Neonatal Myosin Heavy Chain Isoforms Research Articles

Muscle hypertrophy in heavy weight Japanese quail line: Delayed muscle maturation and continued muscle growth with prolonged upregulation of myogenic regulatory factors

The objective of this study was to compare the temporal expression of myosin heavy chain (MyHC) isoforms, Pax7, and myogenic regulatory factors (MRF) between heavy weight (HW) and random bred control (RBC) Japanese quail lines during muscle development to better understand the mechanisms leading to increased skeletal muscle mass in the HW quail line selected for a greater BW at 4 wk of age separated from RBC quail. Expression of neonatal MyHC isoform began at 3 and 7 d posthatch in RBC and HW quail lines, respectively. In the RBC quail line, adult MyHC isoform, as a marker for muscle maturation, was expressed at 28 d posthatch with sustained expression through 75 d posthatch, whereas this protein was detected only at 75 d posthatch in the HW quail line. Moreover, Pax7 expression continued from embryonic ages to 14 d posthatch in the HW quail line and to 7 d posthatch in the RBC quail line. These expression patterns of MyHC isoforms and Pax7 in the HW quail line were accompanied by delayed muscle maturation and prolonged growth compared with the RBC quail line. Temporal expressions of the primary MRF showed that higher expression levels of MyoD and Myf-5 were observed at 9 and 11 d embryo in the HW quail line compared with the RBC quail line (P < 0.05). The HW quail line exhibited approximately 2 times greater average levels of myogenin expression from 7 to 75 d posthatch (P < 0.05) than the RBC quail line. Prolonged upregulation of these primary and secondary MRF during muscle development is associated with delayed maturation and continued muscle growth, which consequently would permit muscle hypertrophic potentials in the HW quail line compared with the RBC quail line.

A new role for the calcineurin/NFAT pathway in neonatal myosin heavy chain expression via the NFATc2/MyoD complex during mouse myogenesis

The calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway is involved in the modulation of the adult muscle fiber type, but its role in the establishment of the muscle phenotype remains elusive. Here, we show that the NFAT member NFATc2 cooperates with the basic helix-loop-helix transcription factor MyoD to induce the expression of a specific myosin heavy chain (MHC) isoform, the neonatal one, during embryogenesis. We found this cooperation to be crucial, as Myod/Nfatc2 double-null mice die at birth, with a dramatic reduction of the major neonatal MHC isoform normally expressed at birth in skeletal muscles, such as limb and intercostal muscles, whereas its expression is unaffected in myofibers mutated for either factor alone. Using gel shift and chromatin immunoprecipitation assays, we identified NFATc2 bound to the neonatal Mhc gene, whereas NFATc1 and NFATc3 would preferentially bind the embryonic Mhc gene. We provide evidence that MyoD synergistically cooperates with NFATc2 at the neonatal Mhc promoter. Altogether, our findings demonstrate that the calcineurin/NFAT pathway plays a new role in establishing the early muscle fiber type in immature myofibers during embryogenesis.

Temporal myosin heavy chain isoform expression transitions faster in broiler chickens compared with Single Comb White Leghorns

Myosin heavy chain (MyHC), one of the major components in the contractile machinery of skeletal muscle fibers, is found in several isoforms during myogenesis. During chicken development, embryonic, neonatal, and adult MyHC isoforms are expressed. Broiler chickens have been selected for fast and large muscle growth, whereas Single Comb White Leghorn (SCWL) chickens have been selected for egg laying capabilities. This has led to an obvious difference in muscle growth and development with broilers being much larger than SCWL. The objective of this study was to determine if differences in muscle growth and development of SCWL and broilers are associated with differences in temporal expression of MyHC isoforms in skeletal muscle between the 2 breeds. Pectoralis major muscle (PM) was collected from SCWL and broilers at embryonic d 15, 17, and 19 and 1, 5, 11, 20, 27, and 33 d posthatch with n = 3 samples per time point and breed. Western blotting using 3 monoclonal antibodies (EB165, 2E9, and AB8) was performed to compare the expression patterns of embryonic/adult, neonatal, and adult isoforms of MyHC, respectively, for all time points in both SCWL and broiler chickens. Both broiler and SCWL chickens began expressing the neonatal MyHC isoform on d 5; however, SCWL chickens expressed the neonatal isoform much longer than broilers. The SCWL chickens had sustained expression of the neonatal MyHC isoform through d 27, whereas in broiler chickens the neonatal isoform was not expressed at d 20. Pectoralis major tissue from broiler chickens expressed the adult MyHC isoform as early as d 20, whereas the SCWL chickens began expressing the adult isoform later. The rate of transition to neonatal and adult MyHC isoforms in broilers and Leghorns is consistent with the faster maturation and growth of broilers relative to Leghorns. This relationship between faster growth of the PM and the rate of transition of MyHC isoforms within the fast skeletal muscle of the PM may indicate a selection marker for improvement of broiler PM growth.

