Muscle-specific Markers Research Articles

Basal Lamina Mimetic Nanofibrous Peptide Networks for Skeletal Myogenesis.

Extracellular matrix (ECM) is crucial for the coordination and regulation of cell adhesion, recruitment, differentiation and death. Therefore, equilibrium between cell-cell and cell-matrix interactions and matrix-associated signals are important for the normal functioning of cells, as well as for regeneration. In this work, we describe importance of adhesive signals for myoblast cells’ growth and differentiation by generating a novel ECM mimetic peptide nanofiber scaffold system. We show that not only structure but also composition of bioactive signals are important for cell adhesion, growth and differentiation by mimicking the compositional and structural properties of native skeletal muscle basal lamina. We conjugated laminin-derived integrin binding peptide sequence, “IKVAV”, and fibronectin-derived well known adhesive sequence, “RGD”, into peptide nanostructures to provide adhesive and myogenic cues on a nanofibrous morphology. The myogenic and adhesive signals exhibited a synergistic effect on model myoblasts, C2C12 cells. Our results showed that self-assembled peptide nanofibers presenting laminin derived epitopes support adhesion, growth and proliferation of the cells and significantly promote the expression of skeletal muscle-specific marker genes. The functional peptide nanofibers used in this study present a biocompatible and biodegradable microenvironment, which is capable of supporting the growth and differentiation of C2C12 myoblasts into myotubes.

The role of demineralized bone particle in a PLGA scaffold designed to create a media equivalent for a tissue engineered blood vessel

Poly(lactic-co-glycolic acid) (PLGA) is a copolymer used for scaffolds in tissue engineering due to its biodegradable and biocompatible properties. Demineralized bone particle (DBP) is a natural material containing bone morphogenic proteins (BMPs). The objective of this study was to determine if DBP induced differentiation of mesenchymal stem cells (MSCs) into a smooth muscle cells (SMCs) phenotype and act in a similar manner when treated with TGF-β1 as SMCs under the same conditions. PLGA scaffolds were fabricated with or without DBP using the solvent casting/salt leaching technique. Proliferation of MSCs and SMCs was measured using the MTT assay, while the expression of the smooth muscle-specific markers α-smooth muscle actin (α-SMA), smooth muscle 22 alpha (SM22α), and calponin was measured using RT-PCR and western blotting. Results showed PLGA with DBP scaffolds increased cell proliferation of SMCs (1 and 3 days) and MSCs (3 and 7 days) compared to the PLGA only scaffolds. Smooth muscle-specific genes were significantly increased in SMCs cultured for 7 days on PLGA with DBP scaffolds compared to PLGA only scaffolds and were also significantly increased when used SMC medium containing TGF-β1. Interestingly, the smooth muscle-specific genes were not altered in MSCs by the addition of TGF-β1 to the PLGA with DBP. However, in the 6-well and PLGA only groups, TGF-β1 addition caused increased levels of SM22α and calponin mRNA. Western blotting results were similar to the mRNA levels. These results suggest that the cell growth and gene expression of SMCs were up-regulated by DBP and TGF-β1 in PLGA scaffold; however, they were inadequate for the shift of MSCs into a SMCs phenotype.

Hepatic Angiomyolipoma

Introduction: Comprehensive analysis is essential in determining the appropriate etiology of clinical manifestations. Pathological analysis and immunohistochemistry can differentiate hepatic angiomyolipoma (AML), a rare yet benign tumor, from other more common liver lesions. Case Description: A 62-year-old male with human immunodeficiency virus and Hepatitis B presented with epigastric pain, nausea, and a 30 pound weight loss over 6 months. Physical examination was significant for mild epigastric and right upper quadrant tenderness. Computed tomography (CT) of the abdomen and pelvis revealed a large, well defined heterogeneous mass of the right hepatic lobe measuring 9.6cm in maximal diameter, with vascular soft tissue intermixed with macroscopic fat. Radiologic differentials included a primary hepatic neoplasm such as hepatocellular carcinoma or hepatic AML. Magnetic resonance imaging (MRI) of the liver with triple phase contrast was performed and the lesion remained suspicious for hepatocellular carcinoma. AFP was 3.7 (normal <8.5 ng/mL). A percutaneous CT-guided biopsy of the mass was performed. Histology showed large cells with eosinophilic and fibrillar cytoplasm, admixed with adipocytes, inflammatory cells and extramedullary hematopoiesis consistent with a hepatic AML. This was confirmed with positive staining for smooth muscle actin, Melan-A and HMB-45. Given the size of the AML and the symptoms, the patient underwent open excision of the mass with partial right hepatic lobectomy. Intraoperatively, the lesion was found to be encapsulated. The resected mass was 9.3 x 7.4 x 4.3cm in size and histology confirmed the initial biopsy findings. There were no postoperative complications and he remained symptom free at his follow-up appointment.Figure 1Figure 2Figure 3Discussion: Hepatic AML is a benign mesenchymal tumor composed of variable proportions of smooth muscle cells, adipocytes, and blood vessels. It is most often a solitary tumor found incidentally, although a minority present with upper abdominal discomfort related to mass effect. MRI is considered to be the imaging study of choice but has been reported to misdiagnose between 48-90% of cases given variability in tumor composition. They can be mistaken for hepatocellular carcinoma, hemangioma or hepatic adenoma. Definitive diagnosis is dependent on tissue histopathology and immunostaining for muscle-specific markers such as smooth muscle actin as well as melanoma markers HMB-45 and melan-A.

