Heavy Myosin Research Articles

Immunofluorescent Identification of Dystrophin, Actin, and Light and Heavy Myosin Chains in Somatic Cells of Earthworm Lumbricus terrestris

Immunofluorescent Identification of Dystrophin, Actin, and Light and Heavy Myosin Chains in Somatic Cells of Earthworm Lumbricus terrestris

Muscular and neuronal control of voice production – forgotten findings, current concepts, and new developments

Voice production has been an area of interest in science since ancient times, and although advancing research has improved our understanding of the anatomy and function of the larynx, there is still little general consensus on these two topics. This review aims to outline the main developments in this field and highlight the areas where further research is needed. The most important hypotheses are presented and discussed highlighting the four main lines of research in the anatomy of the human larynx and their most important findings: (1) the arrangement of the muscle fibers of the thyroarytenoid muscle is not parallel to the vocal folds in the internal part (vocalis muscle), leading to altered properties during contraction; (2) the histological structure of the human vocal cords differs from other striated muscles; (3) there is a specialized type of heavy myosin chains in the larynx; and (4) the neuromuscular system of the larynx has specific structures that form the basis of an intrinsic laryngeal nervous system. These approaches are discussed in the context of current physiological models of vocal fold vibration, and new avenues of investigation are proposed.

Considering Two Aspects of Fish Welfare on African Catfish (Clarias gariepinus) Fillet throughout Postmortem Condition: Efficiency and Mechanisms.

Knowledge about fish welfare and its impact on fish fillet quality is still insufficient. Therefore, the influence of two aspects of fish welfare (slaughtering method: bled and unbled fish; fish stock densities: 90, 120, and 150 kg·m-3) on African catfish fillet quality during postmortem conditions was investigated. The aim of study was to determine (i) the efficiency of bleeding on oxidation progress and (ii) the influence of stock density on fillet quality. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a higher protein loss in the unbled than in the bled groups, especially in the heavy myosin chain (MHC) band. However, density did not show any influence on protein profile. Western blot analysis showed fewer oxidized carbonyls in the bled than in the unbled groups; higher oxidation development, microbial growth, and lower hardness were observed in unbled fillets. Additionally, hardness was higher at 90 and 120 kg·m-3 densities in bled fillet compared to 150 kg·m-3. The first three days of storage showed a higher oxidation rate in unbled fillets than in bled fillets, confirming the contribution of hemoglobin to oxidation development with different mechanisms of protein oxidation. The obtained results revealed the same fillet quality in all aspects at either 90 or 120 (kg·m-3) stock densities, which would suggest 120 kg·m-3 for the fishery industry. However, higher stocking density in this study would not be appropriate for fish welfare.

Role of oxygen exposure on the differentiation of human induced pluripotent stem cells in 2D and 3D cardiac organoids

Human induced pluripotent stem cells (hiPSC) have the ability to differentiate theoritically into any cell type. The development of organoid systems exhibiting the essential features of human organ such as liver and heart is of high interest. Optimizing the culture conditions to obtain the highest cardiac organoids efficacy is crucial. In fact, cardiac differentiation protocols have been established by essentially focusing on specific growth factors on hiPSC differentiation efficiency. However, the optimal environmental factors such as the optimal oxygen exposure to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Yet, the effect of low oxygen exposure on the molecular and functional maturity of the hiPSC-derived cardiomyocytes remains unexplored. We aimed here at comparing the molecular and functional consequences of low (5% O 2 or LOE) and high oxygen exposure (21% O 2 or HOE) on cardiac differentiation of hiPSCs in 2D monolayer and 3D organoids protocols. hiPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed using qRT-PCR and immunofluorescence. The mitochondrial localization and metabolic properties were evaluated by high-resolution respirometry and mitochondrial staining. The intracellular Ca 2+ handling and contractile properties were also monitored using confocal fluorescent microscopy and atomic force microscopy. Our results indicated that the 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac organoids containing hiPSC-CMs in LOE exhibited higher cardiac markers expression such as troponin T (TnTc), RyR2, Serca2a, alpha and beta heavy myosin chains. Moreover, we found enhanced contractile force, hypertrophy and steadier SR Ca 2+ release reflected by a more regular spontaneous Ca 2+ transients associated with a higher maximal amplitude and lower spontaneous Ca 2+ events revealing a better SR Ca 2+ handling in LOE. Similar beat rate, preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac organoids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.