The Effect of Resection on Satellite Cell Activity in Rabbit Extraocular Muscle

A common treatment for motility disorders of the extraocular muscles (EOMs) is a resection procedure in which there is surgical shortening of the muscle. This procedure results in rotation of the globe toward the resected muscle, increased resting tension across the agonist-antagonist pair, and stretching of the elastic components of the muscles. In the rabbit, due to orbital constraints and limited rotation, resection results in more significant stretch of the surgically treated muscle than the antagonist. This surgical preparation allows for the examination of the effects of surgical shortening of one rectus muscle and passive stretch of its ipsilateral antagonist. The insertional 6 mm of the superior rectus muscles of adult rabbits were resected and reattached to the original insertion site. After 7 and 14 days, the animals were injected intraperitoneally with bromodeoxyuridine (BrdU) every 2 hours for 12 hours, followed by a 24-hour BrdU-free period. All superior and inferior rectus muscles from both globes were examined for BrdU incorporation, MyoD expression, neonatal and developmental myosin heavy chain (MyHC) isoform expression, and myofiber cross-sectional area alterations. In the resected muscle and in the passively stretched antagonist muscle, there was a dramatic increase in the number of myofibers positive for neonatal MyHC and in the number of BrdU- and MyoD-positive satellite cells. The addition of BrdU-positive myonuclei increased from 1 per 1000 myofibers in cross sections of control muscles to 2 to 3 per 100 myofibers in the resected muscles. Single myofiber reconstructions showed that multiple BrdU-positive myonuclei were added to individual myofibers. Addition of new myonuclei occurred in random locations along the myofiber length of single fibers. There was no correlation between myofibers with BrdU-positive myonuclei and neonatal MyHC isoform expression. Both active and passive stretch of the rectus muscles produced by strabismus surgery dramatically upregulated the processes of satellite cell activation, integration of new myonuclei into existing myofibers, and concomitant upregulation of immature myosin heavy chain isoforms. Understanding the effects of strabismus surgery on muscle cell biological reactions and myofiber remodeling may suggest new approaches for improving surgical outcomes.

Myosin heavy chain isoform immunolabelling in diabetic rats with peripheral neuropathy

This study evaluated mature and immature myosin heavy chain (MHC) isoform immunolocalisation in soleus muscle of diabetic rats with documented motor neuropathy. Sprague Dawley rats were assigned to one of three groups: control (C), diabetic with insulin (DI), or diabetic without insulin (DNI). Twelve weeks after diabetes induction, soleus muscles were excised and quick-frozen. Cross-sections were labelled immunohistochemically for slow, fast, developmental and neonatal MHC isoforms to determine fiber-type composition. Fiber cross-sectional areas were determined morphometrically. Results revealed that DNI and DI muscles contained greater percentages of myofibers positive for fast MHC compared with controls. DNI animals also showed a lower percentage of myofibers positive for slow MHC compared to the DI group. The number of fibers immunolabelled for developmental MHC isoforms was greater in DNI animals than in the other groups. The differences in slow and fast MHC-labelling appear to indicate a condition of altered neuromuscular activity affecting the diabetic muscles. The increase in developmental MHC-labelling in the DNI muscles could indicate myofiber regeneration or reinnervation that would be more pronounced in the DNI animals in context of their more severe neuropathy. Insulin appeared to influence muscle fiber cross-sectional area and possibly fiber-type grouping frequency; the potential mechanism for these effects was not elucidated.

