Ronald E Allen

Interests

Teaching

Animal Physiology

Research

Skeletal muscle stem cells in domestic animals and man and their role in muscle growth, repair and disease.

Courses

Scholarly Contributions

Journals/Publications

• Dodson, M. V., Allen, R. E., Du, M., Bergen, W. G., Velleman, S. G., Poulos, S. P., Fernyhough-Culver, M., Wheeler, M. B., Duckett, S. K., Young, M. R., Voy, B. H., Jiang, Z., & Hausman, G. J. (2015). INVITED REVIEW: Evolution of meat animal growth research during the past 50 years: Adipose and muscle stem cells. Journal of animal science, 93(2), 457-81.
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  If one were to compare today's animal growth research to research from a mere 50 yr ago, one would see programs with few similarities. The evolution of this research from whole-animal through cell-based and finally molecular and genomic studies has been enhanced by the identification, isolation, and in vitro evaluation of adipose- and muscle-derived stem cells. This paper will highlight the struggles and the milestones that make this evolving area of research what it is today. The contribution of adipose and muscle stem cell research to development and growth, tissue regeneration, and final carcass composition are reviewed.
• Flann, K. L., Rathbone, C. R., Cole, L. C., Liu, X., Allen, R. E., & Rhoads, R. P. (2014). Hypoxia simultaneously alters satellite cell-mediated angiogenesis and hepatocyte growth factor expression. Journal of cellular physiology, 229(5).
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  Skeletal muscle regeneration is a multifaceted process requiring the spatial and temporal coordination of myogenesis as well as angiogenesis. Hepatocyte growth factor (HGF) plays a pivotal role in myogenesis by activating satellite cells (SC) in regenerating muscle and likely plays a role as a contributor to revascularization. Moreover, repair of a functional blood supply is critical to ameliorate tissue ischemia and restore skeletal muscle function, however effects of hypoxia on satellite cell-mediated angiogenesis remain unclear. The objective of this study was to examine the role of HGF and effect of hypoxia on the capacity of satellite cells to promote angiogenesis. To characterize the role of HGF, a microvascular fragment (MVF) culture model coupled with satellite cell conditioned media (CM) was employed. The activity of HGF was specifically blocked in SC CM reducing sprout length compared to control CM. In contrast, MVF sprout number did not differ between control or HGF-deficient SC CM media. Next, we cultured MVF in the presence of CM from satellite cells exposed to normoxic (20% O2 ) or hypoxic (1% O2 ) conditions. Hypoxic CM recapitulated a MVF angiogenic response identical to HGF deficient satellite cell CM. Hypoxic conditions increased satellite cell HIF-1α protein abundance and VEGF mRNA abundance but decreased HGF mRNA abundance compared to normoxic satellite cells. Consistent with reduced HGF gene expression, HGF promoter activity decreased during hypoxia. Taken together, this data indicates that hypoxic modulation of satellite cell-mediated angiogenesis involves a reduction in satellite cell HGF expression. J. Cell. Physiol. 229: 572-579, 2014. © 2013 Wiley Periodicals, Inc.
• Pilia, M., McDaniel, J. S., Guda, T., Chen, X. K., Rhoads, R. P., Allen, R. E., Corona, B. T., & Rathbone, C. R. (2014). Transplantation and perfusion of microvascular fragments in a rodent model of volumetric muscle loss injury. European cells & materials, 28, 11-23; discussion 23-4.
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  Few clinical options are available for the treatment of volumetric muscle loss (VML). An important consideration that needs to be addressed for the development of treatments for these injuries is the establishment of a vascular supply sufficient to support skeletal muscle regeneration. The objective of the current study was to evaluate the potential for microvascular fragments (MVFs) harvested from adipose tissue to support tissue perfusion for VML. Tibialis anterior muscle defects in rats were replaced with constructs that were created on the day of surgery containing either (1) collagen only (COL), (2) freshly isolated microvascular fragments in collagen (MVF), or (3) adipose tissue derived stem cells (ASCs) in collagen. Muscles were harvested 7 and 14 days after surgery. Defects treated with MVFs had a vessel density higher than the other groups at both 7 and 14 days, and those treated with ASCs had a higher vessel density than COL by day 14 (p < 0.05). Perfused vessels were observed in both the ASC and MVF treated defects at day 14, as well as at day 7 in the MVF. This study supports the use of MVFs as a platform to improve tissue perfusion to treat large VML defects. The use of freshly isolated MVFs on the day of surgery supports their clinical use and application.
• Yates, D. T., Clarke, D. S., Macko, A. R., Anderson, M. J., Shelton, L. A., Nearing, M., Allen, R. E., Rhoads, R. P., & Limesand, S. W. (2014). Myoblasts from intrauterine growth-restricted sheep fetuses exhibit intrinsic deficiencies in proliferation that contribute to smaller semitendinosus myofibres. The Journal of physiology, 592(Pt 14), 3113-25.
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  Intrauterine growth restriction (IUGR) reduces skeletal muscle mass in fetuses and offspring. Our objective was to determine whether myoblast dysfunction due to intrinsic cellular deficiencies or serum factors reduces myofibre hypertrophy in IUGR fetal sheep. At 134 days, IUGR fetuses weighed 67% less (P < 0.05) than controls and had smaller (P < 0.05) carcasses and semitendinosus myofibre areas. IUGR semitendinosus muscles had similar percentages of pax7-positive nuclei and pax7 mRNA but lower (P < 0.05) percentages of myogenin-positive nuclei (7 ± 2% and 13 ± 2%), less myoD and myogenin mRNA, and fewer (P < 0.05) proliferating myoblasts (PNCA-positive-pax7-positive) than controls (44 ± 2% vs. 52 ± 1%). Primary myoblasts were isolated from hindlimb muscles, and after 3 days in growth media (20% fetal bovine serum, FBS), myoblasts from IUGR fetuses had 34% fewer (P < 0.05) myoD-positive cells than controls and replicated 20% less (P < 0.05) during a 2 h BrdU pulse. IUGR myoblasts also replicated less (P < 0.05) than controls during a BrdU pulse after 3 days in media containing 10% control or IUGR fetal sheep serum (FSS). Both myoblast types replicated less (P < 0.05) with IUGR FSS-supplemented media compared to control FSS-supplemented media. In differentiation-promoting media (2% FBS), IUGR and control myoblasts had similar percentages of myogenin-positive nuclei after 5 days and formed similar-sized myotubes after 7 days. We conclude that intrinsic cellular deficiencies in IUGR myoblasts and factors in IUGR serum diminish myoblast proliferation and myofibre size in IUGR fetuses, but intrinsic myoblast deficiencies do not affect differentiation. Furthermore, the persistent reduction in IUGR myoblast replication shows adaptive deficiencies that explain poor muscle growth in IUGR newborn offspring.
• Elster, J. L., Rathbone, C. R., Liu, Z., Liu, X., Barrett, H. H., Rhoads, R. P., & Allen, R. E. (2013). Skeletal muscle satellite cell migration to injured tissue measured with 111In-oxine and high-resolution SPECT imaging. Journal of muscle research and cell motility, 34(5-6).
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  The delivery of adult skeletal muscle stem cells, called satellite cells, to several injured muscles via the circulation would be useful, however, an improved understanding of cell fate and biodistribution following their delivery is important for this goal to be achieved. The objective of this study was to evaluate the ability of systemically delivered satellite cells to home to injured skeletal muscle using single-photon emission computed tomography (SPECT) imaging of (111)In-labeled satellite cells. Satellite cells labeled with (111)In-oxine and green fluorescent protein (GFP) were injected intravenously after bupivicaine-induced injury to the tibialis anterior muscle. Animals were imaged with a high-resolution SPECT system called FastSPECT II for up to 7 days after transplantation. In vivo FastSPECT II imaging demonstrated a three to five-fold greater number of transplanted satellite cells in bupivicaine-injured muscle as compared to un-injured muscle after transplantation; a finding that was verified through autoradiograph analysis and quantification of GFP expression. Satellite cells also accumulated in other organs including the lung, liver, and spleen, as determined by biodistribution measurements. These data support the ability of satellite cells to home to injured muscle and support the use of SPECT and autoradiograph imaging techniques to track systemically transplanted (111)In labeled satellite cells in vivo, and suggest their homing may be improved by reducing their entrapment in filter organs.
• Elster, J. L., Rathbone, C. R., Liu, Z., Liu, X., Barrett, H. H., Rhoads, R. P., & Allen, R. E. (2013). Skeletal muscle satellite cell migration to injured tissue measured with¹¹¹In-oxine and high-resolution SPECT imaging. Journal of Muscle Research and Cell Motility, 34(5-6), 417-427.
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  Abstract: The delivery of adult skeletal muscle stem cells, called satellite cells, to several injured muscles via the circulation would be useful, however, an improved understanding of cell fate and biodistribution following their delivery is important for this goal to be achieved. The objective of this study was to evaluate the ability of systemically delivered satellite cells to home to injured skeletal muscle using single-photon emission computed tomography (SPECT) imaging of 111In-labeled satellite cells. Satellite cells labeled with 111In-oxine and green fluorescent protein (GFP) were injected intravenously after bupivicaine-induced injury to the tibialis anterior muscle. Animals were imaged with a high-resolution SPECT system called FastSPECT II for up to 7 days after transplantation. In vivo FastSPECT II imaging demonstrated a three to five-fold greater number of transplanted satellite cells in bupivicaine-injured muscle as compared to un-injured muscle after transplantation; a finding that was verified through autoradiograph analysis and quantification of GFP expression. Satellite cells also accumulated in other organs including the lung, liver, and spleen, as determined by biodistribution measurements. These data support the ability of satellite cells to home to injured muscle and support the use of SPECT and autoradiograph imaging techniques to track systemically transplanted 111In labeled satellite cells in vivo, and suggest their homing may be improved by reducing their entrapment in filter organs. © 2013 Springer Science+Business Media Dordrecht.
• George, R. M., Biressi, S., Beres, B. J., Rogers, E., Mulia, A. K., Allen, R. E., Rawls, A., Rando, T. A., & Wilson-Rawls, J. (2013). Numb-deficient satellite cells have regeneration and proliferation defects. Proceedings of the National Academy of Sciences of the United States of America, 110(46), 18549-18555.
