Fast Myosin Heavy Chain Research Articles

The relationship between slow and fast myosin heavy chain content, calpastatin and meat tenderness in different ovine skeletal muscles

The present study investigated the relationship between fibre type distribution and slow (MHC-s) and fast (MHC-f) myosin heavy chain content on calpastatin and meat tenderness in longissimus dorsi (LD), tensor fasciae latae (TFL), semitendinosus (ST), trapezius (TZ) and supraspinatus (SS) muscles from six Mule × Charolais rams. Samples taken at slaughter were frozen either in liquid N 2 for analysis of MHC-s and MHC-f by immunoblotting, or in cooled isopentane for histochemical fibre typing. Calpastatin activity and an immunoreactive 135 kDa calpastatin band were analysed in samples taken 24 h postmortem. Shear force was determined on muscle chops taken at 24 h postmortem and conditioned until day 14. The intensity of MHC-s and MHC-f immunopositive bands correlated with %Type I and %Type II fibres identified histochemically ( r 2 = 0.612 and 0.366, respectively, p < 0.001). Muscle specific differences were observed in MHC-s and MHC-f immunoreactivity, fibre type distribution, calpastatin activity, calpastatin 135 kDa immunoreactivity and shear force. MHC-s correlated positively with calpastatin activity ( r 2 = 0.725, p < 0.001) and 135 kDa calpastatin ( r 2 = 0.228, p < 0.01) across all muscle types. The data show that detection of MHC-s can be used to identify fibre type differences between ovine muscles and that this correlates with differences in calpastatin content and inhibitory activity, but not tenderness.

Altered excitation-contraction coupling with skeletal muscle specific FKBP12 deficiency.

The immunophilin FKBP12 binds the skeletal muscle Ca2+ release channel or ryanodine receptor (RyR1), but the functional consequences of this interaction are not known. In this study, we have generated skeletal muscle specific FKBP12-deficient mice to investigate the role of FKBP12 in skeletal muscle. Primary myotubes from these mice show no obvious change in either Ca2+ stores or resting Ca2+ levels but display decreased voltage-gated intracellular Ca2+ release and increased L-type Ca2+ currents. Consistent with the decreased voltage-gated Ca2+ release, maximal tetanic force production is decreased and the force frequency curves are shifted to the right in extensor digitorum longus (EDL) muscles of the mutant mice. In contrast, there is no decrease in maximal tetanic force production in the mutant diaphragm or soleus muscle. The force frequency curve is shifted to the left in the FKBP12-deficient diaphragm muscle compared with controls. No changes in myosin heavy chain (MHC) phenotype are observed in EDL or soleus muscle of the FKBP12-deficient mice, but diaphragm muscle displays an increased ratio of slow to fast MHC isoforms. Also, calcineurin levels are increased in the diaphragm of the mutant mice but not in the soleus or EDL. In summary, FKBP12 deficiency alters both orthograde and retrograde coupling between the L-type Ca2+ channel and RyR1 and the consequences of these changes depend on muscle type and activity. In highly used muscles such as the diaphragm, adaptation to the loss of FKBP12 occurs, possibly due to the increased Ca2+ influx.

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.

Muscle molecular phenotype after stroke is associated with gait speed.

The disability of patients after stroke is generally attributed to upper motor neuron defects, but secondary changes in paretic muscle may enhance the disability. We analyzed the molecular phenotype and metabolic profile of the paretic and nonparetic vastus lateralis (VL) and we measured the severity of gait deficit in 13 patients at least 6 months after ischemic stroke. The results showed a significant increase in the proportion of fast myosin heavy chain (MHC, 68 +/- 14%) in the paretic compared to the nonparetic VL (50 +/- 13%). The specific activity of citrate synthase and glyceraldehyde phosphodehydrogenase was not significantly different between the two sides. The proportion of fast MHC was inversely associated with severity of gait deficit indexed by self-selected walking speed in the paretic leg, but not the nonparetic leg. Our findings demonstrate significant and potentially modifiable secondary biologic changes in hemiparetic muscle phenotype that may contribute to the disability of stroke.

C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression.