Adult human pharyngeal muscle fibers express some unusual myosin-heavy chain isoforms

Problem: Fiber type and myosin-heavy chain (MHC) composition in the human pharyngeal constrictor (PC) and upper esophageal sphincter (UES) muscles were analyzed to better understand the muscle functions. Methods: Twelve adult human PC and UES muscles obtained from autopsies were studied using immunohistochemical and immunoblotting techniques. For comparison, masseter and biceps brachii muscles of the same subjects served as controls. Serial cross-sections were incubated with a panel of isoform-specific anti-MHC monoclonal antibodies. Results: The human PC and UES muscles were composed of 2 histochemically defined fiber layers, a slow inner layer (SIL) (78% type I) and a fast outer layer (FOL) (59% type II). The adult PC and UES fibers expressed unusual MHC isoforms (ie, slow-tonic, alpha-cardiac, embryonic, and neonatal) that were also identified in the masseter, but not in the biceps brachii. These unusual MHC isoforms demonstrated by immunocytochemistry were confirmed by electrophoretic immunoblotting. The slow-tonic and alpha-cardiac fibers were concentrated mainly in the SIL, whereas the developmental MHC-containing fibers were located primarily in the FOL. The unusual MHC isoforms were coexpressed with the major MHC isoforms (ie, slow type I and fast type II), thus forming various major/unusual (m/u) MHC hybrid fiber types. The slow-tonic and alpha-cardiac MHC isoforms coexisted mainly with type I MHC, whereas the embryonic and neonatal MHC isoforms were coexpressed primarily with type IIa MHC. At least 8 m/u MHC hybrid fiber types were identified in these muscles. Conclusion: The multiplicity of MHC isoforms in the PC and UES fibers is believed to be related to embryonic origin, innervation, and unique functional requirements. Significance: Age-related changes in the fiber type and MHC composition in the human PCs and UES may be associated with pathogeneses of dysphagia and obstructive sleep apnea that are commonly seen in the elderly. Support: None reported.

Adult human mylohyoid muscle fibers express slow-tonic, alpha-cardiac, and developmental myosin heavy-chain isoforms.

Some adult cranial muscles have been reported to contain unusual myosin heavy-chain (MHC) isoforms (i.e., slow-tonic, alpha-cardiac, embryonic, and neonatal), which exhibit distinct contractile properties. In this study, adult human mylohyoid (MH) muscles obtained from autopsies were investigated to detect the unusual MHC isoforms. For comparison, the biceps brachii and masseter muscles of the same subjects were also examined. Serial cross-sections from the muscles studied were incubated with a panel of isoform-specific anti-MHC monoclonal antibodies that distinguish major and unusual MHC isoforms. On average, the slow type I and fast type II MHC-containing fibers in the MH muscle accounted for 54% and 46% of the fibers, respectively. In contrast to limb and trunk muscles, the adult human MH muscle was characterized by a large proportion of hybrid fibers (85%) and a small percentage of pure fibers (15%; P < 0.01). Of the fast fiber types, the proportion of the type IIa MHC-containing fibers (92%) was much greater than that of the type IIx MHC-containing fibers (8%; P < 0.01). Our data demonstrated that the adult human MH fibers expressed the unusual MHC isoforms that were also identified in the masseter, but not in the biceps brachii. These isoforms were demonstrated by immunocytochemistry and confirmed by electrophoretic immunoblotting. Fiber-to-fiber comparisons showed that the unusual MHC isoforms were coexpressed with the major MHC isoforms (i.e., MHCI, IIa, and IIx), thus forming various major/unusual (or m/u) MHC hybrid fiber types. Interestingly, the unusual MHC isoforms were expressed in a fiber type-specific manner. The slow-tonic and alpha-cardiac MHC isoforms were coexpressed predominantly with slow type I MHC isoform, whereas the developmental MHC isoforms (i.e., embryonic and neonatal) coexisted primarily with fast type IIa MHC isoform. There were no MH fibers that expressed exclusively unusual MHC isoforms. Approximately 81% of the slow type I MHC-containing fibers expressed slow-tonic and alpha-cardiac MHC isoforms, whereas 80% of the fast type IIa MHC-containing fibers expressed neonatal MHC isoform. The m/u hybrid fibers (82% of the total fiber population) were found to constitute the predominant fiber types in the adult human MH muscle. At least seven m/u MHC hybrid fiber types were identified in the adult human MH muscle. The most common m/u hybrid fiber types were found to be the MHCI/slow-tonic/alpha-cardiac and MHCIIa/neonatal, which accounted for 39% and 33% of the total fiber population, respectively. The multiplicity of MHC isoforms in the adult MH fibers is believed to be related to embryonic origin, innervation pattern, and unique functional requirements.

