Critical role of Frizzled1 in age‐related alterations of Wnt/β‐catenin signal in myogenic cells during differentiation

Activation of Wnt/β‐catenin signal in muscle satellite cells (mSCs) of aged mice during myogenic differentiation has been appreciated as an important age‐related feature of the skeletal muscles, resulting in impairment of their regenerative ability following muscle injury. However, it remains elusive about molecules involved in this age‐related alteration of Wnt/β‐catenin signal in myogenic cells. To clarify this issue, we carried out expression analyses of Wnt receptor genes using real‐time RT‐PCR in mSCs isolated from the skeletal muscles of young and aged mice. Here, we show that expression of Frizzled1 (Fzd1) was detected at high levels in mSCs of aged mice. Higher expression levels of Fzd1 were also detected in mSC‐derived myogenic cells from aged mice and associated with activation of Wnt/β‐catenin signal during their myogenic differentiation in vitro. We also provide evidence that suppressed expression of Fzd1 in myogenic cells from aged mice results in a significant increase in myogenic differentiation, and its forced expression in those from young mice results in its drastic inhibition. These findings indicate the critical role of Fzd1 in altered myogenic differentiation associated with aging.     

Cadherin-11 is highly expressed in rhabdomyosarcomas and during differentiation of myoblasts in vitro

Rhabdomyosarcomas bear a morphological and genetic resemblance to developing skeletal muscle. Apart from myogenic marker genes (bHLH factors, myosin, actin), cell adhesion molecules such as N-cadherin and N-CAM have been reported to be expressed both in rhabdomyosarcomas and during myogenesis. The present study demonstrates the expression of another cadherin, cadherin-11, in rhabdomyosarcomas and during differentiation of myoblasts in vitro: cadherin-11, a predominantly mesenchymal cell adhesion molecule, is highly expressed in embryonal rhabdomyosarcomas and alveolar rhabdomyosarcomas, which do not bear the Pax-3–FKHR fusion previously described. Cadherin-11 is down-regulated in normal skeletal muscle and after myotube formation in vitro. The results of this study suggest that cadherin-11 might be involved in myogenesis and that rhabdomyosarcomas may re-express or fail to down-regulate cadherin-11. Since alveolar rhabdomyosarcomas bearing the t(2;13) translocation do not express cadherin-11, it is postulated that Pax-3 and cadherin-11 might be linked and involved in the same myogenic pathway. Copyright © 1999 John Wiley & Sons, Ltd.     

Basic fibroblast growth factor has a differential effect on MyoD conversion of cultured aortic smooth muscle cells from newborn and adult rats.

MyoD is a master regulatory gene for myogenesis that also converts many mesoderm-derived cells into the skeletal muscle phenotype. Rat aortic smooth muscle cells do not contain MyoD homologous mRNA. However, expression of an exogenously supplied MyoD gene in aortic smooth muscle cells cultured from newborn and adult animals converts these cells to elongated myoblasts and myotubes expressing the skeletal muscle genes for titin, nebulin, myosin, and skeletal alpha-actin. The presence of basic fibroblast growth factor during growth and serum starvation completely inhibits MyoD-mediated conversion in cultures of newborn smooth muscle cells. However, in smooth muscle cell cultures derived from adult rats the presence of fibroblast growth factor increases the conversion frequency. The differential response of exogenous MyoD suggests that the two morphological types of aortic smooth muscle cells, one typical for the newborn rat, the other for the adult rat, represent two distinctive states of differentiation.     

Expression and organization of muscle specific proteins during the early developmental stages of the rabbit heart

Summary The expression and intracellular distribution patterns of muscle-specific proteins were studied during rabbit embryo development (7–13 dpc) using monoclonal antibodies against titin, myosin, tropomyosin and actin, as well as the intermediate filament proteins desmin, keratin and vimentin. From our panel, titin appeared to be the first muscle-specific protein to be exclusively expressed in the embryonic rabbit heart. Upon differentiation (myocyte and myotube formation), titin reorganizes from dot-like aggregates into a cross-striated pattern (in 9- to 30-somite embryos) via a transiently filamentous distribution. When the expression and organization of the other muscle proteins was studied in relation to titin, it became apparent that tropomyosin followed upon titin with respect to its exclusive expression in the heart anlagen and its organization into a striated pattern. Myosin and desmin were organized into cross-striated patterns after titin and tropomyosin, but this arrangement had not reached its final form in 13-dpc embryos. Actin, keratin and vimentin were distributed in cytoplasmic filaments in the embryonic stages we investigated. Since the first pulsations are already detected in 3-somite embryos, we conclude that the organization of titin, tropomyosin, myosin and desmin into a striated pattern does not seem to be essential for the initiation of muscle cell contraction in the heart anlagen. Furthermore, this study shows that, in comparison with studies on mouse, chick and rat, the sequence of expression of muscle-specific and intermediate filament proteins during cardiomyogenesis is species-dependent, and that their expression and organization varies in time in different regions of the developing heart.Abbreviations IFP intermediate filament proteins - PBS phosphate-buffered saline - FITC fluorescein isothiocyanate - TRITC tetramethylrhodamine isothiocyanate - TxRd texas red - dpc days post conception     

Epithelial cell differentiation in organotypic cultures of fetal rat lung

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbonstripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbonstripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that (1) the hormones extracted from bovine fetal calf serum by carbonstripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and (2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.     