We describe a simple culture method for obtaining highly differentiated clonal C2C12 myotubes using a feeder layer of confluent fibroblasts, and document the expression of contractile protein expression and aspects of myofibre morphology using this system. Traditional culture methods using collagen- or laminin-coated tissue-culture plastic typically results in a cyclic pattern of detachment and reformation of myotubes, rarely producing myotubes of a mature adult phenotype. C2C12 co-culture on a fibroblast substratum facilitates the sustained culture of contractile myotubes, resulting in a mature sarcomeric register with evidence for peripherally migrating nuclei. Immunoblot analysis demonstrates that desmin, tropomyosin, sarcomeric actin, alpha-actinin-2 and slow myosin are detected throughout myogenic differentiation, whereas adult fast myosin heavy chain isoforms, members of the dystrophin-associated complex, and alpha-actinin-3 are not expressed at significant levels until >6 days of differentiation, coincident with the onset of contractile activity. Electrical stimulation of mature myotubes reveals typical and reproducible calcium transients, demonstrating functional maturation with respect to calcium handling proteins. Immunocytochemical staining demonstrates a well-defined sarcomeric register throughout the majority of myotubes (70-80%) and a striated staining pattern is observed for desmin, indicating alignment of the intermediate filament network with the sarcomeric register. We report that culture volume affects the fusion index and rate of sarcomeric development in developing myotubes and propose that a fibroblast feeder layer provides an elastic substratum to support contractile activity and likely secretes growth factors and extracellular matrix proteins that assist myotube development.

Differentiation of rat skeletal muscle fibres during development and ageing

The purpose of the present study was to determine at which point in the period from embryonic day 21 up to postnatal day (PD) 75, the different fibre types and subtypes are detectable in rat extensor digitorum longus, soleus and gastrocnemius muscles using immunohistochemical, enzyme histochemical and cytophotometrical methods. Moreover, fibre type-specific changes in metabolic profile and changes in fibre type population during postnatal development were analysed. Before birth, no clear differentiation of fibre types was found. At PD 1, slow and fast fibres were typed by antibodies against neonatal, slow and fast myosin heavy chains (MHCs). At PD 8, the different ATPase activities of slow and fast MHCs after alkaline preincubation were detected histochemically. At PD 21, differences in acid stability of ATPase activity of fast MHC isoforms revealed the fast subtypes IIA and IIB (including IIX). At this age, also differences in metabolic properties (oxidative and glycolytic enzyme activities) of fibres were detected for the first time by cytophotometry classifying the fibres into SO, FOG I, FOG II and FG. Before the age of 21 days, the fast fibres were metabolically undifferentiated. During further development and ageing, the population of FG fibres with high glycolytic activity increased at the expense of FOG fibres suggesting FOG to FG transformation. Cytophotometrical measurements revealed that the muscle fibres developed their highest contractile, oxidative and glycolytic activity at PD 21, the time of weaning. In this way, muscle fibres may be prepared for the higher demands for posture and mobility after leaving the nest.

Effect of Dietary Creatine on Skeletal Muscle Myosin Heavy Chain Isoform Expression in an Animal Model of Uremia

Background: Chronic renal failure is accompanied with muscle dysfunction and myopathy, characterized by muscle weakness and increased fatigue. Myosin heavy chain (MHC) is the principal structural protein that controls the intrinsic contractile properties of striated muscle. Creatine is widely used as an ergogenic nutritional supplement in sportsmen. This study investigates the effect of creatine supplementation on MHC expression in the setting of uremic myopathy. Methods: Male Wistar rats were either sham operated or subtotally nephrectomized and received a control diet or creatine (2% w/w)-supplemented diet. After 4 weeks of treatment, serum creatinine, creatine, urea and creatinine clearances were determined. MHC isoforms were determined electrophoretically in the extensor digitorum longus and soleus muscles. Results: Creatinine clearances were lower in nephrectomized animals, but similar in creatine-supplemented and control diet animals. Nephrectomized animals had significantly higher MHC IIb and lower MHC IIx isoform expression in the extensor digitorum longus muscle compared to sham-operated animals. In the soleus muscle, MHC IIb expression was increased in nephrectomized animals. Creatine supplementation reversed the MHC transitions observed in uremia in the soleus muscle, but not in the extensor digitorum longus muscle. Conclusion: We observed altered expression of MHC isoforms in uremia. In uremic animals, fast MHC IIb isoforms were increased, whereas MHC I and IIx isoforms predominate in control animals. Dietary creatine supplementation reversed the altered MHC expression during uremia in slow-twitch, but not in fast-twitch muscles.

