Mouse Muscle Cell Line Research Articles

Effect of electrochemotherapy on myogenesis of mouse C2C12 cells in vitro

Electrochemotherapy (ECT) is a local ablative therapy for the treatment of different skin and subcutaneous tumors and certain tumors in internal organs. Skeletal muscle represents a major tumor- surrounding tissue, exposed to side effects of ECT. At the cellular level, side-effects of ECT on skeletal muscle and underlying mechanisms have not been examined yet. Thus, we aimed to determine the effect of ECT in the mouse muscle cell line C2C12 during in vitro myogenesis. We evaluated the electroporation efficiency and viability of C2C12 myotubes at increasing voltages (200–1300 V/cm) using propidium iodide (PI). Permeabilization of PI into the cells was voltage-dependent accounting up to 97 % efficiency at the highest voltage. High cell viability and myotube integrity were maintained until 4 days after electroporation. ECT with the cytostatic drugs bleomycin and cisplatin decreased the viability of C2C12 myoblasts and myotubes in a dose-dependent manner. However, myoblasts were more sensitive to ECT than myotubes. Increased secretion of IL-6, observed 3 days after ECT, confirming its effects on early myogenesis. Only minor effects of ECT were observed in treated myotubes. These results contribute to the safety profile of ECT in tumor treatment.

Role of Exosomes Derived from Adipose Tissue under Obese Conditions in Skeletal Muscle and Liver Cells: Commonalities and Differences.

To determine the correlation between obesity and insulin resistance in skeletal muscle and liver tissues, this study isolates exosomes from adipose tissue under obese conditions and investigates the effect of adipose tissue-derived exosomes (Ad-exosomes) in mouse muscle (C2C12 cells) and liver cell lines (AML12 cells). The study isolates exosomes from the adipose tissue of normal diet-fed mice or high-fat diet (HFD)-fed obese mice and confirms the uptake into differentiated C2C12 and AML12 cells. Ad-exosomes from HFD-fed mice induce insulin resistance, triglyceride (TG) accumulation, endoplasmic reticulum stress, and inflammation in both C2C12 and AML12 cells. Interestingly, the study finds that the TG accumulation induces by Ad-exosomes from HFD-fed obese mice is dramatically increased in AML12 cells compared with that in the differentiated C2C12 cells, and glucose uptake following the same treatment is decreased in C2C12 cells and increased in AML12 cells. In addition, Ad-exosomes from HFD-fed obese mice cause not only TG accumulation but also lipogenesis in AML12 cells. The results suggest that Ad-exosomes from HFD-fed obese mice cause insulin resistance in both the muscles and liver, but their effects on metabolism during the development of insulin resistance vary between tissues.

Loss of full-length dystrophin expression results in major cell-autonomous abnormalities in proliferating myoblasts.

Duchenne muscular dystrophy (DMD) affects myofibers and muscle stem cells, causing progressive muscle degeneration and repair defects. It was unknown whether dystrophic myoblasts-the effector cells of muscle growth and regeneration-are affected. Using transcriptomic, genome-scale metabolic modelling and functional analyses, we demonstrate, for the first time, convergent abnormalities in primary mouse and human dystrophic myoblasts. In Dmdmdx myoblasts lacking full-length dystrophin, the expression of 170 genes was significantly altered. Myod1 and key genes controlled by MyoD (Myog, Mymk, Mymx, epigenetic regulators, ECM interactors, calcium signalling and fibrosis genes) were significantly downregulated. Gene ontology analysis indicated enrichment in genes involved in muscle development and function. Functionally, we found increased myoblast proliferation, reduced chemotaxis and accelerated differentiation, which are all essential for myoregeneration. The defects were caused by the loss of expression of full-length dystrophin, as similar and not exacerbated alterations were observed in dystrophin-null Dmdmdx-βgeo myoblasts. Corresponding abnormalities were identified in human DMD primary myoblasts and a dystrophic mouse muscle cell line, confirming the cross-species and cell-autonomous nature of these defects. The genome-scale metabolic analysis in human DMD myoblasts showed alterations in the rate of glycolysis/gluconeogenesis, leukotriene metabolism, and mitochondrial beta-oxidation of various fatty acids. These results reveal the disease continuum: DMD defects in satellite cells, the myoblast dysfunction affecting muscle regeneration, which is insufficient to counteract muscle loss due to myofiber instability. Contrary to the established belief, our data demonstrate that DMD abnormalities occur in myoblasts, making these cells a novel therapeutic target for the treatment of this lethal disease.