PKCε as a novel promoter of skeletal muscle differentiation and regeneration

IntroductionSatellite cells are muscle resident stem cells and are responsible for muscle regeneration. In this study we investigate the involvement of PKCε during muscle stem cell differentiation in vitro and in vivo. Here, we describe the identification of a previously unrecognized role for the PKCε–HMGA1 signaling axis in myoblast differentiation and regeneration processes. MethodsPKCε expression was modulated in the C2C12 cell line and primary murine satellite cells in vitro, as well as in an in vivo model of muscle regeneration. Immunohistochemistry and immunofluorescence, RT-PCR and shRNA silencing techniques were used to determine the role of PKCε and HMGA1 in myogenic differentiation. ResultsPKCε expression increases and subsequently re-localizes to the nucleus during skeletal muscle cell differentiation. In the nucleus, PKCε blocks Hmga1 expression to promote Myogenin and Mrf4 accumulation and myoblast formation. Following in vivo muscle injury, PKCε accumulates in regenerating, centrally-nucleated myofibers. Pharmacological inhibition of PKCε impairs the expression of two crucial markers of muscle differentiation, namely MyoD and Myogenin, during injury induced muscle regeneration. ConclusionThis work identifies the PKCε–HMGA1 signaling axis as a positive regulator of skeletal muscle differentiation.

Protective effect of hydroxyfasudil, a Rho kinase inhibitor, on ventral prostatic hyperplasia in the spontaneously hypertensive rat.

Rho kinase (ROCK) pathway is associated with various cellular functions, such as smooth muscle contraction, inflammatory response, and cell proliferation. The spontaneously hypertensive rat (SHR) is commonly used genetically hypertensive rat model which develops hyperplastic morphological abnormalities in the ventral prostate. We investigated whether administration of hydroxyfasudil, a ROCK inhibitor, could reduce the levels of growth factors, inflammatory markers, and morphological abnormalities in the ventral prostate of the SHR. Twelve-week-old SHRs were treated with hydroxyfasudil (1 mg/kg/day, i.p.) or vehicle once daily for another 6 weeks. Wistar Kyoto (WKY) rats treated with vehicle were used as normotensive controls. At 18 weeks of age, blood pressure and heart rate were measured by the tail cuff method. Then the rats were sacrificed, and the ventral prostates were removed. The levels of ROCK activity, growth factors (TGF-β1 and bFGF), a smooth muscle differentiation marker (α-SMA) and an inflammatory cytokine (IL-6) in the ventral prostate were measured by ELISA and western blot. A histological evaluation in each group was also performed. There were significant increases in blood pressure, prostate weight, prostate body weight ratio, and tissue levels of ROCK activity, TGF-β1, bFGF, α-SMA, and IL-6 in the SHR compared to the WKY rat. Histological examination of the ventral prostate showed morphological abnormalities such as a higher degree of proliferation in the glandular epithelial and stromal area in the SHR compared to the WKY rat. Treatment with hydroxyfasudil reduced the elevated ROCK activity, TGF-β1, bFGF, α-SMA, and IL-6 found in the ventral prostate of the SHR. Moreover, treatment with hydroxyfasudil decreased the morphological abnormalies in the SHR ventral prostate. Treatment with hydroxyfasudil decreased the growth factors, an inflammatory cytokine, and morphological abnormalies in the SHR ventral prostate. These results suggest that chronic treatment with hydroxyfasudil may inhibit the progression of prostatic hyperplasia in the SHR.