A single-fibre study of the relationship between MHC and TnC isoform composition in rat skeletal muscle.

In the present study, we investigated the possibility that MHC (myosin heavy chain) and TnC (troponin C) isoforms exist in specific combinations in rat-skeletal-muscle fibres. Single fibres (numbering 245) from soleus (predominantly slow-twitch) and sternomastoid (predominantly fast-twitch) muscles of adult rats were analysed for MHC and TnC isoform composition, using alanine-SDS/PAGE for separating MHC isoforms, and a novel method (based on the previously reported influence of Ca2+ on the mobility of Ca2+-binding proteins in SDS gels) for unequivocal identification of TnC isoforms in single-fibre segments. In this study, all fibres that contained only one MHC isoform (slow or fast) contained only the matching TnC isoform and all fibres that contained multiple fast MHC isoforms contained only the fast TnC isoform. Fibres expressing both slow and fast MHC isoforms displayed either both TnC isoforms or only one TnC isoform of a type depending on the relative proportion of fast/slow MHC present. Our results suggest a close relationship between MHC and TnC isoform composition in non-transforming skeletal muscles of adult rat.

Differential expression of myosin heavy chain isoforms between abductor and adductor muscles in the human larynx

Objective This study examines the differential expression of myosin heavy chain (MyHC) components in human laryngeal muscle groups. Study design A battery of monospecific monoclonal antibodies in Western blots was used to determine expression of IIX, extraocular-specific (EOM), and IIB MyHCs for the thyroarytenoid (TA), vocalis (VOC), lateral cricoarytenoid (LCA), cricothyroid (CT), and posterior cricoarytenoid (PCA) muscles obtained from fresh cadaver specimens. Results Fast IIX MyHC was only expressed in the TA, VOC, and LCA muscles. Fast IIA and slow MyHCs were expressed in all laryngeal muscles including the CT and PCA. The CT with mixed phonatory and respiratory function and the PCA with respiratory function did not express IIX MyHC. The 2 MyHC isoforms associated with the highest speeds of contraction in rat laryngeal muscle, namely, the EOM MyHC and IIB MyHC, were not detected in human laryngeal muscles. Novel MyHC bands were not detected in SDS-PAGE gels or Western blots using a broad specificity MyHC antibody. Conclusion The profile of MyHC expression in human laryngeal muscles differs from that observed in human extraocular and masticator muscles, and other mammalian species. Our data demonstrate that IIX MyHC expression is associated primarily with muscles affecting glottic closure and is absent in CT and PCA. Significance A higher percentage of IIX MyHC is expected to impart a high speed of shortening to the TA and LCA muscles. The absence of IIX MyHC in muscles with respiratory (PCA) and mixed respiratory/phonatory function (CT) further supports the inference that the physiologic difference between laryngeal muscles is reflected in the molecular composition of contractile protein.

Different time course of changes in sarcoplasmic reticulum and myosin isoforms in rat soleus muscle at early stage of hyperthyroidism.

We studied changes in the expression of myosin and sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and in Ca2+ uptake and release rates by the SR in rat slow-twitch soleus muscles treated with thyroid hormone (3,5,3'-triiodo-l-thyronine, T3) for 1, 3, 7 and 21 days. The content of fast SR Ca2+-ATPase isoform SERCA1a protein significantly increased as early as 3 days of T3 treatment, while a rise in fast myosin heavy chain isoforms was found only 21 days after the onset of T3 administration. T3-induced slow-to-fast transitions were much less pronounced in myosin light chain than in myosin heavy chain. Moreover, in vitro measurements of SR Ca2+-handling capacity with indo-1 indicated that increases in uptake rate preceded those of release rate (7 vs. 21 days). These data suggest that changes in the myosin and SERCA expressions seem not to occur in a coordinated manner at the early stage of hyperthyroidism and that intervention with T3 may temporarily produce slow-twitch type I fibres which contain, in addition to slow SERCA2a isoform, the appreciable amounts of fast SERCA1a isoform. Moreover, it is speculated that some fibres in the hyperthyroid soleus muscles may temporarily acquire unique contractile properties distinct from normal fast and slow fibres.