MCU-complex-mediated mitochondrial calcium signaling is impaired in Barth syndrome.

Calcium signaling via mitochondrial calcium uniporter (MCU) complex coordinates mitochondrial bioenergetics with cellular energy demands. Emerging studies show that the stability and activity of the pore-forming subunit of the complex, MCU, is dependent on the mitochondrial phospholipid, cardiolipin (CL), but how this impacts calcium-dependent mitochondrial bioenergetics in CL-deficiency disorder like Barth syndrome (BTHS) is not known. Here we utilized multiple models of BTHS including yeast, mouse muscle cell line, as well as BTHS patient cells and cardiac tissue to show that CL is required for the abundance and stability of the MCU-complex regulatory subunit MICU1. Interestingly, the reduction in MICU1 abundance in BTHS mitochondria is independent of MCU. Unlike MCU and MICU1/MICU2, other subunit and associated factor of the uniporter complex, EMRE and MCUR1, respectively, are not affected in BTHS models. Consistent with the decrease in MICU1 levels, we show that the kinetics of MICU1-dependent mitochondrial calcium uptake is perturbed and acute stimulation of mitochondrial calcium signaling in BTHS myoblasts fails to activate pyruvate dehydrogenase, which in turn impairs the generation of reducing equivalents and blunts mitochondrial bioenergetics. Taken together, our findings suggest that defects in mitochondrial calcium signaling could contribute to cardiac and skeletal muscle pathologies observed in BTHS patients.

Knockdown of CNN3 Impairs Myoblast Proliferation, Differentiation, and Protein Synthesis via the mTOR Pathway.

BackgroundMyogenesis is a complex process that requires optimal outside–in substrate–cell signaling. Calponin 3 (CNN3) plays an important role in regulating myogenic differentiation and muscle regeneration; however, the precise function of CNN3 in myogenesis regulation remains poorly understood. Here, we investigated the role of CNN3 in a knockdown model in the mouse muscle cell line C2C12.MethodsMyoblast proliferation, migration, differentiation, fusion, and protein synthesis were examined in CNN3 knockdown C2C12 mouse muscle cells. Involvement of the mTOR pathway in CNN3 signaling was explored by treating cells with the mTOR activator MHY1485. The regulatory mechanisms of CNN3 in myogenesis were further examined by RNA sequencing and subsequent gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA).ResultsDuring proliferation, CNN3 knockdown caused a decrease in cell proliferation and migration. During differentiation, CNN3 knockdown inhibited myogenic differentiation, fusion, and protein synthesis in C2C12 cells via the AKT/mTOR and AMPK/mTOR pathways; this effect was reversed by MHY1485 treatment. Finally, KEGG and GSEA indicated that the NOD-like receptor signaling pathway is affected in CNN3 knockdown cell lines.ConclusionCNN3 may promote C2C12 cell growth by regulating AKT/mTOR and AMPK/mTOR signaling. The KEGG and GSEA indicated that inhibiting CNN3 may activate several pathways, including the NOD-like receptor pathway and pathways involved in necroptosis, apoptosis, and inflammation.

Trim33 (Tif1\u03b3) is not required for skeletal muscle development or regeneration but suppresses cholecystokinin expression

The expression of Trim33 (Tif1γ) increases in skeletal muscles during regeneration and decreases upon maturation. Although Trim33 is required for the normal development of other tissues, its role in skeletal muscle is unknown. The current study aimed to define the role of Trim33 in muscle development and regeneration. We generated mice with muscle-specific conditional knockout of Trim33 by combining floxed Trim33 and Cre recombinase under the Pax7 promoter. Muscle regeneration was induced by injuring mouse muscles with cardiotoxin. We studied the consequences of Trim33 knockdown on viability, body weight, skeletal muscle histology, muscle regeneration, and gene expression. We also studied the effect of Trim33 silencing in satellite cells and the C2C12 mouse muscle cell line. Although Trim33 knockdown mice weighed less than control mice, their skeletal muscles were histologically unremarkable and regenerated normally following injury. Unexpectedly, RNAseq analysis revealed dramatically increased expression of cholecystokinin (CCK) in regenerating muscle from Trim33 knockout mice, satellite cells from Trim33 knockout mice, and C2C12 cells treated with Trim33 siRNA. Trim33 knockdown had no demonstrable effect on muscle differentiation or regeneration. However, Trim33 knockdown induced CCK expression in muscle, suggesting that suppression of CCK expression requires Trim33.

