21 research outputs found
Muscle satellite cell proliferation and association: new insights from myofiber time-lapse imaging
<p>Abstract</p> <p>Background</p> <p>As the resident stem cells of skeletal muscle, satellite cells are activated by extracellular cues associated with local damage. Once activated, satellite cells will re-enter the cell cycle to proliferate and supply a population of myoblasts, which will repair or replace damaged myofibers by differentiating and fusing either with an existing myofiber or with each other. There is also evidence that the orientation of cell division with respect to the myofiber may indicate or convey asymmetry in the two daughter cells. Our recent studies with time-lapse imaging of myofiber-associated satellite cells <it>in vitro </it>have yielded new data on the timing and orientation of satellite cell divisions, and revealed persistent differences in the behavior of daughter cells from planar versus vertical divisions.</p> <p>Results</p> <p>We analyzed 244 individual fiber-associated satellite cells in time-lapse video from 24 to 48 hours after myofiber harvest. We found that initial cell division in fiber culture is not synchronous, although presumably all cells were activated by the initial trauma of harvest; that cell cycling time is significantly shorter than previously thought (as short as 4.8 hours, averaging 10 hours between the first and second divisions and eight hours between the second and third); and that timing of subsequent divisions is not strongly correlated with timing of the initial division. Approximately 65% of first and 80% of second cell divisions occur parallel to the axis of the myofiber, whereas the remainder occur outside the plane of the fiber surface (vertical division). We previously demonstrated that daughter cells frequently remain associated with each other after division or reassociate after a brief separation, and that unrelated cells may also associate for significant periods of time. We show in this paper that daughter cells resulting from a vertical division remain associated with one another several times longer than do daughters from a horizontal division. However, the total average time of association between sister cells is not significantly different from the total average time of association between unrelated cells.</p> <p>Conclusions</p> <p>These longitudinal characterizations of satellite cell behavior shortly after activation provide new insights into cell proliferation and association as a function of relatedness, and indicate significant and consistent heterogeneity within the population based on these metrics.</p
Cell culture, sex determination and single cell cloning of ovine transgenic satellite cells in vitro
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A role for RNA post-transcriptional regulation in satellite cell activation
Background: Satellite cells are resident skeletal muscle stem cells responsible for muscle maintenance and repair. In resting muscle, satellite cells are maintained in a quiescent state. Satellite cell activation induces the myogenic commitment factor, MyoD, and cell cycle entry to facilitate transition to a population of proliferating myoblasts that eventually exit the cycle and regenerate muscle tissue. The molecular mechanism involved in the transition of a quiescent satellite cell to a transit-amplifying myoblast is poorly understood. Methods: Satellite cells isolated by FACS from uninjured skeletal muscle and 12 h post-muscle injury from wild type and Syndecan-4 null mice were probed using Affymetrix 430v2 gene chips and analyzed by Spotfiretmand Ingenuity Pathway analysis to identify gene expression changes and networks associated with satellite cell activation, respectively. Additional analyses of target genes identify miRNAs exhibiting dynamic changes in expression during satellite cell activation. The function of the miRNAs was assessed using miRIDIAN hairpin inhibitors.Results: An unbiased gene expression screen identified over 4,000 genes differentially expressed in satellite cells in vivo within 12 h following muscle damage and more than 50% of these decrease dramatically. RNA binding proteins and genes involved in post-transcriptional regulation were significantly over-represented whereas splicing factors were preferentially downregulated and mRNA stability genes preferentially upregulated. Furthermore, six computationally identified miRNAs demonstrated novel expression through muscle regeneration and in satellite cells. Three of the six miRNAs were found to regulate satellite cell fate.Conclusions: The quiescent satellite cell is actively maintained in a state poised to activate in response to external signals. Satellite cell activation appears to be regulated by post-transcriptional gene regulation. © 2012 Farina et al.; licensee BioMed Central Ltd
A role for RNA post-transcriptional regulation in satellite cell activation
Abstract Background Satellite cells are resident skeletal muscle stem cells responsible for muscle maintenance and repair. In resting muscle, satellite cells are maintained in a quiescent state. Satellite cell activation induces the myogenic commitment factor, MyoD, and cell cycle entry to facilitate transition to a population of proliferating myoblasts that eventually exit the cycle and regenerate muscle tissue. The molecular mechanism involved in the transition of a quiescent satellite cell to a transit-amplifying myoblast is poorly understood. Methods Satellite cells isolated by FACS from uninjured skeletal muscle and 12 h post-muscle injury from wild type and Syndecan-4 null mice were probed using Affymetrix 430v2 gene chips and analyzed by Spotfiretm and Ingenuity Pathway analysis to identify gene expression changes and networks associated with satellite cell activation, respectively. Additional analyses of target genes identify miRNAs exhibiting dynamic changes in expression during satellite cell activation. The function of the miRNAs was assessed using miRIDIAN hairpin inhibitors. Results An unbiased gene expression screen identified over 4,000 genes differentially expressed in satellite cells in vivo within 12 h following muscle damage and more than 50% of these decrease dramatically. RNA binding proteins and genes involved in post-transcriptional regulation were significantly over-represented whereas splicing factors were preferentially downregulated and mRNA stability genes preferentially upregulated. Furthermore, six computationally identified miRNAs demonstrated novel expression through muscle regeneration and in satellite cells. Three of the six miRNAs were found to regulate satellite cell fate. Conclusions The quiescent satellite cell is actively maintained in a state poised to activate in response to external signals. Satellite cell activation appears to be regulated by post-transcriptional gene regulation
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Muscle spindle alterations precede onset of sensorimotor deficits in Charcot-Marie-Tooth type 2E.