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.

Improved sphincter contractility after allogenic muscle-derived progenitor cell injection into the denervated rat urethra

Objectives To study the physiologic outcome of allogenic transplant of muscle-derived progenitor cells (MDPCs) in the denervated female rat urethra. Methods MDPCs were isolated from muscle biopsies of normal 6-week-old Sprague-Dawley rats and purified using the preplate technique. Sciatic nerve-transected rats were used as a model of stress urinary incontinence. The experimental group was divided into three subgroups: control, denervated plus 20 μL saline injection, and denervated plus allogenic MDPCs (1 to 1.5 × 10 6 cells) injection. Two weeks after injection, urethral muscle strips were prepared and underwent electrical field stimulation. The pharmacologic effects of d-tubocurare, phentolamine, and tetrodotoxin on the urethral strips were assessed by contractions induced by electrical field stimulation. The urethral tissues also underwent immunohistochemical staining for fast myosin heavy chain and CD4-activated lymphocytes. Results Urethral denervation resulted in a significant decrease of the maximal fast-twitch muscle contraction amplitude to only 8.77% of the normal urethra and partial impairment of smooth muscle contractility. Injection of MDPCs into the denervated sphincter significantly improved the fast-twitch muscle contraction amplitude to 87.02% of normal animals. Immunohistochemistry revealed a large amount of new skeletal muscle fiber formation at the injection site of the urethra with minimal inflammation. CD4 staining showed minimal lymphocyte infiltration around the MDPC injection sites. Conclusions Urethral denervation resulted in near-total abolishment of the skeletal muscle and partial impairment of smooth muscle contractility. Allogenic MDPCs survived 2 weeks in sciatic nerve-transected urethra with minimal inflammation. This is the first report of the restoration of deficient urethral sphincter function through muscle-derived progenitor cell tissue engineering. MDPC-mediated cellular urethral myoplasty warrants additional investigation as a new method to treat stress urinary incontinence.

Method of isolation, rate of postmortem metabolism, and myosin heavy chain isoform composition influence ATPase activity of isolated porcine myofibrils

The objective of this study was to determine the influence of myofibril isolation procedures and myosin heavy chain (MyHC) isoform composition on myofibrillar ATPase activity as related to postmortem muscle metabolism. Myofibrils from the red (RST) and white (WST) portions of semitendinosus muscles were isolated using two different methods (A and B) at 3 min and 24 h postmortem in control (NS) and electrically stimulated (ES) pork carcasses. Comparison of the relative MyHC isoform profiles between the two different myofibril isolation methods and myosin extracts from the RST and WST at 3 min showed that method B myofibrils were more similar to the myosin extract than method A. Myofibrillar ATPase activity remained constant or increased ( P<0.01) from 3 min to 24 h postmortem in NS carcasses and decreased ( P<0.0001) in ES carcasses. From the RST, method A myofibrils had higher ( P<0.0001) ATPase activity compared to method B across sampling time and carcass treatment. In the WST, method A myofibrils had lower ( P<0.01) activity at 3 min, were not different at 24 h in NS carcasses, but had higher ( P<0.05) activity at 24 h in ES carcasses versus method B myofibrils. Compared to method B, isolation method A biased the isoform profile of myofibril samples more towards faster MyHC (2A and 2X) in the RST and towards MyHC 2X in the WST. Results suggest that the ATPase activity and MyHC isoform profile of isolated myofibril samples are influenced by method of myofibril isolation, postmortem sampling time, and the rate of postmortem metabolism. Thus, differences in MyHC isoform profile and method of myofibril isolation must be taken into account to determine accurately the relationship between myofibrillar ATPase activity and rate of postmortem metabolism.

Distribution of SERCA isoforms in human intrafusal fibers.