358. AAV-MitoTimer to Asses Mitochondrial Turnover

Mitochondrial turnover is a key marker in the process of cellular senescence. In order to track mitochondrial turnover rates in various cell lines, an indicator is necessary to identify the formation of new mitochondria along with the degradation of old mitochondria. This measure is critical for the study of disease processes in which there is an up or down regulation of mitochondrial turnover rates. One dynamic tool to measure this balance is the pTRE-Tight-MitoTimer plasmid. The MitoTimer transgene combines the red fluorescent protein, dsRed, and a human cytochrome c oxidase subunit VIII (Cox 8) mitochondrial localization sequence to traffic the protein to the mitochondria where the color shifts from green to red over time (approximately 24 hours). Our goal is to use this as a tool to assess the increase or decrease in mitochondrial turnover as a result of mutations that cause mitochondrial dysfunction. We successfully developed two plasmids through the ligation of the MitoTimer transgene into a double stranded AAV-ITR containing plasmid with a 1) Desmin promoter pds-AAV-Des-MitoTimer to restrict expression to striated muscle and 2) CMV promoter pds-AAV-CMV-MitoTimer to enable ubiquitous expression among many cell types. Once cloned, these novel plasmids were then transfected into an immortalized C2C12 mouse muscle cell line to confirm successful generation of functional plasmids. An immediate application for these novel plasmids in our laboratory is the characterization of mitochondrial turnover rates in Barth Syndrome (BTHS) patient derived cells. BTHS is an X-linked mitochondrial disease that results from mutations of the gene TAZ, which encodes tafazzin. This gene serves to orchestrate the production of mature cardiolipin (CL) within the mitochondrial membrane. Mutations in the TAZ gene result in altered CL maturation, with implications on mitochondrial function. Thus far, mitochondrial turnover rates have not been carefully assessed in this disorder. As a preliminary experiment using human patient derived BTHS fibroblasts (harboring an exon 10: 748G &gt T nonsense mutation), pds-AAV-CMV-MitoTimer was successfully transfected and displayed variation in green and red fluorescence ratios as compared to a healthy control cell line. This demonstration of the impact of BTHS on mitochondrial turnover dynamics supports future use of these plasmids in BTHS induced pluripotent stem cells (iPSCs) that have been differentiated into cardiomyocytes or skeletal myotubes - the two cell-types most affected in BTHS. This will enable future comparisons of mitochondrial turnover in clinically relevant BTHS cells harboring a variety of different mutations and provide another metric by which to measure the success of pre-clinical therapeutics for BTHS. We are preparing an AAV Quad-mutant serotype for in vitro testing, and other serotypes will be used for in vivo models of mitochondrial dysfunction. AAV-MitoTimer will continue to be used as a measure of mitochondrial turnover, and provide an effective characterization method for metabolic diseases.

PEDF attenuates insulin-dependent molecular pathways of glucose homeostasis in skeletal myocytes

Pigment epithelium-derived factor (PEDF) is an anti-angiogenic serpin associated with insulin resistance in metabolic disorders such as diabetes, metabolic syndrome, obesity and polycystic ovarian syndrome. While the mechanism of PEDF induced-insulin resistance of metabolic disorders has been attributed to its inflammatory and lipolytic effects, little evidence exists to support a direct role of PEDF in mediating insulin resistance. Here, we seminally provide evidence that PEDF can inhibit insulin signal transduction governing glucose homeostasis from the receptor to the effector phosphorylation through Akt/PKB-dependent and -independent pathways in mouse and human skeletal muscle cell lines. PEDF attenuates the insulin-dependent molecular axes of glucose metabolism. Exposure of skeletal myocytes to PEDF attenuates insulin-dependent insulin receptor autophosphorylation, tyrosine phosphorylation of insulin receptor substrate 1, and dual loop phosphorylation-activation of Akt. PEDF significantly inhibits the downstream effector - glycogen synthase kinase (and thereby the glycogenic axis of insulin signalling). PEDF turned off both the molecular switches of GLUT4 translocation: IRS-Akt/PKB-AS160 mediated and IR-pCbl-dependent GLUT4 translocation (the molecular axis of glucose uptake). These findings implicate a direct effect of PEDF on multiple insulin-dependent molecular mechanisms of glucose homeostasis in skeletal muscle cells, thereby enabling it to contribute to peripheral insulin resistance at the cellular level.

