The Experts below are selected from a list of 87 Experts worldwide ranked by ideXlab platform
Buel D. Rodgers - One of the best experts on this subject based on the ideXlab platform.
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1 MYOSTATIN INHIBITS Myosatellite Cell PROLIFERATION AND CONSEQUENTLY 1 ACTIVATES DIFFERENTIATION: EVIDENCE FOR ENDOCRINE-REGULATED 2 TRANSCRIPT PROCESSING 3
2016Co-Authors: Dilip K Garikipati, Buel D. Rodgers, Ph. DAbstract:Abstract 24 Myostatin is a potent negative regulator of muscle growth in mammals. Despite high 25 structural conservation, functional conservation in non-mammalian species is only assumed. 26 This is particularly true for the fishes due to the presence of several myostatin paralogs; two in 27 most species and four in salmonids (MSTN-1a,-1b,-2a,-2b). Rainbow trout are a rich source 28 of primary Myosatellite Cells as hyperplastic muscle growth occurs even in adult fish. These 29 Cells were therefore used to determine myostatin’s effects on proliferation whereas our earlier 30 studies reported its effects on quiescent Cells. As in mammals, recombinant myostatin sup-31 pressed proliferation with no changes in Cell morphology. Expression of MSTN-1a was several 32 fold higher than the other paralogs and was autoregulated by myostatin, which also upregulated 33 the expression of key differentiation markers: Myf5, MyoD1, myogenin and myosin light chain. 34 Thus, myostatin-stimulated Cellular growth inhibition activates rather than represses differentia-35 tion. IGF-I stimulated proliferation, but had minimal and delayed effects on differentiation and its 36 actions were suppressed by myostatin. However, IGF-I upregulated MSTN-2a expression and 37 the processing of its transcript, which is normally unprocessed. Myostatin therefore appears to 3
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Myostatin stimulates, not inihibits, C2C12 myoblast proliferation.
Endocrinology, 2014Co-Authors: Buel D. Rodgers, Benjamin D. Wiedeback, Knut E. Hoversten, Melissa F. Jackson, Ryan G. Walker, Thomas B. ThompsonAbstract:The immortal C2C12 Cell line originates from dystrophic mouse thigh muscle and has been used to study the endocrine control of muscle Cell growth, development, and function, including those actions regulated by myostatin. Previous studies suggest that high concentrations of recombinant myostatin generated in bacteria inhibit C2C12 proliferation and differentiation. Recombinant myostatin generated in eukaryotic systems similarly inhibits the proliferation of primary Myosatellite Cells, but consequently initiates, rather than inhibits, their differentiation and is bioactive at far lower concentrations. Our studies indicate that 2 different sources of recombinant myostatin made in eukaryotes stimulate, not inhibit, C2C12 proliferation. This effect occurred at different Cell densities and serum concentrations and in the presence of IGF-I, a potent myoblast mitogen. This stimulatory effect was comparable to that obtained with TGFβ1, a related factor that also inhibits primary Myosatellite Cell proliferation. Attenuating the myostatin/activin (ie, Acvr2b) and TGFβ1 receptor signaling pathways with the Alk4/5 and Alk5 inhibitors, SB431542 and SB505142, respectively, similarly attenuated proliferation induced by serum, myostatin or TGFβ1 and in a dose-dependent manner. In serum-free medium, both myostatin and TGFβ1 stimulated Smad2 phosphorylation, but not that of Smad3, and a Smad3 inhibitor (SIS3) only inhibited proliferation in Cells cultured in high serum. Thus, myostatin and TGFβ1 stimulate C2C12 proliferation primarily via Smad2. These results together question the physiological relevance of the C2C12 model and previous studies using recombinant myostatin generated in bacteria. They also support the alternative use of primary Myosatellite Cells and recombinant myostatin generated in eukaryotes.
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Genetic manipulation of myoblasts and a novel primary Myosatellite Cell culture system: comparing and optimizing approaches.