Myogenesis in hemopoietic tissue mesenchymal stem cell culture

The myogenic differentiation capacity of prenatal mesenchymal stem cells from the main sites of hemopoiesis (bone marrow, thymus, liver, and spleen) was studied. Myogenesis was observed in all studied cell cultures except splenic mesenchymal stem cells. Differentiating cells from the thymus, bone marrow, and liver were positively stained for skeletal muscle markers (myogenin and MyoD). Autonomously contracting structures positively stained for cardiotroponin I and slow muscle myosin, were detected in the same cultures. Our experiments revealed differences in differentiation of mesenchymal stem cells from hemopoietic organs depending on the source of cells. __________ Translated from Kletochnye Tekhnologii v Biologii i Medicine, No. 2, pp. 63–69, April, 2006     

Monoclonal antibodies to titin in conjunction with antibodies to desmin separate rhabdomyosarcomas from other tumor types

Titin is a major constituent protein of sarcomeric muscles and is thought to give rise to an elastic filament component underlying the myofibrillar organization. Monoclonal antibodies to titin have been characterized on normal and pathological human material and on human cell lines in culture. A positive immunocytochemical reaction was restricted to sarcomeric muscles and did not occur on visceral or vascular smooth muscles or on various nonmuscle tissues. When different tumor types were examined titin antibodies reacted solely with rhabdomyosarcomas and did not react with leiomyosarcoma or leiomyoma, or with the nonmuscle tumor types tested. In rhabdomyosarcomas a noticeably smaller population of cells were positive with antibodies to titin than with antibodies to desmin showing that individual cells within a rhabdomyosarcoma achieve different degrees of myogenic differentiation. The results reinforce the use of desmin as a marker for muscle sarcomas and show that a positive identification of rhabdomyosarcoma can be achieved by immunocytochemistry with the parallel use of desmin and titin antibodies.     

BMP regulation of myogenesis in zebrafish

In amniotes, BMP signaling from lateral plate and dorsal neural tube inhibits differentiation of muscle precursors in the dermomyotome. Here, we show that BMPs are expressed adjacent to the dermomyotome during and after segmentation in zebrafish. In addition, downstream BMP pathway members are expressed within the somite during dermomyotome development. We also show that zebrafish dermomyotome is responsive to BMP throughout its development. Ectopic overexpression of Bmp2b increases expression of the muscle precursor marker pax3, and changes the time course of myoD expression. At later stages, overexpression increases the number of Pax7+ myogenic precursors, and delays muscle differentiation, as indicated by decreased numbers of MEF2+ nuclei, decreased number of multi‐nucleated muscle fibers, and an increased myotome angle. In addition, we show that while BMP overexpression is sufficient to delay myogenic differentiation, inhibition of BMP does not detectably affect this process, suggesting that other factors redundantly inhibit myogenic differentiation. Developmental Dynamics 239:806–817, 2010. © 2010 Wiley‐Liss, Inc.     

Adapted physical exercise enhances activation and differentiation potential of satellite cells in the skeletal muscle of old mice

During ageing, a progressive loss of skeletal muscle mass and a decrease in muscle strength and endurance take place, in the condition termed sarcopenia. The mechanisms of sarcopenia are complex and still unclear; however, it is known that muscle atrophy is associated with a decline in the number and/or efficiency of satellite cells, the main contributors to muscle regeneration. Physical exercise proved beneficial in sarcopenia; however, knowledge of the effect of adapted physical exercise on the myogenic properties of satellite cells in aged muscles is limited. In this study the amount and activation state of satellite cells as well as their proliferation and differentiation potential were assessed in situ by morphology, morphometry and immunocytochemistry at light and transmission electron microscopy on 28‐month‐old mice submitted to adapted aerobic physical exercise on a treadmill. Sedentary age‐matched mice served as controls, and sedentary adult mice were used as a reference for an unperturbed control at an age when the capability of muscle regeneration is still high. The effect of physical exercise in aged muscles was further analysed by comparing the myogenic potential of satellite cells isolated from old running and old sedentary mice using an in vitro system that allows observation of the differentiation process under controlled experimental conditions. The results of this ex vivo and in vitro study demonstrated that adapted physical exercise increases the number and activation of satellite cells as well as their capability to differentiate into structurally and functionally correct myotubes (even though the age‐related impairment in myotube formation is not fully reversed): this evidence further supports adapted physical exercise as a powerful, non‐pharmacological approach to counteract sarcopenia and the age‐related deterioration of satellite cell capabilities even at very advanced age.     