Expression of Myosin Heavy Chain and Some Energy Metabolism-Related Genes in the Longissimus Dorsi Muscle of Krškopolje Pigs: Effect of the Production System.

The Slovenian Krškopolje pig is the only preserved local autochthonous breed, appreciated mainly for its good meat quality and considered more appropriate for processing into dry-cured products. However, the biological characteristics of the skeletal myofibers of the Krškopolje breed, specifically the heavy myosin chain-based contractile and metabolic phenotypes that could affect meat quality, have not been established under different husbandry systems. The breed is generally maintained in either conventional indoor or organic systems. In the present study, the morphological, contractile, and metabolic properties of myofibers of the longissimus dorsi muscle were compared between animals reared in either an organic or a conventional indoor system. The myofibers were studied using immunohistochemical and succinate dehydrogenase (SDH) activity-based classification, histomorphometric assessment, and qPCR. Results revealed that the organic production system influenced the composition of the longissimus dorsi myofiber type, characterized by a smaller myofiber cross-sectional area, a shift toward oxidative (SDH-positive) myofiber types, increased relative expression of myosin heavy chain (MyHC) isoforms I, IIa, and IIx, and downregulation of MyHC IIb. On the contrary, no apparent effect was observed on the metabolic phenotype of the myofiber as assessed through relative mRNA expression of energy metabolism-related genes [peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1α), peroxisome proliferator-activated receptor gamma (PPARγ), lipoprotein-lipase (LPL), carnitine palmitoyltransferase 1B (CPT1B), glycogen synthase 1 (GYS1), hexokinase 2 (HK2), and fatty acid synthase (FASN)]. Differences in MyHC expression were largely corroborated by the histochemical classification, indicating that the contractile protein content is directly regulated by the MyHC genes. A correlation between the muscle contractile and metabolic phenotypes was not established, except for that between the HK2 and MyHC I genes. In conclusion, the present study showed an evident effect of rearing on the longissimus dorsi myofiber contractile phenotype but not the metabolic phenotype. Moreover, obtained data suggest that rearing the Krškopolje pig breed in a conventional system would result in an increased fiber size and a greater proportion of type IIb myofibers, which are known to be negatively correlated with some meat quality traits.

Effect of citric acid on physicochemical properties and protein structure of low-salt restructured tilapia (Oreochromis mossambicus) meat products.

The growing consumer demand for healthy products has encouraged the development of low-salt meat products. In this study, to develop low-salt restructured tilapia (Oreochromis mossambicus) meat products, citric acid was used to improve the properties of restructured tilapia products. In comparison with control restructured fish products (RP) and surimi products (SP), 0.2% citric acid-treated restructured fish products (RPC) and surimi products (SPC) showed a significant decrease in expressible water and water activity and a remarkable increase in whiteness, dry matter, hardness, chewiness, gumminess, and acceptability. Mechanistic studies suggested that citric acid significantly changed the content of total protein and myofibrillar proteins and promoted degradation of heavy myosin chains. Fourier-transform infrared and Raman spectra revealed the citric acid-mediated alteration in the peak intensities of amide I and amide II bands, which changed the secondary structures of RPC and SPC. It is feasible to prepare low-salt restructured tilapia meat products using citric acid, which offers a means of using muscle by-products and exploiting new functional products with an added commercial value. © 2020 Society of Chemical Industry.