The FEBS journal, 2013Co-Authors: Melissa F. Jackson, Knut E. Hoversten, John M. Powers, Grant D. Trobridge, Buel D. RodgersAbstract:The genetic manipulation of skeletal muscle Cells in vitro is notoriously difficult, especially when using undifferentiated muscle Cell lines (myoblasts) or primary muscle stem Cells (Myosatellites). We therefore optimized methods of gene transfer by overexpressing green fluorescent protein (GFP) in mouse C2C12 Cells and in a novel system, primary rainbow trout Myosatellite Cells. A common lipid-based transfection reagent was used (Lipofectamine 2000) along with three different viral vectors: adeno-associated virus serotype 2 (AAV2), baculovirus (BAC) and lentivirus. Maximal transfection efficiencies of 49% were obtained in C2C12 Cells after optimizing Cell density and reagent : DNA ratio, although the GFP signal rapidly dissipated with proliferation and was not maintained with differentiation. The transduction efficiency of AAV2 was optimized to 65% by extending incubation time and decreasing Cell density, although only 30% of Cells retained expression after passing. A viral comparison revealed that lentivirus was most efficient at transducing C2C12 myoblasts as 97% of Cells were transduced with only 10(6) viral genomes (vg) compared to 54% with 10(8) vg AAV2 and 23% with 10(9) vg BAC. Lentivirus also transduced 90% of primary trout Myosatellites compared to 1-10% with AAV2 and BAC. The phosphoglycerate kinase 1 (pgk) promoter was 10-fold more active than the cytomegalovirus immediate-early promoter in C2C12 Cells and both were effective in trout Myosatellites. Maximal transduction of C2C12 myotubes was achieved by differentiating myoblasts previously transduced with lentivirus and the pgk promoter. Thus, our optimized protocol proved highly effective in diverse muscle Cell systems and could therefore help overcome a common technological barrier.
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Myostatin inhibits Myosatellite Cell proliferation and consequently activates differentiation: evidence for endocrine-regulated transcript processing
The Journal of endocrinology, 2012Co-Authors: Dilip K Garikipati, Buel D. RodgersAbstract:Myostatin is a potent negative regulator of muscle growth in mammals. Despite high structural conservation, functional conservation in nonmammalian species is only assumed. This is particularly true for fish due to the presence of several myostatin paralogs: two in most species and four in salmonids (MSTN-1a, -1b, -2a, and -2b). Rainbow trout are a rich source of primary Myosatellite Cells as hyperplastic muscle growth occurs even in adult fish. These Cells were therefore used to determine myostatin's effects on proliferation whereas our earlier studies reported its effects on quiescent Cells. As in mammals, recombinant myostatin suppressed proliferation with no changes in Cell morphology. Expression of MSTN-1a was several fold higher than the other paralogs and was autoregulated by myostatin, which also upregulated the expression of key differentiation markers: Myf5, MyoD1, myogenin, and myosin light chain. Thus, myostatin-stimulated Cellular growth inhibition activates rather than represses differentiation. IGF-1 stimulated proliferation but had minimal and delayed effects on differentiation and its actions were suppressed by myostatin. However, IGF-1 upregulated MSTN-2a expression and the processing of its transcript, which is normally unprocessed. Myostatin therefore appears to partly mediate IGF-stimulated Myosatellite differentiation in rainbow trout. This also occurs in mammals, although the IGF-stimulated processing of MSTN-2a transcripts is highly unique and is indicative of subfunctionalization within the gene family. These studies also suggest that the myokine's actions, including its antagonistic relationship with IGF-1, are conserved and that the salmonid gene family is functionally diverging.
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myostatin stimulates Myosatellite Cell differentiation in a novel model system evidence for gene subfunctionalization
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2012Co-Authors: Dilip K Garikipati, Buel D. RodgersAbstract:Myosatellite Cells play an important role in mammalian muscle regeneration as they differentiate and fuse with mature fibers. In fish, they also contribute to postnatal growth and the formation of ...
Dilip K Garikipati - One of the best experts on this subject based on the ideXlab platform.