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  PMID: 24170859;PMCID: PMC3831958;Abstract: The adaptor protein Numb has been implicated in the switch between cell proliferation and differentiation made by satellite cells during muscle repair. Using two genetic approaches to ablate Numb, we determined that, in its absence, muscle regeneration in response to injury was impaired. Single myofiber cultures demonstrated a lack of satellite cell proliferation in the absence of Numb, and the proliferation defect was confirmed in satellite cell cultures. Quantitative RT-PCR from Numb-deficient satellite cells demonstrated highly up-regulated expression of p21 and Myostatin, both inhibitors of myoblast proliferation. Transfection with Myostatin-specific siRNA rescued the proliferation defect of Numb-deficient satellite cells. Furthermore, overexpression of Numb in satellite cells inhibited Myostatin expression. These data indicate a unique function for Numb during the initial activation and proliferation of satellite cells in response to muscle injury.
• George, R. M., Biressi, S., Beres, B. J., Rogers, E., Mulia, A. K., Allen, R. E., Rawls, A., Rando, T. A., & Wilson-Rawls, J. (2013). Numb-deficient satellite cells have regeneration and proliferation defects. Proceedings of the National Academy of Sciences of the United States of America, 110(46).
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  The adaptor protein Numb has been implicated in the switch between cell proliferation and differentiation made by satellite cells during muscle repair. Using two genetic approaches to ablate Numb, we determined that, in its absence, muscle regeneration in response to injury was impaired. Single myofiber cultures demonstrated a lack of satellite cell proliferation in the absence of Numb, and the proliferation defect was confirmed in satellite cell cultures. Quantitative RT-PCR from Numb-deficient satellite cells demonstrated highly up-regulated expression of p21 and Myostatin, both inhibitors of myoblast proliferation. Transfection with Myostatin-specific siRNA rescued the proliferation defect of Numb-deficient satellite cells. Furthermore, overexpression of Numb in satellite cells inhibited Myostatin expression. These data indicate a unique function for Numb during the initial activation and proliferation of satellite cells in response to muscle injury.
• Penrod, L. V., Allen, R. E., Rhoads, M. L., Limesand, S. W., & Arns, M. J. (2013). Oxytocin stimulated release of PGF2α and its inhibition by a cyclooxygenase inhibitor and an oxytocin receptor antagonist from equine endometrial cultures. Animal reproduction science, 139(1-4).
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  Uterine inflammation results in a poor uterine environment and early embryonic loss in the mare due to an inhibition of maternal recognition of pregnancy caused from increased prostaglandin F2α (PGF2α). Oxytocin binds to endometrial cell receptors to activate prostaglandin synthesis. An oxytocin receptor antagonist (Atosiban) and a cyclooxygenase inhibitor (indomethacin) both decrease PGF2α production. The aim of this study was to evaluate the in vitro effects of Atosiban and indomethacin on equine uterine prostaglandin secretion. Equine endometrial explants were harvested on day two of behavioral estrus. Endometrial explant cultures were challenged with oxytocin (250nM) and PGF2α concentrations were measured over time. Explants were also cultured with Atosiban and indomethacin for 6h to determine the influence on PGF2α secretion. When endometrial explants were challenged with oxytocin, PGF2α concentrations were greater (P
• Penrod, L. V., Allen, R. E., Rhoads, M. L., Limesand, S. W., & Arns, M. J. (2013). Oxytocin stimulated release of PGF₂α and its inhibition by a cyclooxygenase inhibitor and an oxytocin receptor antagonist from equine endometrial cultures. Animal Reproduction Science, 139(1-4), 69-75.
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  PMID: 23664650;Abstract: Uterine inflammation results in a poor uterine environment and early embryonic loss in the mare due to an inhibition of maternal recognition of pregnancy caused from increased prostaglandin F2α (PGF2α). Oxytocin binds to endometrial cell receptors to activate prostaglandin synthesis. An oxytocin receptor antagonist (Atosiban) and a cyclooxygenase inhibitor (indomethacin) both decrease PGF2α production. The aim of this study was to evaluate the in vitro effects of Atosiban and indomethacin on equine uterine prostaglandin secretion. Equine endometrial explants were harvested on day two of behavioral estrus. Endometrial explant cultures were challenged with oxytocin (250nM) and PGF2α concentrations were measured over time. Explants were also cultured with Atosiban and indomethacin for 6h to determine the influence on PGF2α secretion. When endometrial explants were challenged with oxytocin, PGF2α concentrations were greater (P
• Penrod, L. V., Allen, R. E., Turner, J. L., Limesand, S. W., & Arns, M. J. (2013). Effects of oxytocin, lipopolysaccharide (LPS), and polyunsaturated fatty acids on prostaglandin secretion and gene expression in equine endometrial explant cultures. Domestic Animal Endocrinology, 44(1), 46-55.
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  PMID: 23063410;Abstract: Increased secretion of prostaglandin F2α (PGF2α) within the uterus because of uterine inflammation can cause luteolysis and result in early embryonic loss. Supplementation with polyunsaturated fatty acids (PUFAs) has been shown to influence PG production in many species, although the effects on the mare remain unknown. The present study aimed to determine fatty acid uptake in equine endometrial explants and evaluate their influence on PG secretion and expression of enzymes involved in PG synthesis in vitro. Equine endometrial explants were treated with 100 M arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid and then challenged with oxytocin (250 nM) or lipopolysaccharide (LPS; 1g/mL). Production of F2α and PG E2 (PGE2) was measured, and mRNA expression of enzymes involved in PG synthesis was determined with quantitative real-time PCR. Media concentrations of F2α and PGE2 were higher (P
• Penrod, L. V., Allen, R. E., Turner, J. L., Limesand, S. W., & Arns, M. J. (2013). Effects of oxytocin, lipopolysaccharide (LPS), and polyunsaturated fatty acids on prostaglandin secretion and gene expression in equine endometrial explant cultures. Domestic animal endocrinology, 44(1).
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  Increased secretion of prostaglandin F(2)α (PGF(2)α) within the uterus because of uterine inflammation can cause luteolysis and result in early embryonic loss. Supplementation with polyunsaturated fatty acids (PUFAs) has been shown to influence PG production in many species, although the effects on the mare remain unknown. The present study aimed to determine fatty acid uptake in equine endometrial explants and evaluate their influence on PG secretion and expression of enzymes involved in PG synthesis in vitro. Equine endometrial explants were treated with 100 μM arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid and then challenged with oxytocin (250 nM) or lipopolysaccharide (LPS; 1 μg/mL). Production of PGF(2)α and PG E(2) (PGE(2)) was measured, and mRNA expression of enzymes involved in PG synthesis was determined with quantitative real-time PCR. Media concentrations of PGF(2)α and PGE(2) were higher (P < 0.0001) from endometrial explants challenged with oxytocin or LPS compared with controls despite which fatty acid was added. Only DHA lowered (P < 0.0001) media concentrations of PGF(2)α and PGE(2) from explants. Endometrial explants stimulated with oxytocin had increased expression of PG-endoperoxide synthase 1 (PTGS1; P < 0.02), PG-endoperoxide synthase 2 (PTGS2; P < 0.001), PG F(2)α synthase (PGFS; P < 0.01), PG E(2) synthase (PGES; P < 0.01), and phospholipase A(2) (PLA(2); P < 0.005) compared with controls and regardless of fatty acid treatment; whereas stimulation with LPS increased expression of PTGS2 (P < 0.004), PGFS (P < 0.03), PGES (P < 0.01), and PLA(2) (P < 0.01) compared with controls and regardless of fatty acid treatment. Treatment with PUFAs, specifically DHA, can influence PG secretion in vitro through mechanisms other than enzyme expression.
• Rhoads, R. P., Flann, K. L., Cardinal, T. R., Rathbone, C. R., Liu, X., & Allen, R. E. (2013). Satellite cells isolated from aged or dystrophic muscle exhibit a reduced capacity to promote angiogenesis in vitro. Biochemical and Biophysical Research Communications, 440(3), 399-404.
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  PMID: 24070607;Abstract: Deficits in skeletal muscle function exist during aging and muscular dystrophy, and suboptimal function has been related to factors such as atrophy, excessive inflammation and fibrosis. Ineffective muscle regeneration underlies each condition and has been attributed to a deficit in myogenic potential of resident stem cells or satellite cells. In addition to reduced myogenic activity, satellite cells may also lose the ability to communicate with vascular cells for coordination of myogenesis and angiogenesis and restoration of proper muscle function. Objectives of the current study were to determine the angiogenic-promoting capacity of satellite cells from two states characterized by dysfunctional skeletal muscle repair, aging and Duchenne muscular dystrophy. An in vitro culture model composed of satellite cells or their conditioned media and rat adipose tissue microvascular fragments (MVF) was used to examine this relationship. Microvascular fragments cultured in the presence of rat satellite cells from adult muscle donors (9-12. month of age) exhibited greater indices of angiogenesis (endothelial cell sprouting, tubule formation and extensive branching) than MVF co-cultured with satellite cells from aged muscle donors (24. month of age). We sought to determine if the differential degree of angiogenesis we observed in the co-culture setting was due to soluble factors produced by each satellite cell age group. Similar to the co-culture experiment, conditioned media produced by adult satellite cells promoted greater angiogenesis than that of aged satellite cells. Next, we examined differences in angiogenesis-stimulating ability of satellite cells from 12 mo old MDX mice or age-matched wild-type mice. A reduction in angiogenesis activity of media conditioned by satellite cells from dystrophic muscle was observed as compared to healthy muscle. Finally, we found reduced gene expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in both aged and dystrophic satellite cells compared to their adult and normal counterparts, respectively. These results indicate that functional deficits in satellite cell activities during aging and diseased muscle may extend to their ability to communicate with other cells in their environment, in this case cells involved in angiogenesis. © 2013 The Authors.
• Rhoads, R. P., Flann, K. L., Cardinal, T. R., Rathbone, C. R., Liu, X., & Allen, R. E. (2013). Satellite cells isolated from aged or dystrophic muscle exhibit a reduced capacity to promote angiogenesis in vitro. Biochemical and biophysical research communications, 440(3).
