Ion channels in a skeletal muscle cell line from a Duchenne muscular dystrophy patient

A cell line (RCDMD), derived from a muscle biopsy taken from a 7-year-old patient with Duchenne muscular dystrophy (DMD), was established in vitro using conditioned media from the UCHT1 thyroid cell line as described elsewhere (Biochim Biophys Acta 1992;1134:247-255). Unlike other cell lines established by the same procedure, RCDMD cells were highly refractory to transformation and the resulting cell line grew slowly with a doubling time of approximately 72 h. Further, cells continue to grow after more than 20 doublings and 15 passages. Some of the characteristics of the cell line include lack of reaction with antidystrophin antibodies and the presence of receptors for the dihydropyridine PN200-110 (K-d) = 0.3 +/- 0.05 nmol/L and B-max = 1.06 +/- 0.03 pmol/mg protein) and for alpha-bungarotoxin (K-d = 1.02 +/- 0.17 nmol/L and B-max = 4.2 +/- 0.37 pmol/mg protein). Patch clamped cells in the voltage clamp configuration lack ion currents when growing in complete medium with high serum, but they can be induced to differentiate by serum deprivation and addition of hormones and trace elements. After 5 days in differentiating medium, noninactivating, delayed rectifier potassium currents are seen. At day 12, A-type, inactivating potassium currents as well as transient inward currents are seen. In conditions in which sodium and potassium currents are absent, a very fast activating and fast inactivating calcium current was evident. The cell line offers the possibility of studying cellular mechanisms in the pathophysiology of DMD

Cell lines as in vitro models for drug screening and toxicity studies

Cell culture is highly desirable, as it provides systems for ready, direct access and evaluation of tissues. The use of tissue culture is a valuable tool to study problems of clinical relevance, especially those related to diseases, screening, and studies of cell toxicity mechanisms. Ready access to the cells provides the possibility for easy studies of cellular mechanisms that may suggest new potential drug targets and, in the case of pathological-derived tissue, it has an 'Interesting application in the evaluation of therapeutic agents that potentially may treat the dysfunction. However, special considerations must be addressed to establish stable in vitro function. In primary culture, these factors are primarily linked to greater demands of tissue to adequately survive and develop differentiated conditions in vitro. Additional requirements include the use of special substrates (collagen, laminin, extracellular matrix preparations, etc.), growth factors and soluble media supplements, some of which can be quite complex in their composition. These demands, along with difficulties in obtaining adequate tissue amounts, have prompted interest in developing immortalized cell lines which can provide unlimited tissue amounts. However, cell lines tend to exhibit problems in stability and/ or viability, though they serve as a feasible alternative, especially regarding new potential applications in cell transplant therapy. In this regard, stem cells may also be a source for the generation of various cell types in vitro. This review will address aspects of cell culture system application, with focus on immortalized cell lines, in studying cell function and dysfunction with the primary aim being to identify cell targets for drug screening

Knockdown of Myo-Inositol transporter SMIT1 normalizes cholinergic and glutamatergic function in an immortalized cell line established from the cerebral cortex of a trisomy 16 fetal mouse, an animal model of human trisomy 21 (Down Syndrome)

The Na+/myo-inositol cotransporter (SMIT1) is overexpressed in human Down syndrome (DS) and in trisomy 16 fetal mice (Ts16), an animal model of the human condition. SMIT1 overexpression determines increased levels of intracellular myo-inositol, a precursor of phophoinositide synthesis. SMIT1 is overexpressed in CTb cells, an immortalized cell line established from the cerebral cortex of a Ts16 mouse fetus. CTb cells exhibit impaired cytosolic Ca2+ signals in response to glutamatergic and cholinergic stimuli (increased amplitude and delayed time-dependent kinetics in the decay post-stimulation), compared to our CNh cell line, derived from the cerebral cortex of a euploid animal. Considering the role of myo-inositol in intracellular signaling, we normalized SMIT1 expression in CTb cells using specific mRNA antisenses. Forty-eight hours post-transfection, SMIT1 levels in CTb cells reached values comparable to those of CNh cells. At this time, decay kinetics of Ca2+ signals induced by either glutamate, nicotine, or muscarine were accelerated in transfected CTb cells, to values similar to those of CNh cells. The amplitude of glutamate-induced cytosolic Ca2+ signals in CTb cells was also normalized. The results suggest that SMIT1 overexpression contributes to abnormal cholinergic and glutamatergic Ca2+ signals in the trisomic condition, and knockdown of DS-related genes in our Ts16-derived cell line could constitute a relevant tool to study DS-related neuronal dysfunction.Fondecyt (Chile) 1040862 1130241 Univ. of Chile Enlaces ENL 07/05 CNRS/Conicyt Exchange Program Fondation J. Lejeune (France) ICM-ECONOMIA, Chile P09-022-F Millennium Scientific Initiative of the Ministerio de Economia, Fomento y Turism