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1 MYOSTATIN INHIBITS Myosatellite Cell PROLIFERATION AND CONSEQUENTLY 1 ACTIVATES DIFFERENTIATION: EVIDENCE FOR ENDOCRINE-REGULATED 2 TRANSCRIPT PROCESSING 3
2016Co-Authors: Dilip K Garikipati, Buel D. Rodgers, Ph. DAbstract:Abstract 24 Myostatin is a potent negative regulator of muscle growth in mammals. Despite high 25 structural conservation, functional conservation in non-mammalian species is only assumed. 26 This is particularly true for the fishes due to the presence of several myostatin paralogs; two in 27 most species and four in salmonids (MSTN-1a,-1b,-2a,-2b). Rainbow trout are a rich source 28 of primary Myosatellite Cells as hyperplastic muscle growth occurs even in adult fish. These 29 Cells were therefore used to determine myostatin’s effects on proliferation whereas our earlier 30 studies reported its effects on quiescent Cells. As in mammals, recombinant myostatin sup-31 pressed proliferation with no changes in Cell morphology. Expression of MSTN-1a was several 32 fold higher than the other paralogs and was autoregulated by myostatin, which also upregulated 33 the expression of key differentiation markers: Myf5, MyoD1, myogenin and myosin light chain. 34 Thus, myostatin-stimulated Cellular growth inhibition activates rather than represses differentia-35 tion. IGF-I stimulated proliferation, but had minimal and delayed effects on differentiation and its 36 actions were suppressed by myostatin. However, IGF-I upregulated MSTN-2a expression and 37 the processing of its transcript, which is normally unprocessed. Myostatin therefore appears to 3
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Myostatin inhibits Myosatellite Cell proliferation and consequently activates differentiation: evidence for endocrine-regulated transcript processing
The Journal of endocrinology, 2012Co-Authors: Dilip K Garikipati, Buel D. RodgersAbstract:Myostatin is a potent negative regulator of muscle growth in mammals. Despite high structural conservation, functional conservation in nonmammalian species is only assumed. This is particularly true for fish due to the presence of several myostatin paralogs: two in most species and four in salmonids (MSTN-1a, -1b, -2a, and -2b). Rainbow trout are a rich source of primary Myosatellite Cells as hyperplastic muscle growth occurs even in adult fish. These Cells were therefore used to determine myostatin's effects on proliferation whereas our earlier studies reported its effects on quiescent Cells. As in mammals, recombinant myostatin suppressed proliferation with no changes in Cell morphology. Expression of MSTN-1a was several fold higher than the other paralogs and was autoregulated by myostatin, which also upregulated the expression of key differentiation markers: Myf5, MyoD1, myogenin, and myosin light chain. Thus, myostatin-stimulated Cellular growth inhibition activates rather than represses differentiation. IGF-1 stimulated proliferation but had minimal and delayed effects on differentiation and its actions were suppressed by myostatin. However, IGF-1 upregulated MSTN-2a expression and the processing of its transcript, which is normally unprocessed. Myostatin therefore appears to partly mediate IGF-stimulated Myosatellite differentiation in rainbow trout. This also occurs in mammals, although the IGF-stimulated processing of MSTN-2a transcripts is highly unique and is indicative of subfunctionalization within the gene family. These studies also suggest that the myokine's actions, including its antagonistic relationship with IGF-1, are conserved and that the salmonid gene family is functionally diverging.
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myostatin stimulates Myosatellite Cell differentiation in a novel model system evidence for gene subfunctionalization
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2012Co-Authors: Dilip K Garikipati, Buel D. RodgersAbstract:Myosatellite Cells play an important role in mammalian muscle regeneration as they differentiate and fuse with mature fibers. In fish, they also contribute to postnatal growth and the formation of ...
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Myostatin stimulates Myosatellite Cell differentiation in a novel model system: evidence for gene subfunctionalization
American journal of physiology. Regulatory integrative and comparative physiology, 2012Co-Authors: Dilip K Garikipati, Buel D. RodgersAbstract:Myosatellite Cells play an important role in mammalian muscle regeneration as they differentiate and fuse with mature fibers. In fish, they also contribute to postnatal growth and the formation of new fibers. The relative conservation of fish systems, however, is not well known nor are the underlying mechanisms that control Myosatellite Cell differentiation. We therefore characterized this process in primary Cells from rainbow trout and determined the effects of two known regulators in mammalian systems: IGF-I and myostatin. Unlike mammalian Cell lines, subconfluent and proliferating trout Myosatellite Cells differentiated spontaneously and at a rate proportional to serum concentration. The expression of key myogenic markers (Myf5, MyoD1, myogenin, and MLC) and of the different myostatin paralogs (MSTN-1a/1b/2a) increased with serum-stimulated differentiation, although MSTN-1a expression was consistently higher than that of the other paralogs. In addition, MSTN-2a was only expressed as an unprocessed transcript. In low serum, where differentiation is normally suppressed, recombinant myostatin stimulated myogenic marker expression over time. The opposite was true for IGF-I as it stimulated proliferation, not differentiation, and additionally antagonized myostatin. This includes myostatin's effects on marker expression and on the autoregulation of MSTN-1a and -1b expression. These results conflict with studies using mammalian Cell lines and suggest, alternatively, that myostatin is a positive, not negative, regulator of Myosatellite Cell differentiation. Mammalian myoblasts differentiate when confluent and with serum withdrawal, which differs considerably from how Myosatellite Cells differentiate in vivo. Thus the primary rainbow trout Myosatellite Cell culture system appears to be more physiologically relevant.