The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein that plays a crucial role in muscle relaxation by transporting cytosolic Ca2+ into the lumen of the sarco/endoplasmic reticulum. In this study, the presence of SERCA1 and SERCA2 was investigated in human intrafusal fibers by immunocytochemistry. Nuclear bag1 fibers contained both SERCA1 and SERCA2 isoforms, with predominant staining seen with SERCA2 in the A and B regions. Most nuclear bag2 fibers also contained SERCA1 and SERCA2 isoforms and their coexistence frequently occurred in the A region. SERCA1 was present whereas SERCA2 was generally absent in the nuclear chain fibers. The staining intensity seen with the SERCA1 monoclonal antibody varied in the order of chain>bag1>bag2. The expression of SERCA1 isoform was found to correlate with the presence of fast myosin heavy chain (MyHC) isoform in nuclear chain fibers, whereas for nuclear bag fibers there was no such apparent correlation between patterns of expression of SERCA and MyHC isoforms. The phenotype revealed for the human bag fibers was very sophisticated and adapted to attain a very wide range of contraction and relaxation velocities.

Differential expression of equine myosin heavy-chain mRNA and protein isoforms in a limb muscle.

The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei.

The striated urethral sphincter in female rats.

The aim of this study was to give a microscopic description of the organization, the innervation and the slow or fast type of the striated fibers of the external urethral sphincter in the female rat. Conventional methods for photonic microscopy and immunochemistry were applied to cross and longitudinal sections of snap-frozen urethra. With hematoxylin-eosin stained cross sections, striated fibers are of small diameter and attached directly to the surrounding connective tissue. They are innervated by cholinergic endplates as shown by acetylcholinesterase techniques and alpha-bungarotoxin binding. The histological aspects of the cross sections as well as the distribution of endplates along the length of the sphincter suggest an organization of the fibers in four bundles, possibly acting as a photographic diaphragm does. Like striated skeletal muscle fibers, the fibers bind monoclonal antibodies against dystrophin with subsarcolemmal distribution and against desmin which visualizes striations. All the fibers express fast myosin heavy chains and very few co-express slow myosin heavy chains as determined by immunocytochemistry. We are taking advantage of the diaphragmatic organization of the striated sphincter to develop a longitudinal section as a model of chronic incontinence to test the efficiency of grafted myoblasts provided by fast striated skeletal muscle.

Regulation of myosin expression during myotome formation.

The first skeletal muscle fibers to form in vertebrate embryos appear in the somitic myotome. PCR analysis and in situ hybridization with isoform-specific probes reveal differences in the temporal appearance and spatial distribution of fast and slow myosin heavy chain mRNA transcripts within myotomal fibers. Embryonic fast myosin heavy chain was the first isoform expressed, followed rapidly by slow myosin heavy chains 1 and 3, with slow myosin heavy chain 2 appearing several hours later. Neonatal fast myosin heavy chain is not expressed in myotomal fibers. Although transcripts of embryonic fast myosin heavy chain were always distributed throughout the length of myotomal fibers, the mRNA for each slow myosin heavy chain isoform was initially restricted to the centrally located myotomal fiber nuclei. As development proceeded, slow myosin heavy chain transcripts spread throughout the length of myotomal fibers in order of their appearance. Explants of segments from embryos containing neural tube, notochord and somites 7-10, when incubated overnight, become innervated by motor neurons from the neural tube and express all four myosin heavy chain genes. Removal of the neural tube and/or notochord from explants prior to incubation or addition of d-tubocurare to intact explants prevented expression of slow myosin chain 2 but expression of genes encoding the other myosin heavy chain isoforms was unaffected. Thus, expression of slow myosin heavy chain 2 is dependent on functional innervation, whereas expression of embryonic fast and slow myosin heavy chain 1 and 3 are innervation independent. Implantation of sonic-hedgehog-soaked beads in vivo increased the accumulation of both fast and slow myosin heavy chain transcripts, as well as overall myotome size and individual fiber size, but had no effect on myotomal fiber phenotype. Transcripts encoding embryonic fast myosin heavy chain first appear ventrolaterally in the myotome, whereas slow myosin heavy chain transcripts first appear in fibers positioned midway between the ventrolateral and dorsomedial lips of the myotome. Therefore, models of epaxial myotome formation must account for the positioning of the oldest fibers in the more ventral-lateral region of the myotome and the youngest fibers in the dorsomedial region.