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  Deficits in skeletal muscle function exist during aging and muscular dystrophy, and suboptimal function has been related to factors such as atrophy, excessive inflammation and fibrosis. Ineffective muscle regeneration underlies each condition and has been attributed to a deficit in myogenic potential of resident stem cells or satellite cells. In addition to reduced myogenic activity, satellite cells may also lose the ability to communicate with vascular cells for coordination of myogenesis and angiogenesis and restoration of proper muscle function. Objectives of the current study were to determine the angiogenic-promoting capacity of satellite cells from two states characterized by dysfunctional skeletal muscle repair, aging and Duchenne muscular dystrophy. An in vitro culture model composed of satellite cells or their conditioned media and rat adipose tissue microvascular fragments (MVF) was used to examine this relationship. Microvascular fragments cultured in the presence of rat satellite cells from adult muscle donors (9-12 month of age) exhibited greater indices of angiogenesis (endothelial cell sprouting, tubule formation and extensive branching) than MVF co-cultured with satellite cells from aged muscle donors (24 month of age). We sought to determine if the differential degree of angiogenesis we observed in the co-culture setting was due to soluble factors produced by each satellite cell age group. Similar to the co-culture experiment, conditioned media produced by adult satellite cells promoted greater angiogenesis than that of aged satellite cells. Next, we examined differences in angiogenesis-stimulating ability of satellite cells from 12 mo old MDX mice or age-matched wild-type mice. A reduction in angiogenesis activity of media conditioned by satellite cells from dystrophic muscle was observed as compared to healthy muscle. Finally, we found reduced gene expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in both aged and dystrophic satellite cells compared to their adult and normal counterparts, respectively. These results indicate that functional deficits in satellite cell activities during aging and diseased muscle may extend to their ability to communicate with other cells in their environment, in this case cells involved in angiogenesis.
• Hara, M., Tabata, K., Suzuki, T., Do, M. Q., Mizunoya, W., Nakamura, M., Nishimura, S., Tabata, S., Ikeuchi, Y., Sunagawa, K., Anderson, J. E., Allen, R. E., & Tatsumi, R. (2012). Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch. American Journal of Physiology - Cell Physiology, 302(12), C1741-C1750.
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  PMID: 22460715;Abstract: When skeletal muscle is stretched or injured, satellite cells, resident myogenic stem cells positioned beneath the basal lamina of mature muscle fibers, are activated to enter the cell cycle. This signaling pathway is a cascade of events including calcium-calmodulin formation, nitric oxide (NO) radical production by NO synthase, matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the receptor c-met, as demonstrated by assays of primary cultures and in vivo experiments. Here, we add evidence that two ion channels, the mechanosensitive cation channel (MS channel) and the long-lasting-type voltage-gated calcium-ion channel (L-VGC channel), mediate the influx of extracellular calcium ions in response to cyclic stretch in satellite cell cultures. When applied to 1-h stretch cultures with individual inhibitors for MS and L-VGC channels (GsMTx-4 and nifedipine, respectively) or with a less specific inhibitor (gadolinium chloride, Gd), satellite cell activation and upstream HGF release were abolished, as revealed by bromodeoxyuridine-incorporation assays and Western blotting of conditioned media, respectively. The inhibition was dose dependent with a maximum at 0.1 μM (GsMTx4), 10 μM (nifedipine), or 100 μM (Gd) and canceled by addition of HGF to the culture media; a potent inhibitor for transient-type VGC channels (NNC55-0396,100 μM) did not show any significant inhibitory effect. The stretch response was also abolished when calcium-chelator EGTA (1.8 mM) was added to the medium, indicating the significance of extracellular free calcium ions in our present activation model. Finally, cation/calcium channel dependencies were further documented by calcium-imaging analyses on stretched cells; results clearly demonstrated that calcium ion influx was abolished by GsMTx4, nifedipine, and EGTA. Therefore, these results provide an additional insight that calcium ions may flow in through L-VGC channels by possible coupling with adjacent MS channel gating that promotes the local depolarization of cell membranes to initiate the satellite cell activation cascade. © 2012 the American Physiological Society.
• Hara, M., Tabata, K., Suzuki, T., Do, M. Q., Mizunoya, W., Nakamura, M., Nishimura, S., Tabata, S., Ikeuchi, Y., Sunagawa, K., Anderson, J. E., Allen, R. E., & Tatsumi, R. (2012). Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch. American journal of physiology. Cell physiology, 302(12), C1741-50.
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  When skeletal muscle is stretched or injured, satellite cells, resident myogenic stem cells positioned beneath the basal lamina of mature muscle fibers, are activated to enter the cell cycle. This signaling pathway is a cascade of events including calcium-calmodulin formation, nitric oxide (NO) radical production by NO synthase, matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the receptor c-met, as demonstrated by assays of primary cultures and in vivo experiments. Here, we add evidence that two ion channels, the mechanosensitive cation channel (MS channel) and the long-lasting-type voltage-gated calcium-ion channel (L-VGC channel), mediate the influx of extracellular calcium ions in response to cyclic stretch in satellite cell cultures. When applied to 1-h stretch cultures with individual inhibitors for MS and L-VGC channels (GsMTx-4 and nifedipine, respectively) or with a less specific inhibitor (gadolinium chloride, Gd), satellite cell activation and upstream HGF release were abolished, as revealed by bromodeoxyuridine-incorporation assays and Western blotting of conditioned media, respectively. The inhibition was dose dependent with a maximum at 0.1 μM (GsMTx-4), 10 μM (nifedipine), or 100 μM (Gd) and canceled by addition of HGF to the culture media; a potent inhibitor for transient-type VGC channels (NNC55-0396, 100 μM) did not show any significant inhibitory effect. The stretch response was also abolished when calcium-chelator EGTA (1.8 mM) was added to the medium, indicating the significance of extracellular free calcium ions in our present activation model. Finally, cation/calcium channel dependencies were further documented by calcium-imaging analyses on stretched cells; results clearly demonstrated that calcium ion influx was abolished by GsMTx-4, nifedipine, and EGTA. Therefore, these results provide an additional insight that calcium ions may flow in through L-VGC channels by possible coupling with adjacent MS channel gating that promotes the local depolarization of cell membranes to initiate the satellite cell activation cascade.
• Rathbone, C. R., Yamanouchi, K., Chen, X. K., Nevoret-Bell, C. J., Rhoads, R. P., & Allen, R. E. (2011). Effects of transforming growth factor-beta (TGF-β1) on satellite cell activation and survival during oxidative stress. Journal of Muscle Research and Cell Motility, 32(2), 99-109.
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  PMID: 21823037;Abstract: The regulation of adult skeletal muscle repair and regeneration is largely due to the contribution of resident adult myogenic precursor cells called satellite cells. The events preceding their participation in muscle repair include activation (exit from quiescence), proliferation, and differentiation. This study examined the effects of transforming growth factor-beta (TGF-β1) on satellite cell activation, determined whether TGF-β1 could maintain quiescence in the presence of hepatocyte growth factor (HGF), and whether the regulation of satellite cell activation with TGF-β1 improves the ability of satellite cells to withstand oxidative stress. The addition of TGF-β1 during early satellite cell activation (0-48 h) or during the proliferative phase (48-96 h) maintained and induced satellite cell quiescence, respectively, as determined by myogenic differentiation (MyoD) protein expression. TGF-β1 also attenuated satellite cell activation when used with HGF. Finally, the role of quiescence in protecting cells against oxidative stress was examined. TGF-β1 treatment and the low pH satellite cell preparation procedure, a technique that forestalls spontaneous activation in vitro, both enhanced survival of cultured satellite cells following hydrogen peroxide treatment. These findings indicate that TGF-β1 is capable of maintaining and inducing satellite cell quiescence and suggest methods to maintain satellite cell quiescence may improve their transplantation efficiency. © 2011 Springer Science+Business Media B.V. (outside the USA).
• Rathbone, C. R., Yamanouchi, K., Chen, X. K., Nevoret-Bell, C. J., Rhoads, R. P., & Allen, R. E. (2011). Effects of transforming growth factor-beta (TGF-β1) on satellite cell activation and survival during oxidative stress. Journal of muscle research and cell motility, 32(2), 99-109.
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  The regulation of adult skeletal muscle repair and regeneration is largely due to the contribution of resident adult myogenic precursor cells called satellite cells. The events preceding their participation in muscle repair include activation (exit from quiescence), proliferation, and differentiation. This study examined the effects of transforming growth factor-beta (TGF-β1) on satellite cell activation, determined whether TGF-β1 could maintain quiescence in the presence of hepatocyte growth factor (HGF), and whether the regulation of satellite cell activation with TGF-β1 improves the ability of satellite cells to withstand oxidative stress. The addition of TGF-β1 during early satellite cell activation (0-48 h) or during the proliferative phase (48-96 h) maintained and induced satellite cell quiescence, respectively, as determined by myogenic differentiation (MyoD) protein expression. TGF-β1 also attenuated satellite cell activation when used with HGF. Finally, the role of quiescence in protecting cells against oxidative stress was examined. TGF-β1 treatment and the low pH satellite cell preparation procedure, a technique that forestalls spontaneous activation in vitro, both enhanced survival of cultured satellite cells following hydrogen peroxide treatment. These findings indicate that TGF-β1 is capable of maintaining and inducing satellite cell quiescence and suggest methods to maintain satellite cell quiescence may improve their transplantation efficiency.
• Rathbone, C., Yamanouchi, K., Chen, X., Nevoret-Bell, C., Rhoads, ., & Allen, R. (2011). J Muscle Res Cell Motil, 32(2), 99-109.
• Yamada, M., Tatsumi, R., Yamanouchi, K., Hosoyama, T., Shiratsuchi, S., Sato, A., Mizunoya, W., Ikeuchi, Y., Furuse, M., & Allen, R. E. (2010). High concentrations of HGF inhibit skeletal muscle satellite cell proliferation in vitro by inducing expression of myostatin: a possible mechanism for reestablishing satellite cell quiescence in vivo. American journal of physiology. Cell physiology, 298(3), C465-76.