Mid Regional Natriuretic Peptide for Predicting Prognosis of Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) is a genetic disorder of cardiac myocytes that is characterized by cardiac hypertrophy, cellular disarray and interstitial fibrosis. Mutation of MYH7 and MYBPC3 encoding proteins β-myosin heavy chain and myosin binding protein C, respectively, are the two most common genes involved, together accounting for about 50% of cases. The present study was conducted to evaluate the prognostic value of MR-proANP in patients with HCM. Subjects and Methods: The present study was conducted at Narayana Medical College & Hospital, Chintareddy Palem, Nellore, Andhra Pradesh from July 2016 to July 2017 on 46 patients of hypertrophic cardiomyopathy of both genders. Serum NT-proBNP was measured by a two-site electrochemiluminescence immunoassay on a Roche Diagnostics E170 analyser. Results: The mean systolic blood pressure was 124.8 mm Hg, diastolic blood pressure was 78 mm Hg, heart rate was 68 beats/minutes, NYHA 1(n=4), NYHA 2 (n=27), NYHA 3–4 (n=15) and atrial fibrillation was 12%. The mean MR-proANP was 106 pmol/L and NT-proBNP was 540 pg/mL at the start of the study. At the end of 12 months of follow up, 15 patients had a primary end point defined as heart failure hospitalisation (n=10), heart transplant (n=3) death(n=2). Both mean MR-proANP and NT-proBNP were strongly associated with the primary end points at the end of study period with values of 1010 pmol/L and 2545 pg/ml respectively. This showed a strong association with P <0.05. Conclusion: Authors found that MR-proANP is a valuable biomarker for the prediction of heart failure related events in patients with HCM.

3D myotube guidance on hierarchically organized anisotropic and conductive fibers for skeletal muscle tissue engineering.

Tissue engineering represents a promising approach for the functional restoration of large volumetric skeletal muscle loss. However, design and fabrication of 3D hierarchically organized scaffolds that closely mimic the natural micro-/nanostructures of skeletal muscle extracellular matrix (ECM) is still an ongoing challenge. Here, we constructed a hierarchically organized, anisotropic and conductive scaffold with microscale melt electrowriting (MEW) grooves parallel aligned on the top of unidirectionally oriented nanofibrous mesh to guide myoblast cell alignment, elongation and differentiation into myotubes. A 7-days study of H9c2 myoblast cells cultured on the scaffolds indicated that the combination of nanoscale and microscale anisotropic surface topography enhanced myogenesis. Specifically, the parallel patterned scaffold effectively enhanced both the elongation and maturation of myotubes, as indicated by the increased myotube length (>600μm), higher heavy myosin chain (MHC) surface coverage and maturation index on the parallel patterned scaffold compared to their pure (2.4-fold MHC surface coverage, 1.6-fold maturation index) and perpendicular patterned counterparts (1.7-fold MHC surface coverage, 1.5-fold maturation index). Furthermore, a gold nanolayer coating on the aligned nanofibrous mesh surface provided electroactive cues to enhance the formation of myotubes. With the increase of Au coating thickness, improved myoblasts alignment (74.6%, 80.5%, 85.6%, 94.9% for Au 0, 10, 40, 70nm respectively) and higher MHC-positive expression (57.5% for Au 0nm, 90.3%, 92.5%, 95.0% for Au10, 40, 70nm respectively) were observed. Finally, the formation and morphology of myotubes were also found dependent on the spacing between microgrooves (100, 200, 300μm), where myotubes formed on 200μm spacing scaffold showed the highest alignment (within 10° along nanofibers) and elongation (>850μm). The hierarchically organized, anisotropic and conductive fibers hold great potential for regeneration of skeletal muscle tissue.

Proteins Features of the Far Eastern Region Bivalve Mollusk Mactra Сhinensis

Bivalve mollusk are protein raw materials, their soft tissues are characterized by a hard-rubbery consistency due to its proteins characteristics and should be considered when processing into food. The proteins fractional composition study of different groups (water-soluble, myofibrillar, and stroma) is an urgent task, since it is possible to justify the processing technology based on the obtained data. The article presents the fractional composition study results of the water-soluble proteins and peptides, the molecular mass distribution of the separate components of myofibrillary proteins and muscle tissue stroma proteins of the Mactra Chinensis mollusk motor muscle. The researchers noted that the total amount of the high-molecular fraction (molecular weight above 10 kDa) is 62.16 %, and the total peptide fraction amount (molecular weight below 10 kDa) is 37.84 %. There are four main components in myofibrillar proteins – myosin, actin, troponin complex components, and collagen. The content of the main myofibrillar protein that is myosin of various polymerization degrees of is 69.3 %; heavy myosin chains – 30.5 % of the total myofibrillar proteins content and 44.0 % of the relative content of the total myosin content; light myosin chains – 38.8 % of the total myofibrillar proteins content and 56.0 % of the relative content of the total myosin content. The authors determined that actin (molecular weight 43 kDa) and troponin complex components (molecular weight from 15 to 30 kDa) are approximately equal in content. The collagen content (molecular weight more than 300 kDa) in the myofibrillar fraction is only 8.2 %. Stroma proteins are represented by two main fractions with molecular weights of more than 250 and 500 kDa. Collagen is the predominant stroma protein, its content account for 62.6 %. There is a high connectin content in stroma proteins.