Charcot-Marie-Tooth (CMT) is the most common inherited peripheral neuropathy, affecting approximately 2.8 million people. The CMT leads to distal neuropathy that is characterized by reduced motor nerve conduction velocity, ataxia, muscle atrophy and sensory loss. We generated a mouse model of CMT type 2E (CMT2E) expressing human neurofilament light E396K (hNF-LE396K ), which develops decreased motor nerve conduction velocity, ataxia and muscle atrophy by 4 months of age. Symptomatic hNF-LE396K mice developed phenotypes that were consistent with proprioceptive sensory defects as well as reduced sensitivity to mechanical stimulation, while thermal sensitivity and auditory brainstem responses were unaltered. Progression from presymptomatic to symptomatic included a 50% loss of large diameter sensory axons within the fifth lumbar dorsal root of hNF-LE396K mice. Owing to proprioceptive deficits and loss of large diameter sensory axons, we analyzed muscle spindle morphology in presymptomatic and symptomatic hNF-LE396K and hNF-L control mice. Muscle spindle cross-sectional area and volume were reduced in all hNF-LE396K mice analyzed, suggesting that alterations in muscle spindle morphology occurred prior to the onset of typical CMT pathology. These data suggested that CMT2E pathology initiated in the muscle spindles altering the proprioceptive sensory system. Early sensory pathology in CMT2E could provide a unifying hypothesis for the convergence of pathology observed in CMT
Natação e aspectos morfológicos do músculo esquelético em processo de reparo após criolesão Swimming and morphology of skeletal muscle repair process after cryoinjury
O objetivo do estudo foi investigar a influência da natação sobre as alterações morfológicas do músculo esquelético em processo de reparo após criolesão. Foram usados 45 ratos divididos em cinco grupos: controle (n=5); sham (n=5), adaptação (n=5), criolesionados e tratados com natação sacrificados após 7, 14 e 21 dias (n=15); criolesionados e sem tratamento aquático sacrificados após 7, 14 e 21 dias (n=15). As sessões de natação foram realizadas 6 vezes por semana com 90 min de duração cada. Ao término do protocolo os animais foram sacrificados e a análise morfológica da área da lesão foi realizada. A análise morfológica semiquantitativa demonstrou que os músculos do grupo controle apresentaram aspecto histológico normal. O grupo sham apresentou edema, mionecrose e infiltrado inflamatório em grau 1. Nos grupos 7, 14 e 21 dias, não existiram diferenças estatisticamente significativas nas 4 etapas de remodelamento tecidual avaliadas (infiltrado inflamatório, edema, necrose e fibras musculares imaturas) entre os grupos lesionados quando comparados aos grupos com lesão e tratamento aquático. Em conclusão, foi possível verificar que a natação não causou alterações morfológicas durante o reparo do músculo esquelético após criolesão.<br>The aim of study was investigate the influence of swimming on the morphological changes in skeletal muscle repair process following cryoinjury. There were used 45 rats divided into 5 groups: control (n=5), sham (n=5), adaptation (n=5), cryolesioned treated with swimming and sacrificed after 7, 14 and 21 days (n=15), untreated and cryolesioned sacrificed after 7, 14, and 21 days (n=15). Animals swan for 90 min/ each session and 6 times a week. At the end of the protocol, the animals were sacrificed and morphological analysis of the lesion area was performed. The semi-quantitative morphological analysis showed that the muscles in the control group exhibited normal histological aspects while the sham group exhibited edema, myonecrosis and inflammatory infiltrate grade 1. In groups 7, 14, and 21 days, the results indicated that there were no statistically significant differences in four stages of tissue remodeling evaluated (inflammatory infiltration, edema, necrosis, and immature muscle fibers) between the injured groups compared to groups with lesion and treated with swimming. In conclusion, it was verified that swimming did not alter morphological aspects of skeletal muscle during the repair process following cryoinjury