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  Skeletal muscle regeneration and work-induced hypertrophy rely on molecular events responsible for activation and quiescence of resident myogenic stem cells, satellite cells. Recent studies demonstrated that hepatocyte growth factor (HGF) triggers activation and entry into the cell cycle in response to mechanical perturbation, and that subsequent expression of myostatin may signal a return to cell quiescence. However, mechanisms responsible for coordinating expression of myostatin after an appropriate time lag following activation and proliferation are not clear. Here we address the possible role of HGF in quiescence through its concentration-dependent negative-feedback mechanism following satellite cell activation and proliferation. When activated/proliferating satellite cell cultures were treated for 24 h beginning 48-h postplating with 10-500 ng/ml HGF, the percentage of bromodeoxyuridine-incorporating cells decreased down to a baseline level comparable to 24-h control cultures in a HGF dose-dependent manner. The high level HGF treatment did not impair the cell viability and differentiation levels, and cells could be reactivated by lowering HGF concentrations to 2.5 ng/ml, a concentration that has been shown to optimally stimulate activation of satellite cells in culture. Coaddition of antimyostatin neutralizing antibody could prevent deactivation and abolish upregulation of cyclin-dependent kinase (Cdk) inhibitor p21. Myostatin mRNA expression was upregulated with high concentrations of HGF, as demonstrated by RT-PCR, and enhanced myostatin protein expression and secretion were revealed by Western blots of the cell lysates and conditioned media. These results indicate that HGF could induce satellite cell quiescence by stimulating myostatin expression. The HGF concentration required (over 10-50 ng/ml), however, is much higher than that for activation, which is initiated by rapid release of HGF from its extracellular association. Considering that HGF is produced by satellite cells and spleen and liver cells in response to muscle damage, local concentrations of HGF bathing satellite cells may reach a threshold sufficient to induce myostatin expression. This time lag may delay action of the quiescence signaling program in proliferating satellite cells during initial phases of muscle regeneration followed by induction of quiescence in a subset of cells during later phases.
• Yamada, M., Tatsumi, R., Yamanouchi, T. K., Hosoyama, O., Shiratsuchi, S., Sato, A., Mizunoya, W., Ikeuchi, Y., Furuse, M., & Allen, R. E. (2010). High concentrations of HGF inhibit skeletal muscle satellite cell proliferation in vitro by inducing expression of myostatin: A possible mechanism for reestablishing satellite cell quiescence in vivo. American Journal of Physiology - Cell Physiology, 298(3), C465-C476.
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  PMID: 20007454;PMCID: PMC2838568;Abstract: Skeletal muscle regeneration and work-induced hypertrophy rely on molecular events responsible for activation and quiescence of resident myogenic stem cells, satellite cells. Recent studies demonstrated that hepatocyte growth factor (HGF) triggers activation and entry into the cell cycle in response to mechanical perturbation, and that subsequent expression of myostatin may signal a return to cell quiescence. However, mechanisms responsible for coordinating expression of myostatin after an appropriate time lag following activation and proliferation are not clear. Here we address the possible role of HGF in quiescence through its concentration-dependent negative-feedback mechanism following satellite cell activation and proliferation. When activated/ proliferating satellite cell cultures were treated for 24 h beginning 48-h postplating with 10-500 ng/ml HGF, the percentage of bromodeoxyuridineincorporating cells decreased down to a baseline level comparable to 24-h control cultures in a HGF dose-dependent manner. The high level HGF treatment did not impair the cell viability and differentiation levels, and cells could be reactivated by lowering HGF concentrations to 2.5 ng/ml, a concentration that has been shown to optimally stimulate activation of satellite cells in culture. Coaddition of antimyostatin neutralizing antibody could prevent deactivation and abolish upregulation of cyclin-dependent kinase (Cdk) inhibitor p21. Myostatin mRNA expression was upregulated with high concentrations of HGF, as demonstrated by RT-PCR, and enhanced myostatin protein expression and secretion were revealed by Western blots of the cell lysates and conditioned media. These results indicate that HGF could induce satellite cell quiescence by stimulating myostatin expression. The HGF concentration required (over 10-50 ng/ml), however, is much higher than that for activation, which is initiated by rapid release of HGF from its extracellular association. Considering that HGF is produced by satellite cells and spleen and liver cells in response to muscle damage, local concentrations of HGF bathing satellite cells may reach a threshold sufficient to induce myostatin expression. This time lag may delay action of the quiescence signaling program in proliferating satellite cells during initial phases of muscle regeneration followed by induction of quiescence in a subset of cells during later phases. Copyright © 2010 American Physiological Society.
• Rhoads, R. P., Johnson, R. M., Rathbone, C. R., Liu, X., Temm-Grove, C., Sheehan, S. M., Hoying, J. B., & Allen, R. E. (2009). Satellite cell-mediated angiogenesis in vitro coincides with a functional hypoxia-inducible factor pathway. American Journal of Physiology - Cell Physiology, 296(6), C1321-C1328.
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  PMID: 19386789;PMCID: PMC2692418;Abstract: Muscle regeneration involves the coordination of myogenesis and revascularization to restore proper muscle function. Myogenesis is driven by resident stem cells termed satellite cells (SC), whereas angiogenesis arises from endothelial cells and perivascular cells of preexisting vascular segments and the collateral vasculature. Communication between myogenic and angiogenic cells seems plausible, especially given the number of growth factors produced by SC. To characterize these interactions, we developed an in vitro coculture model composed of rat skeletal muscle SC and microvascular fragments (MVF). In this system, isolated epididymal MVF suspended in collagen gel are cultured over a rat SC monolayer culture. In the presence of SC, MVF exhibit greater indices of angiogenesis than MVF cultured alone. A positive dose-dependent effect of SC conditioned medium (CM) on MVF growth was observed, suggesting that SC secrete soluble-acting growth factor(s). Next, we specifically blocked VEGF action in SC CM, and this was sufficient to abolish satellite cell-induced angiogenesis. Finally, hypoxia-inducible factor-1α (HIF-1α), a transcriptional regulator of VEGF gene expression, was found to be expressed in cultured SC and in putative SC in sections of in vivo stretch-injured rat muscle. Hypoxic culture conditions increased SC HIF-1α activity, which was positively associated with SC VEGF gene expression and protein levels. Collectively, these initial observations suggest that a heretofore unexplored aspect of satellite cell physiology is the initiation of a proangiogenic program. Copyright © 2009 the American Physiological Society.
• Tatsumi, R., Sankoda, Y., Anderson, J. E., Sato, Y., Mizunoya, W., Shimizu, N., Suzuki, T., Yamada, M., Rhoads Jr., R. P., Ikeuchi, Y., & Allen, R. E. (2009). Possible implication of satellite cells in regenerative motoneuritogenesis: HGF upregulates neural chemorepellent Sema3A during myogenic differentiation. American Journal of Physiology - Cell Physiology, 297(2), C238-C252.
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  PMID: 19515904;Abstract: Regenerative coordination and remodeling of the intramuscular motoneuron network and neuromuscular connections are critical for restoring skeletal muscle function and physiological properties. The regulatory mechanisms of such coordination remain unclear, although both attractive and repulsive axon guidance molecules may be involved in the signaling pathway. Here we show that expression of a neural secreted chemorepellent semaphorin 3A (Sema3A) is remarkably upregulated in satellite cells of resident myogenic stem cells that are positioned beneath the basal lamina of mature muscle fibers, when treated with hepatocyte growth factor (HGF), established as an essential cue in muscle fiber growth and regeneration. When satellite cells were treated with HGF in primary cultures of cells or muscle fibers, Sema3A message and protein were upregulated as revealed by reverse transcriptionpolymerase chain reaction and immunochemical studies. Other growth factors had no inductive effect except for a slight effect of epidermal growth factor treatment. Sema3A upregulation was HGF dose dependent with a maximum (about 7- to 8-fold units relative to the control) at 10-25 ng/ml and occurred exclusively at the early-differentiation stage, as characterized by the level of myogenin expression and proliferation (bromodeoxyuridine incorporation) of the cells. Neutralizing antibody to the HGF-specific receptor, c-met, did not abolish the HGF response, indicating that c-met may not mediate the Sema3A expression signaling. Finally, in vivo Sema3A was upregulated in the differentiation phase of satellite cells isolated from muscle regenerating following crush injury. Overall, the data highlight a heretofore unexplored and active role for satellite cells as a key source of Sema3A expression triggered by HGF, hence suggesting that regenerative activity toward motor innervation may importantly reside in satellite cells and could be a crucial contributor during postnatal myogenesis. Copyright © 2009 the American Physiological Society.
• Tatsumi, R., Wuollet, A. L., Tabata, K., Nishimura, S., Tabata, S., Mizunoya, W., Ikeuchi, Y., & Allen, R. E. (2009). A role for calcium-calmodulin in regulating nitric oxide production during skeletal muscle satellite cell activation. American Journal of Physiology - Cell Physiology, 296(4), C922-C929.
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  PMID: 19158401;Abstract: When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production by NO synthase (NOS), matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the c-met receptor as demonstrated by a primary culture and in vivo assays. We now add evidence that calcium-calmodulin is involved in the satellite cell activation cascade in vitro. Conditioned medium from cultures that were treated with a calcium ionophore (A23187, ionomycin) for 2 h activated cultured satellite cells and contained active HGF, similar to the effect of mechanical stretch or NO donor treatments. The response was abolished by addition of calmodulin inhibitors (calmidazolium, W-13, W-12) or a NOS inhibitor N G-nitro-L-arginine methyl ester hydrochloride but not by its less inactive enantiomer NG-nitro-D-arginine methyl ester hydrochloride. Satellite cells were also shown to express functional calmodulin protein having a calcium-binding activity at 12 h postplating, which is the time at which the calcium ionophore was added in this study and the stretch treatment was applied in our previous experiments. Therefore, results from these experiments provide an additional insight that calcium-calmodulin mediates HGF release from the matrix and that this step in the activation pathway is upstream from NO synthesis. Copyright © 2009 the American Physiological Society.
• Tatsumi, R., & Allen, R. E. (2008). Mechano-biology of resident myogenic stem cells: Molecular mechanism of stretch-induced activation of satellite cells. Animal Science Journal, 79(3), 279-290.