The effect of retinol binding protein on the proteome of C2C12 muscle cells.

Retinol binding protein (RBP) and its membrane receptor, STRA6, are vital for the management of vitamin A in the body. Recently, elevated serum RBP levels have been implicated as a contributing factor to the development of insulin resistance and type 2 diabetes. However, conflicting opinions exist as to how these increased levels can cause insulin resistance. In order to better understand the influences of RBP, a proteomic study was devised to determine the direct effect of RBP on a mouse muscle cell line, because the muscle is the principal site of insulin induced glucose uptake. C2C12 cells were treated with RBP for 16 h and the proteome analysed for alterations in protein abundance and phosphorylation by 2-DE. A number of changes were observed in response to retinol binding protein treatment, of which the most interesting were decreased levels of the phosphatase, protein phosphatase 1 β. This phosphatase is responsible for regulating glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes involved in glycogen storage and utilization. Retinol binding protein treatment resulted in increased phosphorylation and inhibition of glycogen synthase, with detrimental effects on insulin stimulated glycogen production in these cells. The results indicate that RBP may have a negative effect on energy storage in the cell and could contribute to the development of insulin resistance in muscle tissue. Understanding how retinol binding protein influences insulin resistance may reveal novel strategies to target this disease.

The Hcm Mutation R453C in the Human Beta Myosin Motor Domain Alters the ATP-Binding and Hydrolysis Steps

The human hypertrophic cardiomyopathy mutation R453C is associated with a high incidence of sudden cardiac death. We have expressed the motor domain (MD) of human beta-myosin and the human ventricular essential light chain (MLC3) with the R453C mutation using a mouse muscle cell line. We have examined the binding of nucleotides to the MD and actin·MD and compared the kinetic data to our previous study of the wild type MD (Deacon et al 2012). Little change was observed in the binding of ATP or ADP to actin·MD but the two fluorescence changes accompanying ATP binding to MD in the absence of actin were slower. The fast phase, previously assigned to the conformational change accompanying tight binding of ATP to the motor, was slowed by 30%. The slow phase,assigned to the closure of switch 2 and the recovery stroke, associated with the ATP hydrolysis step, were slowed ∼ 3 fold. The R453C mutation is located in a surface loop that links the switch-2 beta strand with the long O-helix that connects to the cardiomyopathy loop in the actin binding site. R453C is therefore in a position to influence both actin binding and switch 2 closure. The surface loop and R453 are conserved only in sarcomeric myosins, suggesting a specific role for the loop in muscle. Supported by NIH grants GM29090 and GM65013 and Wellcome Trust grant 085309.

Regulation of skeletal muscle oxidative phenotype by classical NF-κB signalling

BackgroundImpairments in skeletal muscle oxidative phenotype (OXPHEN) have been linked to the development of insulin resistance, metabolic inflexibility and progression of the metabolic syndrome and have been associated with progressive disability in diseases associated with chronic systemic inflammation. We previously showed that the inflammatory cytokine tumour necrosis factor-α (TNF-α) directly impairs muscle OXPHEN but underlying molecular mechanisms remained unknown. Interestingly, the inflammatory signalling pathway classical nuclear factor-κB (NF-κB) is activated in muscle in abovementioned disorders. Therefore, we hypothesised that muscle activation of classical NF-κB signalling is sufficient and required for inflammation-induced impairment of muscle OXPHEN. MethodsMyotubes from mouse and human muscle cell lines were subjected to activation or blockade of the classical NF-κB pathway. In addition, wild-type and MISR (muscle-specific inhibition of classical NF-κB) mice were injected intra-muscularly with TNF-α. Markers and key regulators of muscle OXPHEN were investigated. ResultsClassical NF-κB activation diminished expression of oxidative phosphorylation (OXPHOS) sub-units, slow myosin heavy chain expression, activity of mitochondrial enzymes and potently reduced intra-cellular ATP levels. Accordingly, PGC-1/PPAR/NRF-1/Tfam signalling, the main pathway controlling muscle OXPHEN, was impaired upon classical NF-κB activation which required intact p65 trans-activation domains and depended on de novo gene transcription. Unlike wild-type myotubes, IκBα-SR myotubes (blocked classical NF-κB signalling) were refractory to TNF-α-induced impairments in OXPHEN and its regulation by the PGC-1/PPAR/NRF-1/Tfam cascade. In line with in vitro data, NF-κB blockade in vivo abrogated TNF-α-induced reductions in PGC-1α expression. ConclusionClassical NF-κB activation impairs skeletal muscle OXPHEN.