Judy E. Anderson - One of the best experts on this subject based on the ideXlab platform.
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Studies of the dynamics of skeletal muscle regeneration: the mouse came back!
Biochemistry and cell biology = Biochimie et biologie cellulaire, 1998Co-Authors: Judy E. AndersonAbstract:Regeneration of skeletal muscle tissue includes sequential processes of muscle Cell proliferation and commitment, Cell fusion, muscle fiber differentiation, and communication between Cells of various tissues of origin. Central to the process is the Myosatellite Cell, a quiescent precursor Cell located between the mature muscle fiber and its sheath of external lamina. To form new fibers in a muscle damaged by disease or direct injury, satellite Cells must be activated, proliferate, and subsequently fuse into an elongated multinucleated Cell. Current investigations in the field concern modulation of the effectiveness of skeletal muscle regeneration, the regeneration-specific role of myogenic regulatory gene expression distinct from expression during development, the impact of growth and scatter factors and their respective receptors in amplifying precursor numbers, and promoting fusion and maturation of new fibers and the ultimate clinical therapeutic applications of such information to alleviate disease. One approach to muscle regeneration integrates observations of muscle gene expression, proliferation, myoblast fusion, and fiber growth in vivo with parallel studies of Cell cycling behaviour, endocrine perturbation, and potential biochemical markers of steps in the disease-repair process detected by magnetic resonance spectroscopy techniques. Experiments on muscles from limb, diaphragm, and heart of the mdx dystrophic mouse, made to parallel clinical trials on human Duchenne muscular dystrophy, help to elucidate mechanisms underlying the positive treatment effects of the glucocorticoid drug deflazacort. This review illustrates an effective combination of in vivo and in vitro experiments to integrate the distinctive complexities of post-natal myogenesis in regeneration of skeletal muscle tissue.
Jorge M. O. Fernandes - One of the best experts on this subject based on the ideXlab platform.
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In vitro and ex vivo models indicate that the molecular clock in fast skeletal muscle of Atlantic cod is not autonomous
Molecular Biology Reports, 2014Co-Authors: Carlo C. Lazado, Hiruni P. S. Kumaratunga, Kazue Nagasawa, Igor Babiak, Christopher Marlowe A. Caipang, Jorge M. O. FernandesAbstract:The notion that the circadian rhythm is exclusively regulated by a central clock has been challenged by the discovery of peripheral oscillators. These peripheral clocks are known to have a direct influence on the biological processes in a tissue or Cell. In fish, several peripheral clocks respond directly to light, thus raising the hypothesis of autonomous regulation. Several clock genes are expressed with daily rhythmicity in Atlantic cod ( Gadus morhua ) fast skeletal muscle. In the present study, Myosatellite Cell culture and short-term cultured fast skeletal muscle explant models were developed and characterized, in order to investigate the autonomy of the clock system in skeletal muscle of Atlantic cod. Myosatellite Cells proliferated and differentiated in vitro, as shown by the changes in Cellular and myogenic gene markers. The high expression of myogenic differentiation 1 during the early days post-isolation implied the commitment to myogenic lineage and the increasing mRNA levels of proliferating Cell nuclear antigen ( pcna ) indicated the proliferation of the Cells in vitro. Transcript levels of myogenic marker genes such as pcna and myogenin increased during 5 days in culture of skeletal muscle explants, indicating that the muscle Cells were proliferating and differentiating under ex vivo conditions. Transcript levels of the clock gene aryl hydrocarbon receptor nuclear translocator - like 2 ( arntl2 ) in Myosatellite Cells showed no daily oscillation regardless of photoperiod manipulation. On the other hand, mRNA levels of the clock gene circadian locomotor output cycles kaput ( clock ) showed circadian rhythmicity in 5-day-old skeletal muscle explant under different photoperiod regimes. The expression of arntl2 , cryptochrome2 ( cry2 ), period 2a ( per2a ) and nuclear receptor subfamily 1, group D, member 1 was not rhythmic in muscle explants but photoperiod manipulation had a significant effect on mRNA levels of cry2 and per2a . Taken together, the lack of rhythmicity of molecular clocks in vitro and ex vivo indicate that the putative peripheral clock in Atlantic cod fast skeletal muscle is not likely to be autonomous.