Postnatal myosin heavy chain isoforms in prenatal porcine skeletal muscles: insights into temporal regulation.

Our knowledge of the temporal expression of postnatal (adult) fast myosin heavy chain (MyHC) isoforms (2a, 2x, and 2b) in prenatal muscles is limited. Using the pig as a target species and large-animal model, we report on the qualitative and quantitative expression of the major post- and prenatal MyHC isoforms during gestation, as determined by TaqMan real-time PCR and immunohistochemistry. We found that postnatal fast MyHC mRNAs and proteins were expressed much earlier in the pig (gestation day 35) than was previously reported in small mammals. There was a high degree of coexpression and colocalisation of pre- and postnatal MyHC mRNAs and proteins in prenatal muscles. During a period of prenatal muscle growth (gestation days 35-77), relative expression of MyHC isoforms (embryonic > 2a > 2x > 2b) correlated with the gene order in the skeletal MyHC cluster, which suggests the possible presence of cis-acting elements on the same side as the MyHC embryonic gene associated with temporal regulation.

Distribution of developmental myosin heavy chains in adult rabbit extraocular muscle: identification of a novel embryonic isoform absent in fetal limb.

To identify embryonic and neonatal/fetal myosin heavy chains (MyHCs) in rabbit extraocular muscle (EOM) by electrophoretic and immunochemical analyses and to describe the distribution of these two MyHC isoforms in the endplate zone (EPZ) and the distal and proximal segments of EOM fibers. SDS-PAGE and Western blot analysis using monoclonal antibodies (mAbs) against embryonic and neonatal/fetal MyHCs were performed on MyHC isoforms from rabbit adult and neonatal EOM and fetal limb muscles. Immunohistochemical analysis was performed along the entire length of the rabbit superior rectus muscles, using these and other mAbs. Western blot analysis showed that adult rabbit EOM had two embryonic MyHC bands: a weakly stained band that comigrated with the embryonic MyHC from fetal limb muscles, and a strongly stained band of lower electrophoretic mobility for which there was no limb counterpart. Three anti-embryonic MyHC mAbs stained muscle fibers, predominantly in the orbital layer, and staining was localized distal and proximal to the EPZ but not in the EPZ itself. There, most fibers expressed the EOM-specific fast MyHC, although some fibers expressed alpha-cardiac MyHC. Anti-neonatal/fetal MyHC mAb failed to stain in Western blot analysis but stained scattered fibers predominantly in the global layer, and there was no specific absence of staining at the EPZ. There are two electrophoretically distinct isoforms of embryonic MyHCs in adult rabbit EOM. These isoforms are expressed in orbital fibers but are excluded from the EPZ, where EOM-specific fast MyHC is strongly expressed. Neonatal and fetal MyHC is weakly expressed in the EOM, but is not excluded from the EPZ.

Compared effects of hindlimb unloading versus terrestrial deafferentation on muscular properties of the rat soleus

Hindlimb unloading is known to induce some neuromuscular changes especially in postural muscles such as the soleus. Our goal was to determine the role of proprioceptive inputs on these modifications by comparing soleus muscle properties of rats being either hindlimb unloaded or terrestrial deafferented. Under deep anesthesia, a first group of rats were submitted to a bilateral deafferentation (DEAF group, n = 6) performed by section of the dorsal roots L 3 to L 5 after laminectomy. A second group of rats was submitted to a hindlimb-unloading period (HU group, n = 6). After 14 days, the morphological and contractile properties as well as the content in myosin heavy-chain (MHC) isoforms were studied in the right soleus muscle in HU and DEAF groups. The results were compared to those obtained in control animals (CON group). After HU versus CON group, the soleus muscle was atrophied and presented a decrease in muscle forces in relation with a slow-to-fast transition characterized by a decrease in kinetic contractile parameters and by an overexpression in the fast MHC isoforms. The DEAF soleus muscle showed both a significant muscle atrophy and a loss of forces when compared with CON rats. The comparison between DEAF and HU rats indicated that some modifications occurring after HU are purely of motor origin (i.e., slow to fast transition), whereas muscle atrophy and decrease in muscle force are partly the result of an afferent silent. Our study underlined the importance of afferent input integrity in the maintenance of muscle characteristics.