Presence of VDR and CYP27B1 in mouse C2C12 cells and skeletal muscle reveal the action of 25(OH)D3 on suppression of myoblast proliferation

1α,25(OH)2D3, an active form of vitamin D3 was demonstrated to regulate myoblast proliferation via its nuclear vitamin D receptor (VDR). Local production of 1α,25(OH)2D3 was reported in the cells that express VDR and CYP27B1, the latter is used to convert 25(OH)D3 to 1α,25(OH)2D3. However, whether skeletal muscle expresses CYP27B1 and 25(OH)D3 can be used as a precursor for the local synthesis of 1α,25(OH)2D3 in skeletal muscle and regulate muscle cell functions are currently unknown. In this study, we investigated 1) VDR and CYP27B1 mRNA transcripts in C2C12 cells (mouse muscle cell line) and skeletal muscle tissue 2) the expression of VDR and CYP27B1 protein in C2C12 cells 3) the effect of 25(OH)D3 on C2C12 cell proliferation. Here, we demonstrated that C2C12 cells and skeletal muscle expressed VDR and CYP27B1 mRNA transcripts. Both genes were significantly up‐regulated at the mRNA level after skeletal muscle was overloaded. Nuclear VDR and mitochondrial CYP27B1 were expressed in C2C12 myoblasts but not myotubes. 25(OH)D3 treatment increased nuclear VDR expression and inhibited C2C12 myoblast proliferation. This effect was abolished by CYP27B1 knockdown, suggesting CYP27B1 is required for 25(OH)D3 action in myoblasts. Taken together, the presence of VDR and CYP27B1 in C2C12 cells and skeletal muscle revealed the possible contribution of 25(OH)D3 on the regulation of skeletal muscle function, in part on controlling of muscle cell proliferation.This work was supported by NIH R01AR45617 to KAE and PGY funds to OKPS

Small RNA-Mediated Epigenetic Myostatin Silencing

Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

Biomedical and pharmacological potential of tetrodotoxin-producing bacteria isolated from marine pufferfish Arothron hispidus (Muller, 1841)

Specimens of the pufferfish Arothron hispidus collected at Parangipettai, on the southeast coast of India, were subjected to bacterial isolation and identification. Three species were identified, namely Bacillus sp., Kytococcus sedentarius and Cellulomonas fimi. Partially-purified microbial filtrates exhibited hemolytic activity on chicken and human erythrocytes of O, B and AB blood groups, with maximum activity of 32 HU. The microbial filtrates also presented ATPase, Mg2+-ATPase, Na+K+-ATPase and AchE enzymatic activities of positive neuromodulation in Kytococcus sedentarius with 1300, 300.1, 1549.98 and 140.55%, in Cellulomonas fimi with 620, 300, 10 and 128.42%, and in Bacillus species with 40, 200, 849.98 and 158.69%, respectively. Toxicity symptoms were observed when the bacterial filtrate was intraperitoneally injected into mice. The bacterial filtrate caused adverse effects on viability of the mouse muscle cell line (L929) and leukemia cell line (P388). Maximum level of inhibition was observed on the growth of L929 cell line. Bacillus lentimorbus inhibited the cell line from 84.03 to 94.43% whereas Bacillus species inhibited the growth in a range between 77.25 and 86.16% at the lowest dilution.