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  Abstract: Satellite cells, resident myogenic stem cells found between the basement membrane and the sarcolemma in postnatal skeletal muscle, are normally quiescent in adult muscles. But when muscle is injured, exercised, overused or mechanically stretched, these cells are activated to enter the cell cycle, divide, differentiate, and fuse with the adjacent muscle fiber. In this way, satellite cells are responsible for regeneration and work-induced hypertrophy of muscle fibers. Therefore, a mechanism must exist to translate mechanical changes in muscle tissue into chemical signals that can activate satellite cells. This mechanism has not been clearly delineated. Recent in vivo studies and studies of satellite cells and single muscle fibers in culture demonstrated the essential role of hepatocyte growth factor (HGF) and nitric oxide (NO) radical in the activation pathway. These experiments also showed that mechanically stretching cultured satellite cells or living skeletal muscles stimulates satellite cell activation. This is achieved by rapid release of HGF from its tethering in the extracellular matrix and its presentation to the c-met receptor. HGF release has been shown to depend on NO radical production by nitric oxide synthase (NOS) in satellite cells and/or muscle fibers, and relies on the subsequent upregulation of matrix metalloproteinase (MMP) activity (possibly achieved by its nitrosylation). These results suggest that the activation mechanism is a cascade of molecular events including calcium-calmodulin formation, NOS activation, NO radical production, MMP activation, HGF release and HGF binding to c-met. An understanding the 'mechano-biology' of satellite cell activation is essential when planning procedures that could enhance muscle growth and repair. This is particularly important for meat-animal agriculture and in human health, disease and aging. © 2008 Japanese Society of Animal Science.
• Yamada, M., Sankoda, Y., Tatsumi, R., Mizunoya, W., Ikeuchi, Y., Sunagawa, K., & Allen, R. E. (2008). Matrix metalloproteinase-2 mediates stretch-induced activation of skeletal muscle satellite cells in a nitric oxide-dependent manner. International Journal of Biochemistry and Cell Biology, 40(10), 2183-2191.
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  PMID: 18403250;Abstract: When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the c-met receptor. Matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, mediate HGF release from the matrix and this step in the pathway is downstream from NO synthesis [Yamada, M., Tatsumi, R., Kikuiri, T., Okamoto, S., Nonoshita, S., Mizunoya, W., et al. (2006). Matrix metalloproteinases are involved in mechanical stretch-induced activation of skeletal muscle satellite cells. Muscle Nerve, 34, 313-319]. Experiments reported herein provide evidence that MMP2 may be involved in the NO-dependent release of HGF in vitro. Whole lysate analyses of satellite cells demonstrated the presence of MMP2 mRNA and the protein. When rat satellite cells were treated with 30 μM sodium nitroprusside a NO donor or mechanical cyclic stretch for 2 h period, inactive proMMP2 (72 kDa) was converted into 52-kDa form and this processing was abolished by adding a NO synthase inhibitor l-NAME (10 μM) to the stretch culture. The 52-kDa species was also generated by treatment of the recombinant MMP2 protein with 1 μM NOC-7 that can spontaneously release NO under physiological conditions without any cofactor, and its activating activity was demonstrated by applying the NOC-7-treated MMP2 to satellite cell culture. HGF release was detected in NOC-7-MMP2-conditioned media by western blotting; very little HGF was found in media that were generated from cultures receiving NOC-7-treated MMP2 (10 ng/ml) plus 250 ng/ml tissue inhibitor-1 of metalloproteinases. Therefore, results from these experiments provide evidence that NO-activated MMP2 may cause release of HGF from the extracellular matrix of satellite cells and contribute to satellite cell activation. © 2008 Elsevier Ltd. All rights reserved.
• Meeson, A. P., Shi, X., Alexander, M. S., Williams, R. S., Allen, R. E., Jiang, N., Adham, I. M., Goetsch, S. C., Hammer, R. E., & Garry, D. J. (2007). Sox15 and Fhl3 transcriptionally coactivate Foxk1 and regulate myogenic progenitor cells. EMBO Journal, 26(7), 1902-1912.
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  PMID: 17363903;PMCID: PMC1847663;Abstract: The regulation of myogenic progenitor cells during muscle regeneration is not clearly understood. We have previously shown that the Foxk1 gene, a member of the forkhead/winged helix family of transcription factors, is expressed in myogenic progenitor cells in adult skeletal muscle. In the present study, we utilize transgenic technology and demonstrate that the 4.6 kb upstream fragment of the Foxk1 gene directs β-galactosidase expression to the myogenic progenitor cell population. We further establish that Sox15 directs Foxk1 expression to the myogenic progenitor cell population, as it binds to an evolutionarily conserved site and recruits Fhl3 to transcriptionally coactivate Foxk1 gene expression. Knockdown of endogenous Sox15 results in perturbed cell cycle kinetics and decreased Foxk1 expression. Furthermore, Sox15 mutant mice display perturbed skeletal muscle regeneration, due in part to decreased numbers of satellite cells and decreased Foxk1 expression. These studies demonstrate that Sox15, Fhl3 and Foxk1 function to coordinately regulate the myogenic progenitor cell population and skeletal muscle regeneration. © 2007 European Molecular Biology Organization | All Rights Reserved.
• Reed, S. A., Ouellette, S. E., Liu, X., Allen, R. E., & Johnson, S. E. (2007). E2F5 and LEK1 translocation to the nucleus is an early event demarcating myoblast quiescence. Journal of Cellular Biochemistry, 101(6), 1394-1408.
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  PMID: 17295207;Abstract: Raf/MEK/ERK signaling in skeletal muscle cells affects several aspects of myogenesis that are correlated with the duration and intensity of the input signal. 23A2RafERDD myoblasts directing elevated levels of Raf kinase for 24 h are mitotically inactive. Removal of the stimulus results in cell cycle re-entry and proliferation. Using a proteomic approach, E2F5 and LEK1 were detected in the nuclei of Raf-arrested myoblasts. Disruption of MEK1 activity prevents phosphorylation of ERK1/2 and nuclear translocation of E2F5 and LEK1. The pocket proteins, p107 and p130, remain in the cytoplasm of growth arrested myoblasts irrespective of Raf/ERK activation while pRb translocates to the nucleus. Importantly, both E2F5 and LEK1 are found in the nuclei of non-dividing satellite cells and myonuclei in vivo and in vitro. Our results indicate that Raf-arrested myoblasts may serve as a model system for satellite cell cycle studies and that E2F5 and LEK1 translocation to the nucleus is an important first step during entry into quiescence. © 2007 Wiley-Liss, Inc.
• Tatsumi, R., Liu, X., Pulido, A., Morales, M., Sakata, T., Dial, S., Hattori, A., Ikeuchi, Y., & Allen, R. E. (2006). Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor. American Journal of Physiology - Cell Physiology, 290(6), C1487-C1494.
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  PMID: 16684931;Abstract: In the present study, we examined the roles of hepatocyte growth factor (HGF) and nitric oxide (NO) in the activation of satellite cells in passively stretched rat skeletal muscle. A hindlimb suspension model was developed in which the vastus, adductor, and gracilis muscles were subjected to stretch for 1 h. Satellite cells were activated by stretch determined on the basis of 5-bromo-2′-deoxyuridine (BrdU) incorporation in vivo. Extracts from stretched muscles stimulated BrdU incorporation in freshly isolated control rat satellite cells in a concentration-dependent manner. Extracts from stretched muscles contained the active form of HGF, and the satellite cell-activating activity could be neutralized by incubation with anti-HGF antibody. The involvement of NO was investigated by administering nitro-L-arginine methyl ester (L-NAME) or the inactive enantiomer NG-nitro-D-arginine methyl ester HCl (D-NAME) before stretch treatment. In vivo activation of satellite cells in stretched muscle was not inhibited by D-NAME but was inhibited by L-NAME. The activity of stretched muscle extract was abolished by L-NAME treatment but could be restored by the addition of HGF, indicating that the extract was not inhibitory. Finally, NO synthase activity in stretched and unstretched muscles was assayed in muscle extracts immediately after 2-h stretch treatment and was found to be elevated in stretched muscle but not in stretched muscle from L-NAME-treated rats. The results of these experiments demonstrate that stretching muscle liberates HGF in a NO-dependent manner, which can activate satellite cells. Copyright © 2006 the American Physiological Society.
• Tatsumi, R., Yamada, M., Katsuki, Y., Okamoto, S., Ishizaki, J., Mizunoya, W., Ikeuchi, Y., Hattori, A., Shimokawa, H., & Allen, R. E. (2006). Low-pH preparation of skeletal muscle satellite cells can be used to study activation in vitro. International Journal of Biochemistry and Cell Biology, 38(10), 1678-1685.
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  PMID: 16750930;Abstract: When skeletal muscle is stretched or injured, satellite cells are activated to enter the cell cycle, and this process could be mediated by hepatocyte growth factor (HGF) and nitric oxide (NO) as revealed by primary culture technique. In this system, which was originally developed by Allen et al. [Allen, R. E., Temm-Grove, C. J., Sheehan, S. M., & Rice, G. (1997). Skeletal muscle satellite cell cultures. Methods Cell Biol., 52, 155-176], however, some populations of satellite cells would receive activation signals during the cell isolation procedure; the high baseline level of activation diminishes the magnitude of the observed effect of HGF and NO. In this study, we modified the cell isolation procedure by lowering pH of muscle and isolation media from 7.2 (original) to 6.5. This modification was designed to block the activation signal generation, based on our previous observations that the satellite cell activation in response to mechanical stimulation only occurred between pH 7.1 and 7.5. Satellite cells prepared at low-pH showed a low baseline level of activation in bromodeoxyuridine incorporation and MyoD expression assays on control cultures, and demonstrated a large activation response to mechanical stretch, exogenous HGF and NO donor. Cell yield and myogenic purity were not affected by the modifications. The low-pH procedure could provide an improved satellite cell model for in vitro activation experiments. © 2006 Elsevier Ltd. All rights reserved.
• Yamada, M., Tatsumi, R., Kikuiri, T., Okamoto, S., Nonoshita, S., Mizunoya, W., Ikeuchi, Y., Shimokawa, H., Sunagawa, K., & Allen, R. E. (2006). Matrix metalloproteinases are involved in mechanical stretch-induced activation of skeletal muscle satellite cells. Muscle and Nerve, 34(3), 313-319.
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  PMID: 16810685;Abstract: When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the c-met receptor. Experiments reported herein provide new evidence that matrix metalloproteinases (MMPs) are involved in the NO-dependent release of HGF in vitro. When rat satellite cells were treated with 10 ng/ml recombinant tissue inhibitor-1 of MMPs (TIMP-1) and subjected to treatments that induce activation in vitro, i.e., sodium nitroprusside (SNP) of an NO donor or mechanical cyclic stretch, the activation response was inhibited. In addition, conditioned medium generated by cultures treated with TIMP-1 plus SNP or mechanical stretch failed to activate cultured satellite cells and did not contain HGF. Moreover, NOx assay demonstrated that TIMP-1 does not impair NO synthase activity of stretched satellite cell cultures. Therefore, results from these experiments provide strong evidence that MMPs mediate HGF release from the matrix and that this step in the pathway is downstream from NO synthesis. © 2006 Wiley Periodicals, Inc.