Angiotensin receptor II is present in dopaminergic cell line of rat substantia nigra and it is down regulated by aminochrome

Angiotensin receptor II mRNA was found to be expressed in dopaminergic neuronal cell line RCSN3 of rat substantia nigra using RT-PCR reaction. Aminochrome (150 μM), a metabolite of the dopamine oxidative pathway, was found to down regulate the expression of angiotensin receptor mRNA in RCSN3 cells by 83% (p < 0.05)

Expression of ion channels during differentiation of a human skeletal muscle cell line

An immortal, cloned cell Line (RCMH), obtained from human skeletal muscle was established in our laboratory and shown to express muscle specific proteins. We measured ligand binding to ion channels, ion currents using whole cell patch clamp and intracellular calcium both in cells grown in complete media and in cells grown for 4-40 days in media supplemented with hormones and nutrients (differentiating media). Markers for differentiated muscle, such as the muscle isoform of creatine kinase and the cytoskeletal proteins alpha-actinin, alpha-sarcomeric actin, myosin and titin were present in early stages. Receptors for gamma toxin from Tityus serrulatus scorpion venom, a specific modulator for voltage dependent sodium channels, were present (0.9-1.0 pmol mg(-1) protein) during stage 1 (0-6 days in culture with differentiating media) and increased by 50% in stage 3 (more than 13 days in differentiating media). High and low affinity dihydropyridine receptors present in stage 1 convert into a single type of high affinity receptors in stage 3. Both intracellular calcium release and InsP(3) receptors were evident in stage 1 but ryanodine receptors were expressed only in stage 3. RCMH cells showed no voltage sensitive currents in stage 1. Between 7 and 10 days in differentiating media (stage 2), an outward potassium current was observed. Small inward currents appeared only in stage 3; we identified both tetrodotoxin sensitive and tetrodotoxin resistant sodium currents as well as calcium currents. This pattern is consistent with the expression of voltage dependent calcium release before appearance of both the action potential and ryanodine receptors

Microencapsulation of cellular aggregates composed of differentiated insulin and glucagon-producing cells from human mesenchymal stem cells derived from adipose tissue

Background: In type I diabetes mellitus (T1DM) pancreatic beta cells are destroyed. Treatment entails exogenous insulin administration and strict diet control, yet optimal glycemic control is hardly attainable. Islet transplant could be an alternative in patients with poor glycemic control, but inefficient islet purification and autoimmune response of patients is still a challenge. For these reasons, it is necessary to explore new cellular sources and immunological isolation methods oriented to develop T1DM cell-based therapies. Aims: We postulate human adipose-derived stem cell (hASC) as an adequate source to generate pancreatic islet cells in vitro, and to produce islet-like structures. Furthermore, we propose microencapsulation of these aggregates as an immunological isolation strategy. Methods hASC obtained from lipoaspirated fat tissue from human donors were differentiated in vitro to insulin (Ins) and glucagon (Gcg) producing cells. Then, insulin producing cells (IPC) and glucagon producing cells (GPC) were cocultured in low adhesion conditions to form cellular aggregates, and later encapsulated in a sodium alginate polymer. Expression of pancreatic lineage markers and secretion of insulin or glucagon in vitro were analyzed. Results: The results show that multipotent hASC efficiently differentiate to IPC and GPC, and express pancreatic markers, including insulin or glucagon hormones which they secrete upon stimulation (fivefold for insulin in IPC, and fourfold for glucagon, compared to undifferentiated cells). In turn, calculation of the Feret diameter and area of cellular aggregates revealed mean diameters of similar to 80 mu m, and 65% of the aggregates reached 4000 mu m(2) at 72 h of formation. IPC/GPC aggregates were then microencapsulated in sodium-alginate polymer microgels, which were found to be more stable when stabilized with Ba2+, yielding average diameters of similar to 300 mu m. Interestingly, Ba2+-microencapsulated aggregates respond to high external glucose with insulin secretion.Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) 21110401 Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) CONICYT FONDECYT 1161450 1201899 ANID/PIA/ACT192144 1130444 1130241 FONDEQUIP EQM160157 EQM170111 Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) CONICYT FONDAP 15130011 Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) ICM-ECONOMIA, Chile P09-022-F CeBiB FB000