LOW TRANSLATION DOWNSTREAM OF TOR ACTIVATES MYOGENE EXPRESSION AND ENHANCES HEALTH OF BODY MUSCLE IN C. ELEGANS

Previous studies reported that DR or low nutrient signaling reduces translation and extends lifespan, while attenuating growth and reproduction. In this study using C. elegans as a model organism, we found that reduced translation in neurons or germ tissues increases survival and primes the HSR against unfolded protein stress in adult animals. Surprisingly, lowering translation in these tissues significantly upregulates transcription of several key muscle maintenance genes, including those encoding the myogenic response factor HLH-1 and heavy myosin chain factor UNC-54 and MYO-3. It also improves muscle maintenance according to motility assays. However, reduced translation in muscle tissue does not activate the same level of myogenic gene expression. Instead, lowering translation in striated body muscle results in increased reproduction and accelerated development. To further investigate, we examined maintenance of muscle health via deconvolution fluorescent microscopy and immunostaining. Using computational analyses involving Fiji ImageJ and MATLAB program, we discovered that lowering translation delays decrepitude in body muscle structure, resulting in improved mitochondrial morphology and increased thermotolerance. Here we report that, combining physiological, molecular, and microscopic technique, muscle is protected during low translation, low TOR signaling, or DR. We hypothesize that results reflect evolutionary responses that preserve function required for foraging during nutrient scarcity and accelerate development during times low energy usage and high nutrient abundance.

Diazepam Alters the Ultrastructure of Fetal Atrium in Rat

Diazepam (DZ) is a synthetic minor tranquilizer, used in patients with psychological and psychiatric disorders. It is a relaxing sedative, anticonvulsant and antipsychotic. DZ crosses the human placental barrier in mouse. Young women who are addicted to the drug, if they become pregnant and continue to use it, particularly during the first trimester, expose their children to psychomotor disorders. The purpose of this study was to investigate whether DZ administered during pregnancy induces ultrastructural alterations of fetal mouse myocardium. The group (DZ) of pregnant female mice of the CD-1 strain was treated with a single daily dose of 1.0 mg / kg / pc / sc of day 6 to 17 and a group (C) that received saline solution. On day 18 females of both groups were anesthetized, the fetuses were perfused by intracardiac route with 1 % paraformaldehyde and 2.5 % glutaraldehyde, the heart was removed, the atrium was dissected, fixed in 1 % OsO4, it was immersed in epoxy resin. The fine sections were contrasted with uranyl acetate and lead citrate and observed in a transmission electron microscope. In the myocytes of the fetuses of the DZ group, the sarcomers of the compact myocardium were shorter than those of the C group. Areas with disorganized myofibrils were observed. The sarcoplasmic reticulum of some myocytes had distended and fragmented cisterns, altered mitochondria, and abundant polyribosomes were observed. The changes may be due to the effect of DZ on the synthesis of actin and heavy myosin and on cytoplasmic organelles mediated by peripheral benzodiazepine receptors present on the outer membrane of the mitochondria and associated with voltage-dependent calcium channels. Ultrastructural alterations of the atrial myocardium of fetuses of mice exposed to DZ in utero may have postnatal effects.

Acceleration of the sliding movement of actin filaments with the use of a non-motile mutant myosin in in vitro motility assays driven by skeletal muscle heavy meromyosin.