• Wagner, K. R., Liu, X., Chang, X., & Allen, R. E. (2005). Muscle regeneration in the prolonged absence of myostatin. Proceedings of the National Academy of Sciences of the United States of America, 102(7), 2519-2524.
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  PMID: 15699335;PMCID: PMC548322;Abstract: Myostatin is an endogenous inhibitor of muscle conserved across diverse species. In the absence of myostatin, there is massive muscle growth in mice, cattle, and humans. Previous studies in the mdx mouse model of muscular dystrophy demonstrate that inhibiting myostatin attenuates several features of dystrophic muscle. These findings have encouraged the development of human therapies to block myostatin. However, little is known of the long-term effects on muscle of myostatin blockade. To evaluate potential sequelae from the prolonged absence of myostatin, senescent myostatin null (mstn-/-) mice were studied. Senescent mstn-/- mice continue to have normal muscle with increased mass and strength relative to controls. Muscles of senescent mstn-/- mice regenerate robustly from both chronic and acute injury. Early markers of regeneration are enhanced in the absence of myostatin, suggesting a mechanism for the attenuation of dystrophic features found in mdx mice lacking myostatin.
• Mendias, C. L., Tatsumi, R., & Allen, R. E. (2004). Role of cyclooxygenase-1 and -2 in satellite cell proliferation, differentiation, and fusion. Muscle and Nerve, 30(4), 497-500.
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  PMID: 15372441;Abstract: Skeletal muscle satellite cells play an important role in muscle regeneration. Previous work has suggested that nonsteroidal anti-inflammatory drugs may inhibit their activity. We cultured skeletal muscle satellite cells from 9-month-old Sprague-Dawley rats and exposed them to naproxen sodium (a nonselective cyclooxygenase inhibitor), NS-398 (a selective cyclooxygenase-2 inhibitor), and SC-560 (a selective cyclooxygenase-1 inhibitor) for 96 h. Cyclooxygenase-2 inhibition alone resulted in decreased satellite cell proliferation, and inhibition of both cyclooxygenase-1 and cyclooxygenase-2 resulted in decreased satellite cell differentiation and fusion. This study suggests that the cyclooxygenase enzymes appear to play an important part in satellite cell proliferation, differentiation, and fusion and that nonsteroidal anti-inflammatory medication may have an adverse effect on muscle regeneration following injury. The use of a selective cyclooxygenase-2 inhibitor over nonspecific cyclooxygenase inhibitors in the treatment of muscle injuries is not supported.
• Tatsumi, R., & Allen, R. E. (2004). Active hepatocyte growth factor is present in skeletal muscle extracellular matrix. Muscle and Nerve, 30(5), 654-658.
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  PMID: 15389661;Abstract: When skeletal muscle is stretched or injured, satellite cells are activated to proliferate, and this process can be mediated by release of the active form of hepatocyte growth factor (HGF) from the extracellular matrix. The objective of these experiments was to determine whether the mechanism of release includes proteolytic activation of pro-HGF. Extracellular HGF in uninjured adult rat extensor digitorum longus muscle was extracted by treatment with 1 M NaCl or heparinases I and III in the presence of a cocktail of serine protease inhibitors. Active HGF heterodimer was the predominant form present, but both pro-HGF and active HGF were extracted when muscle was incubated with Triton X-100 or crush-injured. Incubation of exogenous pro-HGF with uninjured or crush-injured skeletal muscle resulted in cleavage to the active form, indicating that endogenous extracellular proteases are present and capable of rapidly converting pro-HGF to active HGF. Finally, treatment with sodium nitroprusside, a nitric oxide (NO) donor, resulted in liberation of active HGF. These experiments indicate that the active form of HGF is present in the extracellular compartment of uninjured skeletal muscle; therefore, the mechanism of HGF release in response to stretch and NO does not require proteolytic activation of pro-HGF.
• Tatsumi, R., Mitsuhashi, K., Ashida, K., Haruno, A., Hattori, A., Ikeuchi, Y., & Allen, R. E. (2004). Comparative analysis of mechanical stretch-induced activation activity of back and leg muscle satellite cells in vitro. Animal Science Journal, 75(4), 345-351.
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  Abstract: It has previously been shown that mechanical stretch induces activation of cultured quiescent satellite cells by rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c-met receptor. The present study provides evidence that the stretch activation activity varies according to the origin of satellite cells from back and leg skeletal muscles in vitro. Satellite cells were isolated from three muscle groups, back (BK), upper hind limb (UL) and lower hind limb (LL) muscles, of adult male rats and stretch activation activities were compared. In response to stretch, lower hind limb satellite cells showed significantly greater response than upper hind limb and back muscles (LL > UL > BK). Immunoblots of stretched culture media revealed a higher HGF-releasing capacity of lower hind limb satellite cells than back muscle satellite cells. In addition, lower hind limb satellite cells exhibited a greater activation activity in response to exogenous HGF added to culture media than compared to satellite cells from back and upper hind limb (LL > UL > BK). The increased ability to release HGF and the increased cellular responsiveness might account for higher stretch activation activities of lower hind limb satellite cells. Electrophoretic analysis of myosin heavy chain isoforms verified a higher content of slow muscle fibers in lower limb muscles (LL > UL > BK), suggesting a difference in stretch-induced activation activity between satellite cells associated with fast and slow muscle fibers.
• Tatsumi, R., Hattori, A., Ikeuchi, Y., Anderson, J. E., & Allen, R. E. (2002). Release of hepatocyte growth factor from mechanically stretched skeletal muscle satellite cells and role of pH and nitric oxide. Molecular Biology of the Cell, 13(8), 2909-2918.
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  PMID: 12181355;PMCID: PMC117951;Abstract: Application of mechanical stretch to cultured adult rat muscle satellite cells results in release of hepatocyte growth factor (HGF) and accelerated entry into the cell cycle. Stretch activation of cultured rat muscle satellite cells was observed only when medium pH was between 7.1 and 7.5, even though activation of satellite cells was accelerated by exogenous HGF over a pH range from 6.9 to 7.8. Furthermore, HGF was only released in stretched cultures when the pH of the medium was between 7.1 and 7.4. Conditioned medium from stretched satellite cell cultures stimulated activation of unstretched satellite cells, and the addition of anti-HGF neutralizing antibodies to stretch-conditioned medium inhibited the stretch activation response. Conditioned medium from satellite cells that were stretched in the presence of nitric-oxide synthase (NOS) inhibitor Nω-nitro-L-arginine methyl ester hydrochloride did not accelerate activation of unstretched control satellite cells, and HGF was not released into the medium. Conditioned medium from unstretched cells that were treated with a nitric oxide donor, sodium nitroprusside dihydrate, was able to accelerate the activation of satellite cells in vitro, and HGF was found in the conditioned medium. Immunoblot analysis indicated that both neuronal and endothelial NOS isoforms were present in satellite cell cultures. Furthermore, assays of NOS activity in stretched satellite cell cultures demonstrated that NOS is stimulated when satellite cells are stretched in vitro. These experiments indicate that stretch triggers an intracellular cascade of events, including nitric oxide synthesis, which results in HGF release and satellite cell activation.
• Tatsumi, R., Sheehan, S. M., Iwasaki, H., Hattori, A., & Allen, R. E. (2001). Mechanical stretch induces activation of skeletal muscle satellite cells in vitro. Experimental Cell Research, 267(1), 107-114.
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  PMID: 11412043;Abstract: Cultured quiescent satellite cells were subjected to mechanical stretch in a FlexerCell System. In response to stretch, satellite cells entered the cell cycle earlier than if they were under control conditions. Only a brief period of stretch, as short as 2 h, was necessary to stimulate activation. Additionally, conditioned medium from stretched cells could activate unstretched satellite cells. The presence of HGF on c-met-positive myogenic cells was detected by immunofluorescence at 12 h in culture, and immunoblots demonstrated that HGF was released by stretched satellite cells into medium. Also, stretch activation could be abolished by the addition of anti-HGF antibodies to stretched cultures, and activity in conditioned medium from stretched cells could be neutralized by anti-HGF antibodies. In addition, stretch appeared to cause release of preexisting HGF from the extracellular matrix. These experiments suggest that HGF may be involved in linking mechanical perturbation of muscle to satellite cell activation. © 2001 Academic Press.
• Sheehan, S. M., Tatsumi, R., Temm-Grove, C. J., & Allen, R. E. (2000). HGF is an autocrine growth factor for skeletal muscle satellite cells in vitro. Muscle and Nerve, 23(2), 239-245.
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  PMID: 10639617;Abstract: Muscle satellite cell activation following injury is essential for muscle repair, and hepatocyte growth factor/scatter factor (HGF) was the first growth factor shown to be able to stimulate activation and early division of adult satellite cells in culture and in muscle tissue. In addition, HGF was shown to be present in uninjured and injured skeletal muscle. Experiments in this report demonstrate that cultured satellite cells also synthesize and secrete HGF. Reverse transcription-polymerase chain reaction (RT-PCR) was used to demonstrate the presence of HGF mRNA in cultured adult satellite cells as early as 12 h from the time of plating. Message content was detectable at early times in culture and appeared to increase between 36 and 48 h. HGF protein expression was demonstrated during this time period by immunofluorescence localization; HGF was localized to mononucleated cells and multinucleated myotubes. HGF message was not detectable in muscle-derived fibroblast clones, and fibroblast-like cells in satellite cell cultures were negative for HGF by immunofluorescence analysis. Furthermore, Western blot analysis revealed the presence of HGF in satellite cell culture conditioned medium, associated with the cell surface and inside cells. Finally, the addition of neutralizing HGF antibodies during the proliferation phase in culture (42-90 h) significantly reduced cell proliferation. These experiments indicate that HGF is expressed by cultured satellite cells and that endogenous HGF from satellite cells can act in an autocrine fashion. Because HGF plays a central role in satellite cell activation, it is likely that direct administration of HGF into damaged muscle may represent a potentially useful approach for stimulating muscle repair. This approach may also be useful in enhancing the efficiency of myoblast transplantation in vivo.
• Sheehan, S. M., & Allen, R. E. (1999). Skeletal muscle satellite cell proliferation in response to members of the fibroblast growth factor family and hepatocyte growth factor. Journal of Cellular Physiology, 181(3), 499-506.