Expression of unique and developmental myosin heavy chain isoforms in adult human digastric muscle.

Digastric muscle (DGM) is a powerful jaw-opening muscle that participates in chewing, swallowing, breathing, and speech. For better understanding of its contractile properties, five pairs of adult human DGMs were obtained from autopsies and processed with immunocytochemistry and/or immunoblotting. Monoclonal antibodies against alpha-cardiac, slow tonic, neonatal, and embryonic myosin heavy chain (MHC) isoforms were employed to determine whether the DGM fibers contain these MHC isoforms, which have previously been demonstrated in restricted specialized craniocervical skeletal muscles but have not been reported in normal adult human trunk and limb muscles. The results showed expression of all these MHC isoforms in adult human DGMs. About half of the fibers reacted positively to the antibody specific for the alpha-cardiac MHC isoform in DGMs, and the number of these fibers decreased with age. Slow tonic MHC isoform containing fibers accounted for 19% of the total fiber population. Both the alpha-cardiac and slow tonic MHC isoforms were found to coexist mainly with the slow twitch MHC isoform in a fiber. A few DGM fibers expressed the embryonic or neonatal MHC isoform. The findings suggest that human DGM fibers may be specialized to facilitate performance of complex motor behaviors in the upper airway and digestive tract.

Biochemical analyses of muscles from poultry bred for rapid growth

In the current study, commercial broiler breeder hens were mated with either commercial broiler breeder males (B/B) or artificially inseminated with semen from Leghorn cockerels (B/L). Embryos and chicks from each mating were used to study the effects of paternal genotype on breast muscle myosin expression without the confounding effects of differences in egg size and embryo development due to maternal genotype. Specifically, the temporal transitions of myosin heavy chain (MyHC) isoforms within the pectoralis (P.) major and P. minor were measured. The relative concentration of the embryonic MyHC isoform increased from d 17 through 21 in ovo in both genotypes and was higher in B/B embryos than in B/L embryos (P < or = 0.01). At 21 d posthatch, there was an increased proportion of the adult MyHC isoform within the P. major and P. minor in B/B compared with B/L chicks (P < or = 0.01). This result suggests that the B/B chicks were making the transition to mature skeletal muscle more rapidly than the B/L chicks. Although samples taken from the P. minor of B/B and B/ L chicks exhibited an increased proportion of the adult MyHC isoform and lower proportion of the neonatal MyHC isoform at 21 d, the genetic differences were far more pronounced in the larger P. major (P < or = 0.01). In summary, the P. major from the faster growing B/B chicks exhibited earlier temporal transitions of developmental fast MyHC, and these differences were evident as early as 17 d in ovo.

Mechanisms underlying increased force generation by rat diaphragm muscle fibers during development.

It has been found that maximum specific force (F(max); force per cross-sectional area) of rat diaphragm muscle doubles from birth to 84 days (adult). We hypothesize that this developmental change in F(max) reflects an increase in myosin heavy chain (MHC) content per half-sarcomere (an estimate of the number of cross bridges in parallel) and/or a greater force per cross bridge in fibers expressing fast MHC isoforms compared with slow and neonatal MHC isoforms (MHC(slow) and MHC(neo), respectively). Single Triton 100-X-permeabilized fibers were activated at a pCa of 4.0. MHC isoform expression was determined by SDS-PAGE. MHC content per half-sarcomere was determined by densitometric analysis and comparison to a standard curve of known MHC concentrations. MHC content per half-sarcomere progressively increased during early postnatal development. When normalized for MHC content per half-sarcomere, fibers expressing MHC(slow) and coexpressing MHC(neo) produced less force than fibers expressing fast MHC isoforms. We conclude that lower force per cross bridge in fibers expressing MHC(slow) and MHC(neo) contributes to the lower F(max) seen in early postnatal development.