Synaptogenetic mechanisms controlling postsynaptic differentiation of the neuromuscular junction are nerve-dependent in human and nerve-independent in mouse C2C12 muscle cultures

Acetylcholinesterase (EC 3.1.1.7, AChE) is one of the components of the neuromuscular junction (NMJ). Its expression and targeting in the skeletal muscle fiber is therefore under the control of the mechanisms responsible for the formation of the highly complex structure of this synapse. Recently, it has been demonstrated that myotubes of the C2C12 mouse muscle cell line form highly differentiated pretzel-like postsynaptic accumulations of acetylcholine receptors (AChRs) in the complete absence of the nerve if they are cultured on the laminin coating. This finding questions previously stressed importance of the nerve-derived factors in NMJ synaptogenesis and therefore deserves additional testing. The aim of this paper was to test whether the reported nerve-independency can be demonstrated also in the cultured human muscle meaning that the findings on C2C12 cultures can be extrapolated also to the human muscle. In our experiments aneurally cultured human myotubes failed to form AChR clusters on its surface, no matter if they were grown on normal gelatine or laminin coating. However, when innervated by neurons extending from the rat embryonic spinal cord, human myotubes formed AChR clusters with elaborate topography but strictly on the areas contacted by the nerve. One can hypothesize that higher nerve dependency of the NMJ synaptogenesis in humans in comparison to other species reflects species-specific differences in the organization of movement. Humans have the highest “fractionation of movement” capacity which probably requests different, more nerve-controlled development of the motor system including nerve-restricted development of the neuromuscular contacts.

Neurofilament protein aggregation in a cell line model system

Protein aggregates are associated with many diseases and even aggregates of proteins that have no role in disease are inherently toxic to both neuronal and non-neuronal cells. We have developed a model system to explore the mechanism of protein aggregation using a mouse muscle cell line expressing chimeric neurofilament (NF) proteins, a constituent of the protein aggregates in ALS, Lewy body dementia, and Charcot-Marie-Tooth disease. Formation of protein aggregates in these cells leads to reduced cell viability and activated caspases. Aggregates contained both chimeric NF proteins and ubiquitin by immunolocalization and were predominately cytosolic when proteins were expressed at low levels or for shorter periods of time but were present in the nucleus when expression levels increased. This system represents a flexible, new tool to decipher the molecular mechanism of protein aggregation and the contributions of aggregation to cell toxicity.

Studies of acetylcholine receptor subunit gene expression: chromatin structural changes during myogenesis.

Myogenesis proceeds stepwise from pluripotential stem cell to differentiated myotube. The precise number of transitions that occur along the developmental pathway remains to be determined. We examined the myogenic pathway as modelled by mouse mesodermal stem cell and muscle cell lines for stage-specific alterations in the chromatin structure of the acetylcholine receptor delta and gamma subunit genes. We reasoned that such an analysis would allow us to observe either the primary events in the activation of these muscle-specific genes or processes secondary to the binding of muscle-specific regulatory proteins. We probed chromatin structure with DNase I (deoxyribonuclease I) and precisely mapped to the 5' ends of the delta and gamma genes DNase I hypersensitive (DH) sites whose induction is unique to each myogenic stage. Putative mesodermal stem cells have the simplest pattern of DH sites with no sites near the 5' ends of the delta and gamma genes, whereas differentiated myotubes express the most complex pattern; the myoblast pattern is intermediate and of two types. In muscle cell lines where differentiation must be induced the myoblasts have a simple pattern (one more site than stem cells); in muscle lines where differentiation is spontaneous the myoblasts express a complex pattern of DH sites (one less site than myotubes). Inducible myoblasts seem to be arrested in an earlier step in the myogenic pathway than spontaneously differentiating myoblasts. Thus, myogenic activation of acetylcholine receptor subunit genes appears to be a stepwise process that can be detected by chromatin structural changes specific to four distinct stages of muscle development: stem cell, early myoblast, late myoblast, and differentiated myotube.

Autoimmune Channelopathies and Related Neurological Disorders

Ion channels are crucial elements in neuronal signaling and synaptic transmission, and defects in their function are known to underlie rare genetic disorders, including some forms of epilepsy. A second class of channelopathies, characterized by autoantibodies against ligand- and voltage-gated ion channels, cause a variety of defects in peripheral neuromuscular and ganglionic transmission. There is also emerging evidence for autoantibody-mediated mechanisms in subgroups of patients with central nervous system disorders, particularly those involving defects in cognition or sleep and often associated with epilepsy. In all autoimmune channelopathies, the relationship between autoantibody specificity and clinical phenotype is complex. But with this new information, autoimmune channelopathies are detected and treated with increasing success, and future research promises new insights into the mechanisms of dysfunction at neuronal synapses and the determinants of clinical phenotype.

