1f and MLC3f. Thus, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the cross-bridge kinetics.