Repression of myosin isoforms in developing and denervated skeletal muscle fibers originates near motor endplates.

During development of chicken pectoralis muscle, a neonatal myosin heavy-chain isoform is supplanted progressively by an adult isoform. This expression is under neuronal control. In this study we test the hypothesis that developmental myosin transformations are initiated near the motor endplate of each muscle fiber, thereafter progressing toward the fiber ends. By using immunocytochemical methods, pectoralis muscle from chickens aged 1-115 days after hatching were labeled by antibody against neonatal isoform. Ellipse minor axis and mean optical density of labeled and/or unlabeled fiber profiles from each bird were measured by computer image analysis. Acetylcholinesterase (AChE) activity was demonstrated histochemically. Using serial cross sections, we show that smaller fiber profiles are the tapered ends of larger fiber profiles. The largest fiber profiles (central regions of the fibers) were the first to lose their neonatal myosin during development. Motor endplates were localized by AChE activity to the central regions of the fibers. The pectoralis of mature chickens was denervated for 3, 7, 15, or 21 days. After 2 weeks' denervation, neonatal myosin is first reexpressed in the fiber ends. Dev Dyn 2000;217:50-61.

Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I.

The molecular mechanisms underlying myogenic induction by insulin-like growth factor I (IGF-I) are distinct from its proliferative effects on myoblasts. To determine the postmitotic role of IGF-I on muscle cell differentiation, we derived L6E9 muscle cell lines carrying a stably transfected rat IGF-I gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC-IGF-I exclusively in differentiated L6E9 myotubes, which express the embryonic form of myosin heavy chain (MyHC) and no endogenous IGF-I, resulted in pronounced myotube hypertrophy, accompanied by activation of the neonatal MyHC isoform. The hypertrophic myotubes dramatically increased expression of myogenin, muscle creatine kinase, beta-enolase, and IGF binding protein 5 and activated the myocyte enhancer factor 2C gene which is normally silent in this cell line. MLC-IGF-I induction in differentiated L6E9 cells also increased the expression of a transiently transfected LacZ reporter driven by the myogenin promoter, demonstrating activation of the differentiation program at the transcriptional level. Nuclear reorganization, accumulation of skeletal actin protein, and an increased expression of beta1D integrin were also observed. Inhibition of the phosphatidyl inositol (PI) 3-kinase intermediate in IGF-I-mediated signal transduction confirmed that the PI 3-kinase pathway is required only at early stages for IGF-I-mediated hypertrophy and neonatal MyHC induction in these cells. Expression of IGF-I in postmitotic muscle may therefore play an important role in the maturation of the myogenic program.

Doxorubicin chemomyectomy as a treatment for cervical dystonia: histological assessment after direct injection into the sternocleidomastoid muscle.

The sternocleidomastoid muscle (SCM) is one of the major muscles involved in producing abnormal head position in cervical dystonia patients. This study tested whether doxorubicin chemomyectomy, direct injection of doxorubicin into the SCM to permanently remove muscle fibers, has the potential to be a nonsurgical, permanent treatment for cervical dystonia. The right SCM of rabbits was injected with either 1 or 2 mg doxorubicin. Animals were sacrificed 1-2 months postinjection. The SCM was prepared for histological examination of muscle fiber loss and fiber type composition. In all cases, direct injection of doxorubicin resulted in significant decreases in total muscle cross-sectional areas ranging from 75% up to 98%. Individual myofiber cross-sectional areas were smaller than normal after 2 mg doxorubicin treatment, but similar to normal fiber size after 1 mg doxorubicin. There were increased numbers of myofibers that expressed slow and neonatal myosin heavy chain isoforms in these remaining muscle fibers compared to the untreated SCM on the contralateral side. Developmental myosin heavy chain (MHC) was also present in 53% of the remaining myofibers of the treated muscles. The fiber type composition of muscles contralateral to the doxorubicin injections was compared to the fiber type composition of SCM from normal, untreated controls; no difference was seen in the proportions of fast, slow, and neonatal MHC fiber types in these SCM muscles. In summary, the direct injection of doxorubicin into the SCM resulted in significant muscle loss. This supports the use of doxorubicin chemomyectomy as a potential permanent, nonsurgical treatment for cervical dystonia.