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  PMID: 10528236;Abstract: Fibroblast growth factors (FGF) have the ability to regulate satellite cell proliferation in culture and in muscle tissue, but the specific FGF receptors (FGFR) expressed by adult rat muscle satellite cells and the action of members of the FGF family have not been assessed. Therefore, the expression of FGF receptors 1-4 was examined in proliferating satellite cells in culture, and the effects of eight members of the fibroblast growth factor family (FGFs1, 2, 4, 5, 6, 7, 8, and 9) on adult rat muscle satellite cells were evaluated. In addition, the interactions of FGFs with hepatocyte growth factor (HGF) were described. Of the eight FGFs evaluated, 1,2, 4, 6, and 9 significantly (P < 0.05) stimulated proliferation above control. FGFs5, 7, and 8 displayed no mitogenic activity. Furthermore, combinations of HGF with FGFs2, 4, 6, or 9 stimulated satellite cell proliferation above that of optimal concentrations of HGF alone. Expression of four FGFR genes was detected in satellite cell cultures by reverse-transcription-polymerase chain reaction (RT-PCR). FGFR1 and FGFR4 were the most prominent forms expressed, and FGFR2 was only expressed at low levels. FGFR3 was difficult to detect. FGFR1 and FGFR2 were also expressed in muscle-derived fibroblasts, but FGFR4 and FGFR3 were not. In proliferating cultures of satellite cells, HGF, insulin-like growth factor I (IGF-II) and FGF1 stimulated significantly (P < 0.05) higher levels of FGFR1 message content, relative to control conditions, and platelet-derived growth factor-BB (PDGF-BB) and insulin-like growth factor (IGF-II) significantly (P < 0.05) depressed FGFR1 expression. During the activation period of satellite cell growth in culture (0-48 h), FGFR1 message content significantly (P < 0.05) increased from less than 1,000 copies per cell to approximately 5,000 copies per cell between 18 and 48 h, and HGF treatment significantly (P < 0.05) accelerated the accumulation of FGFR1 message during this period.
• Temm-Grove, C. J., Wert, D., Thompson, V. F., Allen, R. E., & Goll, D. E. (1999). Microinjection of calpastatin inhibits fusion in myoblasts. Experimental Cell Research, 247(1), 293-303.
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  PMID: 10047471;Abstract: Rat satellite cells (RSC) were microinjected with purified calpastatin or m-calpain, and myoblasts from a C2C12 mouse line were microinjected with purified calpastatin. Microinjection with calpastatin completely prevented fusion of myoblasts from both sources, whereas microinjection with m-calpain significantly increased the rate of fusion of cultured RSC; 44% of the nuclei of RSC cultures were in multinucleated myotubes within 48 h after microinjection with m-calpain plus labeled dextran, whereas only 15% of the nuclei were in multinucleated myotubes after microinjection with dextran alone. Western analyses indicated that neither RSC nor C2C12 myoblasts contained detectable amounts of μ-calpain before fusion. The levels of calpastatin in C2C12 myoblasts increased as cells passed from the proliferative stage to the onset of fusion, and these levels increased substantially in both the C2C12 and the RSC cells as they progressed to the late or postfusion stage. Both RSC and C2C12 myoblasts contained an 80-kDa polypeptide that was labeled with an anti-m-calpain antibody in Western blots. The results are consistent with a role of the calpain system (m-calpain in these myoblast lines) in remodeling of the cytoskeletal/plasma membrane interactions during cell fusion.
• Tatsumi, R., Anderson, J. E., Nevoret, C. J., Halevy, O., & Allen, R. E. (1998). HGF/SF is present in normal adult skeletal muscle and is capable of activating satellite cells. Developmental Biology, 194(1), 114-128.
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  PMID: 9473336;Abstract: We have shown that hepatocyte growth factor/scatter factor can stimulate activation and early division of adult satellite cells in culture, and that the action of hepatocyte growth factor/scatter factor is similar to the action of the unidentified satellite cell activator found in extracts of crushed muscle. We now provide new evidence that hepatocyte growth factor/scatter factor is present in uninjured adult rat skeletal muscle and that the activating factor in crushed muscle extract is hepatocyte growth factor/scatter factor. Immunoblots of crushed muscle extract demonstrate the presence of hepatocyte growth factor/scatter factor. Furthermore, crushed muscle extract stimulates the scattering of cultured MDCK cells. Immunolocalization studies with adult rat skeletal muscle show the presence of hepatocyte growth factor/scatter factor in the extracellular matrix surrounding muscle fibers; in addition, the receptor for hepatocyte growth factor/scatter factor, c-met, is localized to putative satellite cells. In muscle from mdx mice, hepatocyte growth factor/scatter factor and c-met are colocalized in activated satellite cells in regions of muscle repair. Moreover, the satellite cell-activating activity of crushed muscle extract is abolished by preincubation with anti-hepatocyte growth factor antibodies. Finally, direct injection of hepatocyte growth factor/scatter factor into uninjured tibialis anterior muscle of 12-month-old rats stimulated satellite cell activation. These experiments demonstrate that hepatocyte growth factor/scatter factor is present in muscle, can be released upon injury, and has the ability to activate quiescent satellite cells in vivo.
• Allen, R. E., Temm-Grove, C. J., Sheehan, S. M., & Rice, G. (1997). Skeletal muscle satellite cell cultures. Methods in Cell Biology, 155-176.
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  PMID: 9379949;
• Allen, R. E., Sheehan, S. M., Taylor, R. G., Kendall, T. L., & Rice, G. M. (1995). Hepatocyte growth factor activates quiescent skeletal muscle satellite cells in vitro. Journal of Cellular Physiology, 165(2), 307-312.
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  PMID: 7593208;Abstract: The effect of hepatocyte growth factor (HGF) on the activation of quiescent rat skeletal muscle satellite cells was evaluated in vitro. Satellite cells from 9-month-old adult rats are quiescent in vivo and when cultured, display a protracted lag phase prior to division that is not present in satellite cells from neonatal or regenerating muscle. Under normal growth conditions, satellite cells divide for the first time between 42 and 60 hr. Hepatocyte growth factor increased proliferation in a dose-dependent fashion prior to 48 hr with half-maximal stimulation at approximately 3 ng/ml; in addition, heparin enhanced this activity. The time course of cyclin-D1 and proliferating cell nuclear antigen (PCNA) expression was accelerated in HGF-treated satellite cells, indicating that cells entered the cell cycle earlier. No significant effects on muscle-derived fibroblast proliferation was observed. The signalling receptor for HGF is the product of the c-met protooncogene, and rtPCP analysis of satellite cells 0-72 hr in culture demonstrated the presence of this message throughout this time period. The presence of c-met in quiescent satellite cells, the ability of HGF to stimulate precocious entry into the cell cycle, and the previously described localization of HGF message in regenerating muscle (Jennische et al., 1993) indicate that HGF could act as an activator of quiescent satellite cells in vivo.
• Johnson, S. E., & Allen, R. E. (1995). Activation of skeletal muscle satellite cells and the role of fibroblast growth factor receptors. Experimental Cell Research, 219(2), 449-453.
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  PMID: 7641796;Abstract: Specific, high-affinity binding of FGF2 was evaluated in cultured skeletal muscle satellite cells from young (3- to 4-week-old) and adult (9- to 12- month-old) rats prior to the first division in culture. Specific binding of FGF2 was detected on satellite cells from young rats at 18 h postplating, the earliest time examined, but specific binding was not detectable until 42 h on satellite cells from old rats. This correlates well with the delayed entry into the cell cycle exhibited by adult satellite cells and with the ability of satellite cells from rats of these ages to proliferate in response to FGF2. Patterns of tyrosine phosphorylation in whole cell extracts, following stimulation by FGF2, indicated specific FGF2 phosphorylation of proteins of 150/145, 90, 42, and 35 kDa in cells from both age groups. Several growth factors were evaluated for their ability to stimulate early entry of adult satellite cells into the cell cycle, and none of the following growth factors were able to activate proliferation of these cells: FGF2, IGF-1, IGF-2, PDGF- BB, TGF-β1, or TGF-β2. In addition, specific binding of FGF2 to 48-h cultures of adult satellite cells was not stimulated by FGF2, IGF-1, IGF-2, PDGF-BB, or TGF-β2, and specific binding was significantly decreased (P < 0.05) by FGF2 and TGF-β2. Specific binding was significantly lower in cells treated with PDGF-BB than in cells treated with either form of IGF but was greater than in cells treated with FGF2 or TGF-β2. The results of these experiments suggest that expression of functional FGF receptors on the surface of satellite cells may represent an important step in the activation of quiescent satellite cells.
• Johnson, S. E., & Allen, R. E. (1993). Proliferating cell nuclear antigen (PCNA) is expressed in activated rat skeletal muscle satellite cells. Journal of Cellular Physiology, 154(1), 39-43.
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  PMID: 8093452;Abstract: Skeletal muscle satellite cells from uninjured muscle of adult animals are generally found to be in a quiescent state, and when cultured, they remain quiescent in vitro for a period of time which is directly related to the age of the donor animal. A technique for studying the activation ol satellite cells in primary cultures has been developed and employs proliferating cell nuclear antigen (PCNA) as a marker for entrance into the S phase of the cell cycle. PCNA is a protein involved in DNA replication and is maximally expressed in S phase of the cell cycle. We monitored PCNA expression in satellite cells isolated from young (3 week) and adult (9 month) rats, and our results indicate that satellite cells begin to accumulate PCNA prior to changes in cell number in both age groups. Using ELISA techniques, we demonstrated that addition of an extract of crushed muscle (CME) activated satellite cells and significantly reduced the length of the lag phase in cells from both age groups. Addition of bFGF shortened the lag phase of PCNA synthesis in satellite cells from 3-week-old rats but had no effect on the kinetics of PCNA expression in cells from 9-month-old rats. Based on our experiments, PCNA expression can be used as a marker to follow the entry of satellite cells into the cell cycle in primary mass cultures. © 1933 Wiley-Liss, Inc.
• Kumamoto, T., Kleese, W. C., Cong, J., Goll, D. E., Pierce, P. R., & Allen, R. E. (1992). Localization of the Ca²⁺-dependent proteinases and their inhibitor in normal, fasted, and denervated rat skeletal muscle. Anatomical Record, 232(1), 60-77.