Folic acid promotes the myogenic differentiation of C2C12 murine myoblasts through the Akt signaling pathway.

Folic acid is a water-soluble vitamin in the B-complex group, and an exogenous intake is required for health, growth and development. As a precursor to co-factors, folic acid is required for one-carbon donors in the synthesis of DNA bases and other essential biomolecules. A lack of dietary folic acid can lead to folic acid deficiency and can therefore result in several health problems, including macrocytic anemia, elevated plasma homocysteine levels, cardiovascular disease, birth defects, carcinogenesis, muscle weakness and difficulty in walking. Previous studies have indicated that folic acid exerts a positive effect on skeletal muscle functions. However, the precise role of folic acid in skeletal muscle cell differentiation remains poorly understood. Thus, in the present study, we examined the effects of folic acid on neo-myotube maturation and differentiation using C2C12 murine myoblasts. We found that folic acid promoted the formation of multinucleated myotubes, and increased the fusion index and creatine kinase(CK) activity in a concentration-dependent manner. In addition, western blot analysis revealed that the expression levels of the muscle-specific marker, myosin heavy chain(MyHC), as well as those of the myogenic regulatory factors(MRFs), MyoD and myogenin, were increased in the folic acid-treated myotubes during myogenic differentiation. Folic acid also promoted the activation of the Akt pathway, and this effect was inhibited by treatment of the C2C12 cells with LY294002 (Akt inhibitor). Blocking of the Akt pathway with a specific inhibitor revealed that it was necessary for mediating the stimulatory effects of folic acid on muscle cell differentiation and fusion. Taken together, our data suggest that folic acid promotes the differentiation of C2C12 cells through the activation of the Akt pathway.

Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes

Striated skeletal muscle cells from humans represent a valuable source for in vitro studies of the motoric system as well as for pathophysiological investigations in the clinical settings. Myoblasts can readily be grown from human muscle tissue. However, if muscle tissue is unavailable, myogenic cells can be generated from human induced pluripotent stem cells (hiPSCs) preferably without genetic engineering. Our study aimed to optimize the generation of hiPSCs derived myogenic cells by employing selection of CD34 positive cells and followed by distinct, stepwise culture conditions. Following the expansion of CD34 positive single cells under myogenic cell culture conditions, serum deprived myoblast-like cells finally fused and formed multinucleated striated myotubes that expressed a set of key markers for muscle differentiation. In addition, these myotubes contracted upon electrical stimulation, responded to acetylcholine (Ach) and were able to generate action potentials. Finally, we co-cultured motoneurons and myotubes generated from identical hiPSCs cell lines. We could observe the early aggregation of acetylcholine receptors in muscle cells of immature co-cultures. At later stages, we identified and characterised mature neuromuscular junctions (NMJs). In summary, we describe here the successful generation of an iPS cell derived functional cellular system consisting of two distinct communicating cells types. This in vitro co-culture system could therefore contribute to research on diseases in which the motoneurons and the NMJ are predominantly affected, such as in amyotrophic lateral sclerosis or spinal muscular atrophy.

C3G (RapGEF1), a regulator of actin dynamics promotes survival and myogenic differentiation of mouse mesenchymal cells

RapGEF1 (C3G) is a ubiquitously expressed protein that is essential for mammalian embryonic development. We have shown earlier that C3G regulates cytoskeletal dynamics and is required for neuronal differentiation. To determine if C3G plays a wider role in differentiation of multiple tissue types, we examined its role in skeletal muscle differentiation using the model system of C2C12 cells in culture. C3G protein is highly expressed in mouse skeletal muscle and its transcript and protein levels increase as C2C12 cells are induced to differentiate. Increase in C3G was predominantly seen in the nuclei of myotubes. Ectopic expression of C3G promoted myotube formation when cells were cultured in growth as well as differentiation medium and, enhanced MHC levels were associated with C3G expression. C3G induced differentiation required its catalytic and protein interaction domains and was dependent on the function of cellular R-Ras. Knockdown of cellular C3G using small hairpin RNA reduced expression of muscle specific markers and β-catenin, resulting in impaired differentiation. Disabling C3G function also resulted in enhanced cell death suggesting that cellular C3G is required for cell survival. In cells grown in growth medium, over-expressed C3G increased Akt activity, and C3G knockdown reduced it. C3G expression also suppressed cyclin D1 levels, and induced p27 expression, molecules involved in regulating cell proliferation. Endogenous C3G localizes to focal adhesions in myotubes and C3G expressing cells show distinct stress fibers, elongation and parallel alignment. Expression of a dominant negative construct of C3G, disrupts actin cytoskeleton and formation of focal adhesions resulting in detachment of cells from the substratum and inhibition of differentiation. Our results provide evidence that C3G plays an important role in myogenic differentiation by coordinating cell cycle exit, actin dynamics and survival signaling.