Protein kinase-A phosphorylates titin in human heart muscle and reduces myofibrillar passive tension

Protein kinase-A (PKA) is activated during β-adrenergic stimulation of the heart and is known to phosphorylate several sarcomeric proteins including the giant polypeptide titin. A PKA phosphorylation site on titin is located within the N2B-unique sequence, which is present in the elastic segment of the two major isoforms of cardiac titin, N2B and N2BA, but not in the skeletal-muscle isoforms of the N2A-type. In bovine and rat cardiomyocytes, PKA-mediated phosphorylation decreases passive tension (PT), an effect ascribed to titin phosphorylation. Whether titin is phosphorylated by PKA upon β-adrenergic stimulation in human heart has not been shown to date. Here we report that PKA induces phosphorylation of N2B and N2BA titin isoforms, as well as a characteristic proteolytic fragment of titin, T2, in human donor hearts. The PKA-induced phosphorylation signals were stronger when myofilaments were first de-phosphorylated by protein phosphatase-1, suggesting inherent phosphorylation of titin in human heart. Titin phosphorylation was associated with a reduction in PT of skinned human cardiac strips; the relative decrease was higher at shorter than at longer physiological sarcomere lengths. The PKA-dependent PT drop was substantially larger when fibers were pre-treated with protein phosphatase-1, indicating that inherent phosphorylation of titin is important for the basal myocardial PT level. Mechanical measurements on isolated myofibrils from rat heart confirmed the PKA effect on passive stiffness and also showed a more pronounced effect in the presence of reducing agent, DTT. In contrast, PKA did not alter the PT of single skinned rat diaphragm muscle fibers; however, the kinase was still able to phosphorylate the skeletal N2A-titin isoform, which lacks the N2B-unique sequence. Thus, an additional phosphorylation site in titin may exist outside the cardiac N2B-unique sequence. We conclude that PKA mediates phosphorylation of titin in normal human myocardium. Titin phosphorylation lowers titin-based passive stiffness in heart but not in skeletal muscle.     

Upregulation of HARP during in vitro myogenesis and rat soleus muscle regeneration

Heparin affin regulatory peptide (HARP) is a heparin binding growth factor that belongs to a family of molecule whose biological function in myogenesis has been suspected without formal demonstration. In the present study, we investigated the expression and the distribution of HARP and its mRNA during soleus muscle regeneration using a crushed-induced regeneration model and also during differentiation of muscle satellite cells in primary cultures. We show that HARP mRNA and protein expression are increased during the regeneration process with a peak at day 5 after muscle crushing when new myotubes are formed. In situ hybridization and immunohistochemical studies showed that activated myoblasts expressed HARP at day two after crushing. Five days after muscle lesion, HARP is localised in newly formed myotubes as well as in prefused activated myoblasts. In regenerated myofibers, 15 days after crushing, expression of HARP was reduced. In vitro experiments using primary cultures of rat satellite cells indicated that HARP expression level increased during the differentiation process and peaked on fusion of myoblasts into myotubes. This is the first study demonstrating the presence of HARP in fusing myogenic cells suggests that this growth factor could play a function in myogenic differentiation.     

Both avian and mammalian embryonic myoblasts are intrinsically heterogeneous

Adult skeletal muscles are composed of different fibre types. What initiates the distinctive muscle fibre type-specific specialization in a developing embryo is still controversial. In vitro studies of avian muscles have shown the expression of one of the slow myosin heavy chains, SM2, in only some myotubes. In this report we demonstrate the expression of another slow myosin heavy chain, SM1, restricted to only some chicken myotubes (presumptive slow) in vitro. We also demonstrate that as is the case for avian species, distinct fast and slow myogenic cells are detectable in mammalian species, human and rat, during in vitro development in the absence of innervation. While antibodies to fast myosin heavy chains stained all myotubes dark in these muscle cell cultures, antibodies to slow myosin heavy chains stained only a proportion of the myotubes (presumptive slow). The other myotubes were either unstained or only weakly stained with slow myosin heavy c hain antibodies. The muscle cell cultures prepared from different developmental stages of rat skeletal muscles showed a reduction in the number of slow myosin heavy chain-positive myotubes with advancing foetal growth. It is concluded that embryonic myogenic cells that are likely to form distinct fast or slow muscle fibre types are intrinsically heterogeneous, not only in avian but also in mammalian species, although extrinsic factors reinforce and modify such commitment throughout subsequent development. © Kluwer Academic Publishers.     