Myofibers deficient in connexins 43 and 45 expression protect mice from skeletal muscle and systemic dysfunction promoted by a dysferlin mutation

Dysferlinopathy is a genetic human disease caused by mutations in the gene that encodes the dysferlin protein (DYSF). Dysferlin is believed to play a relevant role in cell membrane repair. However, in dysferlin-deficient (blAJ) mice (a model of dysferlinopathies) the recovery of the membrane resealing function by means of the expression of a mini-dysferlin does not arrest progressive muscular damage, suggesting the participation of other unknown pathogenic mechanisms. Here, we show that proteins called connexins 39, 43 and 45 (Cx39, Cx43 and Cx45, respectively) are expressed by blAJ myofibers and form functional hemichannels (Cx HCs) in the sarcolemma. At rest, Cx HCs increased the sarcolemma permeability to small molecules and the intracellular Ca2+ signal. In addition, skeletal muscles of blAJ mice showed lipid accumulation and lack of dysferlin immunoreactivity. As sign of extensive damage and atrophy, muscles of blAJ mice presented elevated numbers of myofibers with internal nuclei, increased number of myofibers with reduced cross-sectional area and elevated creatine kinase activity in serum. In agreement with the extense muscle damage, mice also showed significantly low motor performance. We generated blAJ mice with myofibers deficient in Cx43 and Cx45 expression and found that all above muscle and systemic alterations were absent, indicating that these two Cxs play a critical role in a novel pathogenic mechanism of dysfernolophaties, which is discussed herein. Therefore, Cx HCs could constitute an attractive target for pharmacologic treatment of dyferlinopathies.Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) CONICYT FONDECYT 11160739 1150291 1192329 Rings grant ACT 1121 Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) CONICYT PIA/BASAL CeBiB FB0001 ICM-ECONOMIA, Chile P09-022-

Proteome Profiling and Ultrastructural Characterization of the Human RCMH Cell Line: Myoblastic Properties and Suitability for Myopathological Studies

Artículo de publicación ISIStudying (neuro)muscular disorders is a major topic in biomedicine with a demand for suitable model systems. Continuous cell culture (in vitro) systems have several technical advantages over in vivo systems and became widely used tools for discovering physiological/ pathophysiological mechanisms in muscle. In particular, myoblast cell lines are suitable model systems to study complex biochemical adaptations occurring in skeletal muscle and cellular responses to altered genetic/ environmental conditions. Whereas most in vitro studies use extensively characterized murine C2C12 cells, a comprehensive description of an equivalent human cell line, not genetically manipulated for immortalization, is lacking. Therefore, we characterized human immortal myoblastic RCMH cells using scanning (SEM) and transmission electron microscopy (TEM) and proteomics. Among more than 6200 identified proteins we confirm the known expression of proteins important for muscle function. Comparing the RCMH proteome with two well-defined nonskeletal muscle cells lines (HeLa, U2OS) revealed a considerable enrichment of proteins important for muscle function. SEM/TEM confirmed the presence of agglomerates of cytoskeletal components/intermediate filaments and a prominent rough ER. In conclusion, our results indicate RMCH as a suitable in vitro model for investigating muscle function-related processes such as mechanical stress burden and mechanotransduction, EC coupling, cytoskeleton, muscle cell metabolism and development, and (ER-associated) myopathic disorders.Ministerium fur Innovation, Wissenschaft and Forschung des Landes Nordrhein-Westfale

A Second Order Multi-Stencil Fast Marching Method with a Non-Constant Local Cost Model

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