980. Specific Immune Response Against the Plasmodium falciparum CS Protein Mediated by Baculovirus Vectors

Top of pageAbstract Malaria is the most common tropical disease worldwide. More than 300 million people are infected and about 2-3 million die annually. The most pathogenic form, Malaria tropica, is caused by Plasmodium falciparum. This enormous world-health problem is compounded by parasites becoming resistant to drugs which are commonly used for treatment and also the complete lack of an effective vaccine. Recombinant baculovirus vectors derived from the Autographa californica nuclear polyhedrosis virus (AcNPV) are commonly used as a tool for high-level expression of recombinant proteins of interest in insect cells. Baculovirus vectors can mediate an immune response against an antigen when it is displayed on the viral surface, or when it is expressed from the baculovirus backbone using promoters which are active in mammalian cells. Their ability to accommodate very large inserts of foreign DNA, the lack of a preexisting/neutralizing immunity to baculoviruses in humans and the easy generation of high titer virus stocks are further clear advantages of this vector system for the delivery of a vaccine. In order to use baculoviruses for an induction of a specific immune response we inserted a P. falciparum circumsporozoite (CS) protein expression cassette, driven by the strong CMV promoter, into the baculoviral genome (AcNPV-CSFVI). An expression of the CS protein in antigen presenting cells (APC) should activate CD8+ T cells via MHC I presentation. The display of a second, recombinant CS peptide in the viral envelope was achieved by fusion of the CS gene to the main baculovirus surface protein gp64 (AcNPV-CS/gp64). This envelope-modified baculovirus mimics the parasite to induce CD4+ T cells and CS specific antibodies following uptake into APC and antigen presentation by MHC II. A third vector (AcNPV-CSFVI-CS/gp64) was constructed to combine expression and presentation of CS antigens in mammalian cells. Expression of the CS protein upon infection of a mouse muscle cell line with AcNPV-CSFVI or AcNPV-CSFVI-CS/gp64 was confirmed by Western blotting with CS specific antibodies. For vaccination studies, BALB/c mice were injected intramuscularly with 5|[times]|108 pfu of AcNPV-CSFVI, AcNPV-CS/gp64, AcNPV-CSFVI-CS/gp64 or 10 ug recombinant CS protein and boosted after 3 weeks with the same dose. Data of IgG1/IgG2a serum ELISAs showed high CS specific antibody titers for mice injected with AcNPV-CS/gp64. Whereas infection with AcNPV-CSFVI could induce only low levels of antibodies, the combined expression and presentation of CS antigens (AcNPV-CSFVI-CS/gp64) could elicit an even higher IgG2a response. Frequencies of CS specific CD4+ and CD8+ T cells will be analyzed by ELIspot and data will be presented. Our current efforts are aimed towards targeting baculoviruses infection to dendritic cells by incorporation of specific receptor ligands into the viral envelope.

The topoisomerase 1-interacting protein BTBD1 is essential for muscle cell differentiation

DNA topoisomerase I (Topo1) contributes to vital biological functions, but its regulation is not clearly understood. The BTBD1 protein was recently cloned on the basis of its interaction with the core domain of Topo1 and is expressed particularly in skeletal muscle. To determine BTBD1 functions in this tissue, the in vitro model used was the C2C12 mouse muscle cell line, which expresses BTBD1 mainly after myotube differentiation. We studied the effects of a stably overexpressed BTBD1 protein truncated of the 108 N-terminal amino-acid residues and harbouring a C-terminal FLAG tag (Delta-BTBD1). The proliferation speed of Delta-BTBD1 C2C12 cells was significantly decreased and no myogenic differentiation was observed, although these cells maintained their capacity to enter adipocyte differentiation. These alterations could be related to Topo1 deregulation. This hypothesis is further supported by the decrease in nuclear Topo1 content in Delta-BTBTD1 proliferative C2C12 cells and the switch from the main peripheral nuclear localization of Topo1 to a mainly nuclear diffuse localization in Delta-BTBTD1 C2C12 cells. Finally, this study demonstrated that BTBD1 is essential for myogenic differentiation.