We examined the movement of an actin filament sliding on a mixture of normal and genetically modified myosin molecules that were attached to a glass surface. For this purpose, we used a Dictyostelium G680V mutant myosin II whose release rates of Pi and ADP were highly suppressed relative to normal myosin, leading to a significantly extended life-time of the strongly bound state with actin and virtually no motility. When the mixing ratio of G680V mutant myosin II to skeletal muscle HMM (heavy myosin) was 0.01%, the actin filaments moved intermittently. When they moved, their sliding velocities were about two-fold faster than the velocity of skeletal HMM alone. Furthermore, sliding movements were also faster when the actin filaments were allowed to slide on skeletal muscle HMM-coated glass surfaces in the motility buffer solution containing G680V HMM. In this case no intermittent movement was observed. When the actin filaments used were copolymerized with a fusion protein consisting of Dictyostelium actin and Dictyostelium G680V myosin II motor domain, similar faster sliding movements were observed on skeletal muscle HMM-coated surfaces. The filament sliding velocities were about two-fold greater than the velocities of normal actin filaments. We found that the velocity of actin filaments sliding on skeletal muscle myosin molecules increased in the presence of a non-motile G680V mutant myosin motor.

Fast-to-Slow Transition of Skeletal Muscle Contractile Function and Corresponding Changes in Myosin Heavy and Light Chain Formation in the R6/2 Mouse Model of Huntington's Disease.

Huntington´s disease (HD) is a hereditary neurodegenerative disease resulting from an expanded polyglutamine sequence (poly-Q) in the protein huntingtin (HTT). Various studies report atrophy and metabolic pathology of skeletal muscle in HD and suggest as part of the process a fast-to-slow fiber type transition that may be caused by the pathological changes in central motor control or/and by mutant HTT in the muscle tissue itself. To investigate muscle pathology in HD, we used R6/2 mice, a common animal model for a rapidly progressing variant of the disease expressing exon 1 of the mutant human gene. We investigated alterations in the extensor digitorum longus (EDL), a typical fast-twitch muscle, and the soleus (SOL), a slow-twitch muscle. We focussed on mechanographic measurements of excised muscles using single and repetitive electrical stimulation and on the expression of the various myosin isoforms (heavy and light chains) using dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole muscle and single fiber preparations. In EDL of R6/2, the functional tests showed a left shift of the force-frequency relation and decrease in specific force. Moreover, the estimated relative contribution of the fastest myosin isoform MyHC IIb decreased, whereas the contribution of the slower MyHC IIx isoform increased. An additional change occurred in the alkali MyLC forms showing a decrease in 3f and an increase in 1f level. In SOL, a shift from fast MyHC IIa to the slow isoform I was detectable in male R6/2 mice only, and there was no evidence of isoform interconversion in the MyLC pattern. These alterations point to a partial remodeling of the contractile apparatus of R6/2 mice towards a slower contractile phenotype, predominantly in fast glycolytic fibers.

Effects of human umbilical mesenchymal stem cell on Wistar rats models of stress urinary incontinence and its possible mechanism

Objective To investigate the effects of human umbilical mesenchymal stem cells (hUCMSCs) on Wistar rat models of stress urinary incontinence and its possible mechanism. Methods hUCMSCs cells were separated and extracted from human umbilical cord of cesarean delivery of full-term pregnancy women. The models of stress urinary incontinence (SUI) of Wistar rats were established by imitating delivery damage and ovary removing surgery, and then randomly divided into three groups: model group, transforming growth factor-β1 (TGF-β1) group, hUCMSCs group, and normal rats as normal group; and every group included ten rats. Maximal bladder capacity (MBC), leak point pressure (LPP), abdominal leak point pressure (ALPP), and sneezing experiment of rats including normal rats, model rats and model rats accepted TGF-β1 or hUCMSCs treatment were examined. When the treatment accomplished, the rats were killed and urethral sphincter were separated and examined by hematoxylin eosin (HE) staining and the proteins of troponin I (TnI), troponin C (TnC), troponin T (TnT), tropomyosin (Tm), actin (AT), myosin heavy chain (MHC) and myosin light chain (MLC) of urethral sphincter was detected with Western blot method. Results The cells of hUCMSCs were extracted and authenticated, SUI models of Wistar rats were successfully established and authenticated. Compared to model group and TGF-β1 group, the MBC, LPP and ALPP of hUCMSCs group dramatically improved (P<0.05), while the positive rate of sneezing experiment significantly dropped (P<0.05). HE staining showed urethral sphincter became attenuation, fracture, sparse and disorder, simultaneously the proteins of TnI, TnC, TnT, Tm, MHC and MLC of urethral sphincter reduced (P<0.05). While the damaged urethral sphincter basically restored the normal structure and the proteins of TnI, TnC, TnT, Tm, MHC and MLC of urethral sphincter up-regulated through hUCMSCs treatment (P<0.05). Conclusions The treatment of hUCMSCs translation could observably improve the clinic symptoms of SUI Wistar rat models, and its mechanism may be related to enhancement of the protein expression of TnI, TnC, TnT, Tm, MHC and MLC of urethral sphincter, and were involved in the rehabilitation and reconstruction of urethral sphincter. Key words: Cord blood stem cell transplantation; Urinary incontinence, stress/SU/PP; Urethra/PP