A differential response to newt regeneration extract by C2C12 and primary mammalian muscle cells.

BackgroundDedifferentiation, a process whereby differentiated cells lose their specialized characteristics and revert to a less differentiated state, plays a key role in the regeneration process in urodele amphibians such as the red spotted newt, Notophthalmus viridescens. Dedifferentiation of fully mature tissues is generally absent in mammalian cells. Previous studies have shown that mouse C2C12 multinucleated myotubes treated with extract derived from regenerating newt forelimbs can re-enter the cell cycle, fragment into mononucleated cells, and proliferate. However, this response has been difficult to replicate.MethodsWe isolated extract from early newt forelimb regenerates and assessed its effects on differentiation of proliferating primary and C2C12 myoblasts. We also treated fully differentiated primary and C2C12 myotube cultures with extract and assessed cell cycle re-entry and myotube fragmentation.ResultsWe have confirmed the results obtained in C2C12 cells and expanded these studies to also examine the effects of newt regeneration extracts on primary muscle cells. Newt extract can block differentiation of both C2C12 and primary myoblasts. Once differentiation is induced, treatment with newt extract causes cell cycle re-entry and fragmentation of C2C12 myotubes. Downregulation of p21 and muscle-specific markers is also induced. Primary myotubes also fragment in response to extract treatment, and the fragmented cells remain viable for long periods of time in culture. However, unlike C2C12 cells, primary muscle cells do not re-enter the cell cycle in response to treatment with newt extracts.ConclusionsDedifferentiation of fully mature muscle occurs during regeneration in the newt forelimb to contribute cells to the regeneration process. Our study shows that extracts derived from regenerating newt forelimbs can induce dedifferentiation, cell cycle re-entry, and fragmentation of mouse C2C12 cells but can only induce fragmentation in primary muscle cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0044-8) contains supplementary material, which is available to authorized users.

Kras activation in p53-deficient myoblasts results in high-grade sarcoma formation with impaired myogenic differentiation.

While genomic studies have improved our ability to classify sarcomas, the molecular mechanisms involved in the formation and progression of many sarcoma subtypes are unknown. To better understand developmental origins and genetic drivers involved in rhabdomyosarcomagenesis, we describe a novel sarcoma model system employing primary murine p53-deficient myoblasts that were isolated and lentivirally transduced with KrasG12D. Myoblast cell lines were characterized and subjected to proliferation, anchorage-independent growth and differentiation assays to assess the effects of transgenic KrasG12D expression. KrasG12D overexpression transformed p53-/- myoblasts as demonstrated by an increased anchorage-independent growth. Induction of differentiation in parental myoblasts resulted in activation of key myogenic regulators. In contrast, Kras-transduced myoblasts had impaired terminal differentiation. p53-/- myoblasts transformed by KrasG12D overexpression resulted in rapid, reproducible tumor formation following orthotopic injection into syngeneic host hindlimbs. Pathological analysis revealed high-grade sarcomas with myogenic differentiation based on the expression of muscle-specific markers, such as Myod1 and Myog. Gene expression patterns of murine sarcomas shared biological pathways with RMS gene sets as determined by gene set enrichment analysis (GSEA) and were 61% similar to human RMS as determined by metagene analysis. Thus, our novel model system is an effective means to model high-grade sarcomas along the RMS spectrum.