PAX3–FOXO1 is essential for tumour initiation and maintenance but not recurrence in a human myoblast model of rhabdomyosarcoma

The PAX3–FOXO1 fusion gene is generated by a 2;13 chromosomal translocation and is a characteristic feature of an aggressive subset of rhabdomyosarcoma (RMS). To dissect the mechanism of oncogene action during RMS tumourigenesis and progression, doxycycline‐inducible PAX3–FOXO1 and constitutive MYCN expression constructs were introduced into immortalized human myoblasts. Although myoblasts expressing PAX3–FOXO1 or MYCN alone were not transformed in focus formation assays, combined PAX3–FOXO1 and MYCN expression resulted in transformation. Following intramuscular injection into immunodeficient mice, myoblasts expressing PAX3–FOXO1 and MYCN formed rapidly growing RMS tumours, whereas myoblasts expressing only PAX3–FOXO1 formed tumours after a longer latency period. Doxycycline withdrawal in myoblasts expressing inducible PAX3–FOXO1 and constitutive MYCN following tumour formation in vivo or focus formation in vitro resulted in tumour regression or smaller foci associated with myogenic differentiation and cell death. Following regression, most tumours recurred in the absence of doxycycline. Analysis of recurrent tumours revealed a subset without PAX3–FOXO1 expression, and cell lines derived from these recurrent tumours showed transformation in the absence of doxycycline. The doxycycline‐independent oncogenicity in these recurrent tumour‐derived lines persisted even after PAX3–FOXO1 was inactivated with a CRISPR/Cas9 editing strategy. Whereas cell lines derived from primary tumours were dependent on PAX3–FOXO1 and differentiated following doxycycline withdrawal, recurrent tumour‐derived cells without PAX3–FOXO1 expression did not differentiate under these conditions. These findings indicate that PAX3–FOXO1 collaborates with MYCN during early RMS tumourigenesis to dysregulate proliferation and inhibit myogenic differentiation and cell death. Although most cells in the primary tumours are dependent on PAX3–FOXO1, recurrent tumours can develop by a PAX3–FOXO1‐independent mechanism, in which rare cells are postulated to acquire secondary transforming events that were activated or selected by initial PAX3–FOXO1 expression. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.     

Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice

 It is commonly accepted, that regenerative capacity of striated muscle is confined to skeletal muscle by activation of satellite cells that normally reside quiescent between the plasmalemma and the basement membrane of muscle fibers. Muscular dystrophies are characterized by repetitive cycles of de- and regeneration of skeletal muscle fibers and by the frequent involvement of the cardiac muscle. Since during the longstanding course of muscular dystrophies there is a permanent demand of myogenic progenitors we hypothesized that this may necessitate a recruitment of additional myogenic precursors from an undifferentiated, permanently renewed cell pool, such as bone marrow (BM) cells. To this end normal and dystrophic (mdx) female mice received bone marrow transplantation (BMT) from normal congenic male donor mice. After 70 days, histological sections of skeletal and cardiac muscle from BMT mice were probed for the donor-derived Y chromosomes. In normal BMT recipients, no Y chromosome-containing myonuclei were detected, either in skeletal or in cardiac muscle. However, in all samples from dystrophic mdx skeletal muscles Y chromosome-specific signals were detected within muscle fiber nuclei, which additionally were found to express the myoregulatory proteins myogenin and myf-5. Moreover, in the hearts of BMT-mdx mice single cardiomyocytes with donor derived nuclei were identified, indicating, that even cardiac muscle cells are able to regenerate by recruitment of circulating BM-derived progenitors. Our findings suggest that further characterization and identification of the BM cells capable of undergoing myogenic differentiation may have an outstanding impact on therapeutic strategies for diseases of skeletal and cardiac muscle. Accepted: 27 October 1998     

Expression of myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit muscle satellite cell cultures

Summary We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.     

In vitro invasiveness of human bladder cancer from cell lines and biopsy specimens

Aggregates prepared from cell lines established from a human transitional cell carcinoma of the urothelium (Hu 456) or from apparently normal urothelium before (Hu 609) and after phenotypic transformation (Hu 609T) were confronted with fragments of embryonic chick cardiac muscle in organ culture. In this assay a correlation was found between in vitro invasiveness of animal cell lines and their capacity to produce invasive tumours in syngeneic animals [1, 10, 11]. The invasiveness of cells from established human urothelial lines was compared to the invasiveness of cells from fresh biopsy specimens of a normal urothelium, a non-invasive papilloma, and a metastasizing transitional cell carcinoma. Cells from all established lines (Hu 609, Hu 609T and HU 456) and from the biopsy specimens of the transitional cell carcinoma occupied and eventually replaced the cardiac muscle by contrast with cells from the normal urothelium or from the non-invasive papilloma. We concluded that the organ culture assay for invasiveness might be used to define malignancy of human bladder cell lines and to follow the various steps during the acquisition of invasiveness in vitro.