Tyrosine phosphorylation/dephosphorylation of myosin II essential light chains of Entamoeba histolytica trophozoites regulates their motility

Entamoeba histolytica trophozoites dwell in the human intestine as comensals although under still unclear circumstances become invasive and destroy the host tissues. For these activities, trophozoites relay on remarkable motility provided by the cytoskeleton organization. Amebic actin and some of its actin-associated proteins are well known, while components of the myosin II molecule, although predicted from the E. histolytica genome, need biochemical and functional characterization. Recently, an amebic essential light myosin II chain, named EhMLCI, was identified and reported to be phosphorylated in tyrosines. The phosphorylated form of the protein was associated with the soluble assembly incompetent conformation of the heavy myosin chains, while the non-phosphorylated protein was identified with filamentous heavy chains, organized in an assembly competent conformation. It was postulated that EhMLCI tyrosine phosphorylation could act as a negative regulator of myosin II activity by its phosphorylation/dephosphorylation cycles. To test this hypothesis, we constructed an expression vector containing an EhMLCI DNA sequence where two tyrosine residues, with strong probability of phosphorylation and fall within the single EF-hand domain that interacts with the N-terminus of myosin II heavy chains, were replaced by phenylalanines. Transfected trophozoites, expressing the mutant MutEhMLCI protein cannot process it, thereby not incorporated into the phosphorylation/dephosphorylation cycles required for myosin II activity, results in motility defective trophozoites.

Potential use of crude extracts from Alaska Pollock muscle as meat tenderizer

The activity of crude extracts from Alaska Pollock muscle responsible for the hydrolysis of myofibrils and perimysia from beef Longissimus Dorsi muscle was investigated. The results showed that myosin heavy chain and myosin light chain were hydrolyzed completely after 12 h of incubation. Tropomyosin, α-actinin, and troponin-T disappeared after 24 h of incubation, whereas the actin was partially degraded. The collagen fibers were so loose that cross-linking among microfibrils occurred after treatment with the crude extracts. Meanwhile, the second structures of myofibrillar proteins were not changed markedly after incubation. The α-helices in perimysia treated with the crude extracts were partially transformed into β-sheets, while the β-turn and the random coil fractions were steady. It was concluded that myofibrils and perimysia from beef Longissimus Dorsi muscle were commendably hydrolyzed by the crude extracts from Alaska Pollock muscle, so that the crude extracts from Alaska Pollock muscle could be utilized as a potential meat tenderizer.

Acetylcholinesterase and agrin: Different functions, similar expression patterns, multiple roles

Acetylcholinesterase (AChE) and agrin play unique functional roles in the neuromuscular junction (NMJ). AChE is a cholinergic and agrin a synaptogenetic component. In spite of their different functions, they share several common features: their targeting is determined by alternative splicing; unlike most other NMJ components they are expressed in both, muscle and motor neuron and both reside on the synaptic basal lamina of the NMJ. Also, both were reported to play various nonjunctional roles. However, while the origin of basal lamina bound agrin is undoubtedly neural, the neural origin of AChE, which is anchored to the basal lamina with collagenic tail ColQ, is elusive. Hypothesizing that motor neuron proteins targeted to the NMJ basal lamina share common temporal pattern of expression, which is coordinated with the formation of basal lamina, we compared expression of agrin isoforms with the expression of AChE-T and ColQ in the developing rat spinal cord at the stages before and after the formation of NMJ basal lamina. Cellular origin of AChE-T and agrin was determined by in situ hybridization and their quantitative levels by RT PCR. We found parallel increase in expression of the synaptogenetic (agrin 8) isoform of agrin and ColQ after the formation of basal lamina supporting the view that ColQ bound AChE and agrin 8 isoform are destined to the basal lamina. Catalytic AChE-T subunit and agrin isoforms 19 and 0 followed different expression patterns. In accordance with the reports of other authors, our investigations also revealed various alternative functions for AChE and agrin. We have already demonstrated participation of AChE in myoblast apoptosis; here we present the evidence that agrin promotes the maturation of heavy myosin chains and the excitation–contraction coupling. These results show that common features of AChE and agrin extend to their capacity to play multiple roles in muscle development.