Electrospun type 1 collagen matrices preserving native ultrastructure using benign binary solvent for cardiac tissue engineering

Electrospinning is a well-established technique that uses a high electric field to fabricate ultrafine fibrous scaffolds from both natural and synthetic polymers to mimic the cellular microenvironment. Collagen is one of the most preferred biopolymers, due to its widespread occurrence in nature and its biocompatibility. Electrospinning of collagen alone has been reported, with fluoroalcohols such as hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE), but the resultant collagen lost its characteristic ultrastructural integrity of D-periodicity 67 nm banding, confirmed by transmission electron microscopy (TEM), and the fluoroalcohols used were toxic to the environment. In this study, we describe the use of glacial acetic acid and DMSO to dissolve collagen and generate electrospun nanofibers of collagen type 1, which is non-toxic and economical. TEM analysis revealed the characteristic feature of native collagen triple helical repeats, showing 67 nm D-periodicity banding pattern and confirming that the ultrastructural integrity of the collagen was maintained. Analysis by scanning electron microscopy (SEM) showed fiber diameters in the range of 200–1100 nm. Biocompatibility of the three-dimensional (3D) scaffolds was established by MTT assays using rat skeletal myoblasts (L6 cell line) and confocal microscopic analysis of immunofluorescent-stained sections of collagen scaffolds for muscle-specific markers such as desmin and actin. Primary neonatal rat ventricular cardiomyocytes (NRVCM) seeded onto the collagen scaffolds were able to maintain their contractile function for a period of 17 days and also expressed higher levels of desmin when compared with 2D cultures. We report for the first time that collagen type 1 can be electrospun without blending with copolymers using the novel benign solvent combination, and the method can be potentially explored for applications in tissue engineering.

Electrospun Type 1 Collagen Matrices Using a Novel Benign Solvent for Cardiac Tissue Engineering.

Electrospinning is a well-established technique that uses a high electric field to fabricate ultra fine fibrous scaffolds from both natural and synthetic polymers to mimic the cellular microenvironment. Collagen is one of the most preferred biopolymers due to its biocompatibility and widespread occurrence in nature. Electrospinning of Collagen alone has been reported with fluoroalcohols such as Hexafluoroisopropanol (HFIP) and Trifluoroethanol (TFE), which are toxic to the environment. In this study we describe the use of a novel benign binary solvent to generate nanofibers of Collagen type 1, which is non-toxic and economical. Transmission electron microscopy (TEM) analysis revealed the characteristic feature of native collagen namely the 67 nm banding pattern, confirming that the triple helical structure was maintained. Scanning Electron Microscopy (SEM) analysis showed the fiber diameters to be in the 200-800 nm range. Biocompatibility of the three dimensional (3D) scaffolds was established by MTT assays using skeletal myoblasts and Confocal Microscopic analysis of immunofluorescent stained sections for muscle specific markers such as Desmin and Actin. Primary neonatal rat ventricular cardiomyocytes seeded onto the scaffolds were able to maintain their contractile function for a period of 17 days. Our work provides evidence that Collagen 1 can be electrospun without combining with other polymers using a novel benign solvent and we are currently exploring the potential of this approach for cardiac and skeletal muscle tissue engineering. This article is protected by copyright. All rights reserved.

Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation.

Vitamin D3, 25‐Hydroxyvitamin D3, and 1α‐Hydroxyvitamin D3 Modulate Fiber Type Distribution in Bicep Femoris of Growing Rats

An experiment was conducted to examine the effects of different types of vitamin D3 on skeletal muscle development using growing rats. Three‐week‐old rats were fed a vitamin D3‐deficient diet for 4 weeks and vitamin D3‐replete diets for the next 4 weeks as each of vitamin D3 (vit D3, 1,000 IU/kg diet), 25‐hydroxyvitamin D3 (25‐OH D3, 400 IU/kg diet), and 1α‐hydroxyvitamin D3 (1α‐OH D3, 4 IU/kg diet) was supplemented. Negative control group was fed a vitamin D3‐deficient diet, and positive control group was fed a vitamin D3‐supplemented diet (1,000 IU/kg diet) for the whole experimental period, 8 weeks. In results, body weight, feed intake, and body composition (lean and fat) were not affected by vitamin D3 dose and type. Quantitative gene expression of skeletal muscle differentiation markers (MyoD, Myogenin, Mrf4, Myf5, and Myostatin) also showed no difference. However, skeletal muscle fiber type distribution (myofibrillar ATPase staining) was changed in bicep femoris (BF) of the rats fed different types of vitamin D3 supplements.In positive control, type I was increased by 2.4 fold (7.22 % vs. 2.95 %) compared to negative control. In the rats fed vit D3‐replete diets followed by 4‐weeks of vitamin D3 deficiency, both type I and IIa were increased only 1.3 fold (3.72% vs. 2.95%, and 20.79 % vs. 15.74 %) compared to negative control. Percentages of type I were 5.0‐ and 6.9‐fold increased in BF of the rats fed 25‐OH D3 (14.63 % vs. 2.95%) and 1α‐OH D3 (20.45 % vs. 2.95 %) respectively, in comparison to negative control. The present study demonstrates that vitamin D3 does not affect lean growth, but it might mediate skeletal muscle fiber type distribution in growing rats depending on the vitamin D3 types.