Muscle fiber type compartmentalization and expression of an immature myosin isoform in the sternocleidomastoid muscle of rabbits and primates

The sternocleidomastoid muscle is located in the neck and is both a neck rotator and flexor. Cervical dystonia, a focal dystonia disorder, is characterized by forceful involuntary contraction of a group of neck muscles, usually including the sternocleidomastoid. Little is known about the fiber type composition, fiber type compartmentalization and innervation patterns in this muscle in rabbit and primates. Sternocleidomastoid muscles from rabbit and monkey were analyzed for muscle fiber type composition and number, muscle fiber cross-sectional area and patterns of innervation. The sternocleidomastoid muscle was composed of two distinct regions, or compartments, with different fiber type compositions: an outer or superficial region composed of mostly type 2 myofibers and an inner deep region composed of both type 2 and type 1 myofibers. Neonatal myosin heavy chain isoform was detected in approximately 25% of the myofibers in both regions of the muscle. Neuromuscular junctions were located in seven endplate bands approximately 1–3 cm apart throughout the length of the muscle. There is clear evidence of anatomical subdivisions within this muscle. Not only is there variation in fiber type composition between superficial and deep regions of the muscle, but unlike most other mature skeletal muscles, it continues to express neonatal myosin heavy chain isoform in the adult. The motor program for neck movements is extremely complex, and the histological complexity plays a role in allowing for a continuum of movements of the head and neck, from maintenance of posture to rapid head movements.

Early myosin switching induced by nerve activity in regenerating slow skeletal muscle.

Muscle regeneration is a potentially useful model for defining the mechanisms responsible for nerve-dependent myosin isogene regulation in skeletal muscle. As a first step towards this goal we have characterized the pattern of expression of the four myosin heavy chain (MHC) genes, MHC-beta/slow, -2A, -2X and -2B isogenes, during early stages of muscle regeneration both in the presence and in the absence of the nerve. Muscle degeneration/regeneration was induced by intramuscular injection of the myotoxic drug, bupivacaine, in the rat slow soleus muscle. MHC transcripts were identified by in situ hybridization with specific riboprobes during the period from day 3 to day 7 after muscle injury. The four genes are not detected at day 3, when the regenerating muscle contains predominantly embryonic and neonatal MHC isoforms. MHC-2X and -2B transcripts are first detected at day 4 in both innervated and denervated muscles. These transcripts remain as major transcripts in denervated muscle whereas they are down-regulated by day 5 and disappear by day 6-7 in the presence of the nerve. Innervation induces strong up-regulation of MHC-2A at day 4 and MHC-beta/slow transcripts at day 5. MHC-2A transcripts are first homogeneously expressed in most fibers then become segregated in a minor population of fibers by day 6-7 while MHC-beta/slow transcripts increase in most fibers. In the absence of the nerve MHC-beta/slow transcripts are never expressed and MHC-2A transcripts are detected in rare fibers at days 5-7.