Myocilin Interacts with Syntrophins and Is Member of Dystrophin-associated Protein Complex

Genetic studies have linked myocilin to open angle glaucoma, but the functions of the protein in the eye and other tissues have remained elusive. The purpose of this investigation was to elucidate myocilin function(s). We identified α1-syntrophin, a component of the dystrophin-associated protein complex (DAPC), as a myocilin-binding candidate. Myocilin interacted with α1-syntrophin via its N-terminal domain and co-immunoprecipitated with α1-syntrophin from C2C12 myotubes and mouse skeletal muscle. Expression of 15-fold higher levels of myocilin in the muscles of transgenic mice led to the elevated association of α1-syntrophin, neuronal nitric-oxide synthase, and α-dystroglycan with DAPC, which increased the binding of laminin to α-dystroglycan and Akt signaling. Phosphorylation of Akt and Forkhead box O-class 3, key regulators of muscle size, was increased more than 3-fold, whereas the expression of muscle-specific RING finger protein-1 and atrogin-1, muscle atrophy markers, was decreased by 79 and 88%, respectively, in the muscles of transgenic mice. Consequently, the average size of muscle fibers of the transgenic mice was increased by 36% relative to controls. We suggest that intracellular myocilin plays a role as a regulator of muscle hypertrophy pathways, acting through the components of DAPC.

Analysis of expression of heavy myosin chains during in vitro differentiation of satellite cells and myoblasts derived from rat skeletal muscles

Differentiation of cultured myogenic progenitor cells (satellite cells and mononucleated myoblasts) derived from hindlimb muscles of rat embryos and newborn animals was studied. Immunocytochemical methods and PCR analysis revealed expression of heavy myosin chains at the earliest stages of myogenesis (in mononucleated myoblasts). Expression of the gene encoding the embryonic form of myosin and a low level of expression of the gene encoding perinatal myosin in cultured progenitor cells derived from embryonic muscles was detected by PCR. Cells derived from muscles of newborn animals also expressed these two myosin forms, though at a lower level. The progenitor cells derived from muscles of rat embryos and newborn animals were found to express myosin 2a, which is characteristic of fast-twitch definitive muscle fibers.

Age‐related phenotypic transformation alters expression of heavy myosin isoforms SM1 and SM2 in ovine cerebral arteries

Vascular smooth muscle cells exhibit multiple stable phenotypes, including migratory, proliferative, synthetic, and contractile. The protein markers characteristic of these phenotypes are poorly defined. The present study explores the hypothesis that the heavy myosin isoforms SM1 and SM2 are reliable indicators of the extent of differentiation and contractile maturation in ovine cerebral arteries. Arteries from normoxic and chronically hypoxic (110 d at 3820 m) term fetal and non‐pregnant adult sheep were probed with antibodies against SM1 and SM2, then analyzed via Westerns and line‐scan fluorescent microscopy to determine absolute abundance of each marker as a function of relative distance from the lumen to the adventitial‐medial border. Postnatal maturation dramatically decreased the ratio of SM1 to SM2 expression approximately 2‐fold. Hypoxic acclimatization appeared to retard the rate of contractile maturation, and enhanced the SM1/SM2 ratio. Hypoxic effects were most prominent in smooth muscle near the lumen, whereas maturational effects were more evident in peri‐adventitial smooth muscle. Thus, expression patterns of SM1 and SM2 reliably reveal the age‐dependent phenotypic status of contractile smooth muscle, and appear to be influenced by independent mitogenic factors acting at the luminal and adventitial borders of ovine cerebral arteries. Supported by USPHS Grants HD31266, &amp; HL64867.