Rhabdomyoblastic Differentiation in Head and Neck Malignancies Other Than Rhabdomyosarcoma.

Rhabdomyosarcoma is a relatively common soft tissue sarcoma that frequently affects children and adolescents and may involve the head and neck. Rhabdomyosarcoma is defined by skeletal muscle differentiation which can be suggested by routine histology and confirmed by immunohistochemistry for the skeletal muscle-specific markers myogenin or myoD1. At the same time, it must be remembered that when it comes to head and neck malignancies, skeletal muscle differentiation is not limited to rhabdomyosarcoma. A lack of awareness of this phenomenon could lead to misdiagnosis and, subsequently, inappropriate therapeutic interventions. This review focuses on malignant neoplasms of the head and neck other than rhabdomyosarcoma that may exhibit rhabdomyoblastic differentiation, with an emphasis on strategies to resolve the diagnostic dilemmas these tumors may present. Axiomatically, no primary central nervous system tumors will be discussed.

Transgelin: a potentially useful diagnostic marker differentially expressed in triple-negative and non-triple-negative breast cancers.

Triple negative (TN) (estrogen receptor [ER], progesterone receptor [PR] and HER2-) are highly aggressive, rapidly growing, hormone-unresponsive tumors diagnosed at later stage that affect younger women with shorter overall survival. Most TN tumors are of the basal type. For the remainder, identification of target markers for effective treatment strategies remains a challenge. Transgelin (TGLN) is a 22-kd actin-binding protein of the calponin family. It is one of the earliest markers of smooth muscle differentiation. TGLN has been shown to have important biologic activities including regulating muscle fiber contractility, cell migration, and tumor suppression. We examined TGLN expression in the different molecular subtypes of breast cancer. TGLN expression was examined as a function of tumor size, grade, histologic type, lymph node status, patients' age and overall survival, ER, PR, HER2, and Ki-67 in 101 tumors that included 35 luminal A, 28 luminal B, 4 HER2, and 34 TN types. TGLN positivity (defined as 2+ or 3+) was associated with more aggressive tumors (10% of grade I/II tumors were TGLN+ versus 53% of grade III tumors; P < .001), high Ki-67 count, and low ER and PR expression (P < .001) but not with tumor size, age, or lymph node metastasis. TN (n = 34) tumors were 7.7 times more likely to be TGLN+ than non-TN (n = 67) tumors (77% versus 10%, respectively; P < .001). TGLN may be an excellent diagnostic marker of TN tumors and could be useful in stratification of patients. TGLN may also prove a potential target for future treatment strategies.

Progressive loss of myogenic differentiation in leiomyosarcoma has prognostic value.

Well-differentiated leiomyosarcomas show morphologically recognizable smooth muscle differentiation, whereas poorly differentiated tumours may form a spectrum with a subset of undifferentiated pleomorphic sarcomas. The expression of certain muscle markers has been reported to have prognostic impact. We investigated the correlation between the morphological spectrum and the muscle marker expression profile of leiomyosarcoma, and the impact of these factors on patient outcomes. Tissue microarrays including 202 non-uterine and 181 uterine leiomyosarcomas with a spectrum of tumour morphologies were evaluated for expression of immunohistochemical markers of muscle differentiation. Poorly differentiated tumours frequently lost one or more conventional smooth muscle markers [smooth muscle actin, desmin, h-caldesmon, and smooth muscle myosin (P<0.0001)], as well as the more recently described markers SLMAP, MYLK, and ACTG2 (P<0.0001). In primary tumours, both desmin and CFL2 expression predicted improved overall survival in multivariate analyses (P=0.0111 and P=0.043, respectively). Patients with muscle marker-enriched tumours (expressing all four conventional markers or any three of ACTG2, CFL2, CASQ2, MYLK, and SLMAP) had improved overall survival (P<0.05) in univariate analyses. Morphologically and immunohistochemically, poorly differentiated leiomyosarcomas can masquerade as undifferentiated pleomorphic sarcomas with progressive loss of muscle markers. The expression of muscle markers has prognostic significance in primary leiomyosarcomas independently of tumour morphology.