Myosin isoforms and muscle fiber characteristics in equine gluteus medius muscle

To date, four different myosin heavy chain (MyHC) isoforms have been identified in adult skeletal muscle of a number of species: types I, IIa, IIx or IId, and IIb. The aim of this study was to investigate the distribution of various MyHC isoforms in the equine gluteus medius and gluteus profundus muscles in relation with several morphometric variables of muscle fibers. Samples from different depths of the gluteus medius muscle (2, 4, 6, and 8 cm) and gluteus profundus muscle of five sedentary horses were examined by MyHC gel electrophoresis, monoclonal antibodies staining against fast, slow and neonatal MyHC isoforms, myosin adenosine triphosphatase (m-ATPase) activity, nicotinamide adenine dinucleotide tetrazolium reductase, alpha-glycerophosphate dehydrogenase, and alpha-amylase-PAS. Data about relative frequencies, sizes, and capillaries of the various histochemical fiber types were collected by morphometry. Three MyHC isoforms were present in the gluteus medius muscle. Two of them comigrated with type I and IIa MyHC isoforms of rat diaphragm (used as a control). The third isoform showed an electrophoretic mobility closer to type IIx than to the IIb MyHC isoform of rat diaphragm. Only two MyHC isoforms (type I and IIa) were detected in the gluteus profundus muscle. In both muscles, type I fibers (high m-ATPase activity at pH 4.5) only reacted with the anti slow-MyHC antibody and both type IIA and IIB fibers (low and moderate m-ATPase activity at pH 4.5, respectively) only reacted with the anti fast-MyHC antibody. No cross-reactivity of fibers positive for both antibodies was found except for the scarce type IIC fibers. Fiber types and capillaries were heterogeneously distributed across the gluteus medius muscle. The deeper regions of this muscle were found to contain a higher percentage of type I fibers, a large number of capillaries and a lower proportion of type IIB fibers compared to the superficial regions of the muscle. The gluteus profundus muscle had more abundant and larger type I fibers than the deepest sampling site of the gluteus medius muscle. These results show the existence of three different MyHC isoforms in the equine gluteus medius muscle and that fiber types and MyHC isoforms are heterogeneously distributed within this muscle. The distribution of slow-twitch and fast-twitch MyHCs among the fibers determined by immunohistochemistry was in agreement with histochemically identified type I and type II fibers, respectively.

Myosin isoforms and muscle fiber characteristics in equine gluteus medius muscle

Background To date, four different myosin heavy chain (MyHC) isoforms have been identified in adult skeletal muscle of a number of species: types I, IIa, IIx or IId, and IIb. The aim of this study was to investigate the distribution of various MyHC isoforms in the equine gluteus medius and gluteus profundus muscles in relation with several morphometric variables of muscle fibers. Methods Samples from different depths of the gluteus medius muscle (2, 4, 6, and 8 cm) and gluteus profundus muscle of five sedentary horses were examined by MyHC gel electrophoresis, monoclonal antibodies staining against fast, slow and neonatal MyHC isoforms, myosin adenosine triphosphatase (m-ATPase) activity, nicotinamide adenine dinucleotide tetrazolium reductase, α-glycerophosphate dehydrogenase, and α-amylase-PAS. Data about relative frequencies, sizes, and capillaries of the various histochemical fiber types were collected by morphometry. Results Three MyHC isoforms were present in the gluteus medius muscle. Two of them comigrated with type I and IIa MyHC isoforms of rat diaphragm (used as a control). The third isoform showed an electrophoretic mobility closer to type IIx than to the IIb MyHC isoform of rat diaphragm. Only two MyHC isoforms (type I and IIa) were detected in the gluteus profundus muscle. In both muscles, type I fibers (high m-ATPase activity at pH 4.5) only reacted with the anti slow-MyHC antibody and both type IIA and IIB fibers (low and moderate m-ATPase activity at pH 4.5, respectively) only reacted with the anti fast-MyHC antibody. No cross-reactivity of fibers positive for both antibodies was found except for the scarce type IIC fibers. Fiber types and capillaries were heterogeneously distributed across the gluteus medius muscle. The deeper regions of this muscle were found to contain a higher percentage of type I fibers, a large number of capillaries and a lower proportion of type IIB fibers compared to the superficial regions of the muscle. The gluteus profundus muscle had more abundant and larger type I fibers than the deepest sampling site of the gluteus medius muscle. Conclusions These results show the existence of three different MyHC isoforms in the equine gluteus medius muscle and that fiber types and MyHC isoforms are heterogeneously distributed within this muscle. The distribution of slow-twitch and fast-twitch MyHCs among the fibers determined by immunohistochemistry was in agreement with histochemically identified type I and type II fibers, respectively. © 1996 Wiley-Liss, Inc.