Melissa F. Jackson - One of the best experts on this subject based on the ideXlab platform.
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Myostatin stimulates, not inihibits, C2C12 myoblast proliferation.
Endocrinology, 2014Co-Authors: Buel D. Rodgers, Benjamin D. Wiedeback, Knut E. Hoversten, Melissa F. Jackson, Ryan G. Walker, Thomas B. ThompsonAbstract:The immortal C2C12 Cell line originates from dystrophic mouse thigh muscle and has been used to study the endocrine control of muscle Cell growth, development, and function, including those actions regulated by myostatin. Previous studies suggest that high concentrations of recombinant myostatin generated in bacteria inhibit C2C12 proliferation and differentiation. Recombinant myostatin generated in eukaryotic systems similarly inhibits the proliferation of primary Myosatellite Cells, but consequently initiates, rather than inhibits, their differentiation and is bioactive at far lower concentrations. Our studies indicate that 2 different sources of recombinant myostatin made in eukaryotes stimulate, not inhibit, C2C12 proliferation. This effect occurred at different Cell densities and serum concentrations and in the presence of IGF-I, a potent myoblast mitogen. This stimulatory effect was comparable to that obtained with TGFβ1, a related factor that also inhibits primary Myosatellite Cell proliferation. Attenuating the myostatin/activin (ie, Acvr2b) and TGFβ1 receptor signaling pathways with the Alk4/5 and Alk5 inhibitors, SB431542 and SB505142, respectively, similarly attenuated proliferation induced by serum, myostatin or TGFβ1 and in a dose-dependent manner. In serum-free medium, both myostatin and TGFβ1 stimulated Smad2 phosphorylation, but not that of Smad3, and a Smad3 inhibitor (SIS3) only inhibited proliferation in Cells cultured in high serum. Thus, myostatin and TGFβ1 stimulate C2C12 proliferation primarily via Smad2. These results together question the physiological relevance of the C2C12 model and previous studies using recombinant myostatin generated in bacteria. They also support the alternative use of primary Myosatellite Cells and recombinant myostatin generated in eukaryotes.
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Genetic manipulation of myoblasts and a novel primary Myosatellite Cell culture system: comparing and optimizing approaches.
The FEBS journal, 2013Co-Authors: Melissa F. Jackson, Knut E. Hoversten, John M. Powers, Grant D. Trobridge, Buel D. RodgersAbstract:The genetic manipulation of skeletal muscle Cells in vitro is notoriously difficult, especially when using undifferentiated muscle Cell lines (myoblasts) or primary muscle stem Cells (Myosatellites). We therefore optimized methods of gene transfer by overexpressing green fluorescent protein (GFP) in mouse C2C12 Cells and in a novel system, primary rainbow trout Myosatellite Cells. A common lipid-based transfection reagent was used (Lipofectamine 2000) along with three different viral vectors: adeno-associated virus serotype 2 (AAV2), baculovirus (BAC) and lentivirus. Maximal transfection efficiencies of 49% were obtained in C2C12 Cells after optimizing Cell density and reagent : DNA ratio, although the GFP signal rapidly dissipated with proliferation and was not maintained with differentiation. The transduction efficiency of AAV2 was optimized to 65% by extending incubation time and decreasing Cell density, although only 30% of Cells retained expression after passing. A viral comparison revealed that lentivirus was most efficient at transducing C2C12 myoblasts as 97% of Cells were transduced with only 10(6) viral genomes (vg) compared to 54% with 10(8) vg AAV2 and 23% with 10(9) vg BAC. Lentivirus also transduced 90% of primary trout Myosatellites compared to 1-10% with AAV2 and BAC. The phosphoglycerate kinase 1 (pgk) promoter was 10-fold more active than the cytomegalovirus immediate-early promoter in C2C12 Cells and both were effective in trout Myosatellites. Maximal transduction of C2C12 myotubes was achieved by differentiating myoblasts previously transduced with lentivirus and the pgk promoter. Thus, our optimized protocol proved highly effective in diverse muscle Cell systems and could therefore help overcome a common technological barrier.