A role for Galgt1 in skeletal muscle regeneration.

BackgroundCell surface glycans are known to play vital roles in muscle membrane stability and muscle disease, but to date, roles for glycans in muscle regeneration have been less well understood. Here, we describe a role for complex gangliosides synthesized by the Galgt1 gene in muscle regeneration.MethodsCardiotoxin-injected wild type (WT) and Galgt1−/− muscles, and mdx and Galgt1−/−mdx muscles, were used to study regeneration in response to acute and chronic injury, respectively. Muscle tissue was analyzed at various time points for morphometric measurements and for gene expression changes in satellite cell and muscle differentiation markers by quantitative real-time polymerase chain reaction (qRT-PCR). Primary cell cultures were used to measure growth rate and myotube formation and to identify Galgt1 expression changes after cardiotoxin by fluorescence-activated cell sorting (FACS). Primary cell culture and tissue sections were also used to quantify satellite cell apoptosis.ResultsA query of a microarray data set of cardiotoxin-induced mouse muscle gene expression changes identified Galgt1 as the most upregulated glycosylation gene immediately after muscle injury. This was validated by qRT-PCR as a 23-fold upregulation in Galgt1 expression 1 day after cardiotoxin administration and a 16-fold upregulation in 6-week-old mdx muscles. These changes correlated with increased expression of Galgt1 protein and GM1 ganglioside in mononuclear muscle cells. In the absence of Galgt1, cardiotoxin-induced injury led to significantly reduced myofiber diameters after 14 and 28 days of regeneration. Myofiber diameters were also significantly reduced in Galgt1-deficient mdx mice compared to age-matched mdx controls, and this was coupled with a significant increase in the loss of muscle tissue. Cardiotoxin-injected Galgt1−/− muscles showed reduced gene expression of the satellite cell marker Pax7 and increased expression of myoblast markers MyoD, Myf5, and Myogenin after injury along with a tenfold increase in apoptosis of Pax7-positive muscle cells. Cultured primary Galgt1−/− muscle cells showed a normal growth rate but demonstrated premature fusion into myofibers, resulting in an overall impairment of myofiber formation coupled with a threefold increase in muscle cell apoptosis.ConclusionsThese experiments demonstrate a role for Galgt1 in skeletal muscle regeneration and suggest that complex gangliosides made by Galgt1 modulate the survival and differentiation of satellite cells.

Successful treatment of multiple cutaneous leiomyomas with carbon dioxide laser ablation.

Successful treatment of multiple cutaneous leiomyomas with carbon dioxide laser ablation.

Exogenous connexin43-expressing autologous skeletal myoblasts ameliorate mechanical function and electrical activity of the rabbit heart after experimental infarction.

Acute myocardial infarction is one of the major causes of mortality worldwide. For regeneration of the rabbit heart after experimentally induced infarction we used autologous skeletal myoblasts (SMs) due to their high proliferative potential, resistance to ischaemia and absence of immunological and ethical concerns. The cells were characterized with muscle-specific and myogenic markers. Cell transplantation was performed by injection of cell suspension (0.5 ml) containing approximately 6 million myoblasts into the infarction zone. The animals were divided into four groups: (i) no injection; (ii) sham injected; (iii) injected with wild-type SMs; and (iv) injected with SMs expressing connexin43 fused with green fluorescent protein (Cx43EGFP). Left ventricular ejection fraction (LVEF) was evaluated by 2D echocardiography in vivo before infarction, when myocardium has stabilized after infarction, and 3 months after infarction. Electrical activity in the healthy and infarction zones of the heart was examined ex vivo in Langendorff-perfused hearts by optical mapping using di-4-ANEPPS, a potential sensitive fluorescent dye. We demonstrate that SMs in the coculture can couple electrically not only to abutted but also to remote acutely isolated allogenic cardiac myocytes through membranous tunnelling tubes. The beneficial effect of cellular therapy on LVEF and electrical activity was observed in the group of animals injected with Cx43EGFP-expressing SMs. L-type Ca(2+) current amplitude was approximately fivefold smaller in the isolated SMs compared to healthy myocytes suggesting that limited recovery of LVEF may be related to inadequate expression or function of L-type Ca(2+) channels in transplanted differentiating SMs.