Effects of growth hormone injection to embryos on growth and myosin heavy chain isoforms in growing turkeys ( Meleagris gallopavo)

Effects of embryonic imprinting with growth hormone (GH) on growth and myosin heavy chain (MyHC) isoforms in pectoralis muscle were determined by injecting turkey embryos with ovine growth hormone (oGH) at a dose of 10 μg three times a day. Injections were made on days 20 and 26 (Treatment 1), days 14 and 20 (Treatment 2) or days 14 and 26 (Treatment 3) of incubation. In Treatement 1 poults, plasma GH concentrations were elevated at 3 days posthatch and in Treatment 3 poults, plasma GH concentrations were elevated at 15 days posthatch, as compared to control poults. At 4 weeks of age, in males, body weights, shank length and weights of pectoralis, gastrocnemius and sartorius muscles were increased in Treatment 3, and in females, body weights, shank length and weights of gastrocnemius muscle of female turkeys were increased in Treatment 1. The growth rate of female turkeys from 4 weeks through 16 weeks was increased by Treatment 1. Treatment 1 resulted in a delay in the transition from the embryonic MyHC isoform to the neonatal MyHC isoform and to the adult MyHC isoform. Treatment 3 induced an earlier appearance of the adult MyHC isoform. No effects on body and muscle growth and MyHC isoforms were observed by Treatment 2.

Expression of cytoskeleton proteins in central core disease

Despite advances in genetics the pathogenesis of central core disease (CCD) is still unknown. We studied muscles from 5 CCD patients by immunocytochemistry using monoclonal antibodies against various cytoskeletal proteins (dystrophin, spectrin, vinculin, desmin, vimentin, myosin heavy chain (MHC) of developmental, neonatal, adult slow and fast types). Dystrophin, spectrin and vinculin immunoreactivity was localized only at sarcolemma as in normal muscle. Vimentin was not present in myofibers. Only sporadic fibers were positive for developmental and neonatal MHC isoforms in adult CCD muscles. A 4-month-old patient had 5% of neonatal MHC-immunoreactive fibers, a finding similar to that of age-matched normal muscle. Desmin intermediate filaments were overexpressed in many core-fibers in extra-core regions, reduced or absent at cores, and greatly increased at the periphery of some cores. Moreover, irregular desmin-positive spots were seen within some cores. On the contrary, in neurogenic muscle atrophy patients, target lesions had increased desmin. These features indicate a possible role of desmin in the pathogenesis of cores, although we do not know if primary or secondary. In addition, they suggest that: (i) cores and targets may be manifestations of different processes; (ii) it is likely that core-fibers are not denervated fibers.

Influence of neonatal motor denervation on expression of myosin heavy chain isoforms in rat muscle spindles

In order to evaluate the effects of fusimotor elimination on the expression of myosin heavy chain (MHC) proteins in intrafusal fibres, we compared the muscle spindles in hind limb muscles of 3- to 6-week-old rats de-efferented at birth with those of their litter-mate controls. Serial sections were labelled with antibodies against slow tonic, slow twitch, fast twitch and neonatal MHC isoforms, against synaptophysin, the neurofilament 68 kD subunit and laminin. We found that de-efferented intrafusal fibres differentiated, as in normal spindles, into nuclear bag and bag fibres both containing predominantly slow MHC, and nuclear chain fibres that contained fast and neonatal MHC. In both de-efferented and control intrafusal fibres the same MHCs were stained; the degree and extent of staining, however, varied. Both types of de-efferented bag fibres displayed a high content of slow tonic and slow twitch MHC along most of the fibre length, in contrast to the prominent regional variation in control bag fibres. In their encapsulated regions, the de-efferented bag fibres were more similar to each other in their reactivity to anti-fast twitch and anti-neonatal MHC antibodies than the control bag fibres. In these aspects they resembled more closely the bag fibres of newborn rats. The differences might be due to an arrest of "specialization" in the regional expression of the different MHC isoforms. Chain fibres developed MHC patterns identical to those of control spindles with all the antibodies used, even though they differentiated from the beginning in the absence of motor innervation. The structural differentiation of the capsule and sensory innervation in de-efferented muscle spindles, as shown by anti-laminin, anti-synaptophysin and anti-neurofilament staining, did not differ from the controls. We conclude, in agreement with previous studies, that the sensory innervation plays a key role in inducing and supporting the differentiation of intrafusal fibres and the specific expression of their MHC. However, we also show that motor innervation and/or muscle function seem to be necessary for the diversity in the expression and distribution of different slow and fast MHC isoforms in the bag and bag fibres.