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  PMID: 1536466;Abstract: Immunofluorescence and immunogold localization studies show that the two Ca2+-dependent proteinases (μ-calpain for the micromolar Ca2+-requiring proteinase and m-calpain for the millimolar Ca2+-requiring proteinase) and their protein inhibitor (calpastatin) are located exclusively intracellularly in normal rat soleus muscle. Quantitative immunogold studies indicate that binding of antibodies to both calpains and to calpastatin is approximately two times greater at the Z-disk of myofibrils than it is at the I-band or A-band regions. Mitochondria and nuclei in muscle cells contain both calpains and calpastatin at concentrations approximately one-tenth and one-fifth, respectively, of the concentration at the Z-disk, as estimated by antibody binding. Very little calpain or calpastatin was seen in the cytoplasmic intermyofibrillar spaces, and most of the calpain and calpastatin in muscle cells is associated with intracellular structures. Immunofluorescence results suggest that concentration of m-calpain but not μ-calpain or calpastatin is, in some instances, slightly higher near the intracellular surface of the plasma membrane than elsewhere in the muscle cell. Most m-calpain, however, is distributed throughout the interior of mature rat skeletal muscle cells. Denervation, or fasting and refeeding increases the concentration of the calpains and calpastatin in the muscle cell but does not change their distribution. Some μ- and m-calpain and calpastatin is found extracellular is denervated soleus muscle or soleus muscle from fasting rats, but the extracellular calpains and calpastatin seem to originate from ''leakage'' of these proteins out of the cell because serum creatine kinase levels are much higher than normal in denervated or fasting rats.
• Allen, R. E., Rankin, L. L., Greene, E. A., Boxhorn, L. K., Johnson, S. E., Taylor, R. G., & Pierce, P. R. (1991). Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells. Journal of Cellular Physiology, 149(3), 525-535.
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  PMID: 1744177;Abstract: The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that >94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2′-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivicaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells. The effects of combinations of insulin-like growth factor I (IGF-I), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGF-β) on rat satellite cell proliferation and differentiation were assessed by desmin staining, and results were found to be consistent with results obtained previously using conventional cell staining and counting techniques (Allen and Boxhorn, 1989). Our experiments indicate that the pattern of desmin expression in satellite cells differs between rat and bovine and that desmin can be a useful marker for cultured rat satellite cells.
• Greene, E. A., & Allen, R. E. (1991). Growth factor regulation of bovine satellite cell growth in vitro.. Journal of animal science, 69(1), 146-152.
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  PMID: 2005007;Abstract: This study examined the effects of basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I) and transforming growth factor beta (TGF-beta) on the proliferation and differentiation of primary bovine satellite cells (BSC) in vitro. Individually, these three factors had the following effects on satellite cells: bFGF stimulated proliferation (P less than .01) but inhibited differentiation (P less than .05); IGF-I had no effect on proliferation but stimulated differentiation (P less than .01); and TGF-beta inhibited both proliferation and differentiation (P less than .01). When combined, the following effects were observed: maximum stimulation of proliferation (P less than .01) occurred in the presence of bFGF and IGF-I and differentiation was not stimulated; TGF-beta and bFGF continued to inhibit differentiation (P less than .01), but in the presence of bFGF, TGF-beta stimulated proliferation (P less than .01). No stimulation was observed in the presence of TGF-beta and IGF-I. Bovine satellite cells respond to these three growth factors that have been shown to regulate the activity of other myogenic cells, and in most instances, the responses among cells from various species are similar. These experiments indicate that the interactions of growth factors may be critical in regulating bovine satellite cell activity.
• Allen, R. E., & Rankin, L. L. (1990). Regulation of satellite cells during skeletal muscle growth and development. Proceedings of the Society for Experimental Biology and Medicine, 194(2), 81-86.
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  PMID: 2190237;Abstract: Satellite cells are myogenic cells attributed with the role of postnatal growth and regeneration in skeletal muscle. Following proliferation and subsequent differentiation, these cells will fuse with one another or with the adjacent muscle fiber, thereby increasing myonuclei numbers for fiber growth and repair. The potential factors which could regulate this process are many, including exercise, trauma, passive stretch, innervation, and soluble growth factors. Three classes of growth factors in particular (fibroblast growth factor, insulin-like growth factor, and transforming growth factor-β) have been studied extensively with respect to their effects on satellite cell proliferation and differentiation in culture. Fibroblast growth factor has been shown to stimulate proliferation but depress differentiation. Insulin-like growth factor stimulates both proliferation and differentiation, although the latter to a much greater degree. Transforming growth factor-β slightly depresses proliferation but inhibits differentiation. When administered in combination, these factor can induce satellite cell activities in culture which mimic those typical of satellite cells found in vivo in growing, regenerating, or healthy mature muscle. Alterations in the concentrations of these growth factors in the muscle environment as well as alterations in the cell's sensitivity or responsiveness to these factors represent potential mechanisms for regulating satellite cell activity in situ.
• Johnson, S. E., & Allen, R. E. (1990). The effects of bFGF, IGF-I, and TGF-β on RMo skeletal muscle cell proliferation and differentiation. Experimental Cell Research, 187(2), 250-254.
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  PMID: 2180733;Abstract: A new skeletal muscle cell line, rat myoblast ω or RMo, has been characterized with regard to the effects of three growth factors: basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I), and transforming growth factor β (TGF-β). Results indicate a differential response of these factors on both cell proliferation and differentiation. Exposure to bFGF and IGF-I stimulate proliferation, while TGF-β has no effect on cell number. RMo cell differentiation, as indicated by skeletal myosin synthesis, is enhanced by IGF-I, whereas both bFGF and TGF-β suppress differentiation. These responses are in agreement with the effects of bFGF, IGF-I, and TGF-β on myogenic cells cultured from fetal and postnatal muscle, thereby suggesting that RMo cells can serve as a model system for the study of growth factor effects on skeletal muscle cells. © 1990 Academic Press, Inc.
• Allen, R. E., & Boxhorn, L. K. (1989). Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor-beta, insulin-like growth factor I, and fibroblast growth factor. Journal of Cellular Physiology, 138(2), 311-315.
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  PMID: 2918032;Abstract: Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with various combinations of transforming growth factor (TGF)-beta, fibroblast growth factor (FGF), and insulin-like growth factor I (IGF-I). In serum-free defined medium the following observations were made: TGF-beta depressed proliferation and inhibited differentiation; FGF stimulated proliferation and depressed differentiation; IGF-I stimulated proliferation to a small degree but demonstrated a more pronounced stimulation of differentiation. In evaluating combinations of these three factors, the differentiation inhibiting effect of TGF-beta could not be counteracted by any combination of IGF-I or FGF. The proliferation-depressing activity of TGF-beta, however, could not inhibit the mitogenic activity of FGF. Maximum stimulation of proliferation was observed in the presence of both FGF and IGF-I. The highest percentage fusion was also observed under these conditions, but differentiation with minimal proliferation resulted from treatment with IGF-I, alone. By altering the concentrations of TGF-beta, FGF, and IGF-I, satellite cells can be induced to proliferate, differentiate, or to remain quiescent.
• Thompson, S. H., Boxhorn, L. K., Kong, W., & Allen, R. E. (1989). Trenbolone alters the responsiveness of skeletal muscle satellite cells to fibroblast growth factor and insulin-like growth factor I. Endocrinology, 124(5), 2110-2117.
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  PMID: 2707149;Abstract: The potential role of satellite cells in mediating the effect of trenbolone (17β-hydroxyestra-4,9-11-trien-3-one (TBOH)] on skeletal muscle hypertrophy was examined. Young female Sprague-Dawley rats received TBOH injections daily for 2 weeks; growth, body composition, and the composition of selected muscles were assessed. Treated rats grew more rapidly and deposited less body lipid and more protein. The semimembranosus muscle from treated rats was larger and had approximately 60% more DNA per muscle than muscles from control rats. The addition of trenbolone directly to the medium of cultured satellite cells did not stimulate cell proliferation, nor did it augment the stimulatory response of these cells to fibroblast growth factor (FGF) or insulin-like growth factor I (IGF-I). In contrast, satellite cells cultured from TBOH-treated rats exhibited greater proliferative responses to FGF and IGF-I than satellite cells from control rats. In addition, serum from TBOH-treated rats stimulated greater cell proliferation in satellite cell cultures than serum from control rats. These experiments suggest that one possible mechanism responsible for the ability of TBOH to stimulate skeletal muscle hypertrophy may be through enhanced proliferation and differentiation of satellite cells as a result of the increased sensitivity of these cells to IGF-I and FGF.
• Allen, R. A., & Boxhorn, L. K. (1987). Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor-beta. Journal of Cellular Physiology, 133(3), 567-572.
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  PMID: 3480289;
• Allen, R. E. (1987). Muscle cell culture as a tool in animal growth research.. Federation Proceedings, 46(2), 290-294.
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  PMID: 3803619;Abstract: Muscle cell culture techniques have been used for several years in research on muscle growth and development. Several types of culture systems have been devised, including primary cultures from embryonic or postnatal muscle and myogenic cell lines. In addition, serum-free and serum-containing media have been developed to address specific muscle development questions. Many of these questions center around muscle cell differentiation and muscle cell physiology; and, more recently, muscle cell cultures have been used as bioassay tools for examining growth physiology in domestic animals. In our laboratory, skeletal muscle satellite cells have been studied in vitro to evaluate the effect of several protein hormones and growth factors on satellite cell proliferation and differentiation. Of the hormones examined, only the insulin-like growth factors/somatomedins and fibroblast growth factor have been shown to have a stimulatory effect on proliferation that could be physiologically significant. None of the major anterior pituitary hormones interacted directly with satellite cells to stimulate proliferation. With advances in serum-free medium formulations and cell separation techniques, more information can be obtained from experiments with muscle cell cultures. With appropriate design and interpretation, our knowledge of muscle growth in domestic animals will be expanded.
• Dodson, M. V., & Allen, R. E. (1987). Interaction of multiplication stimulating activity/rat insulin-like growth factor II with skeletal muscle satellite cells during aging. Mechanisms of Ageing and Development, 39(2), 121-128.
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  PMID: 3626637;Abstract: Satellite cells were isolated from the skeletal muscle of 3-, 12- and 24-month-old Fischer 344 rats. In vitro growth of these cells was evaluated in serum-containing medium and in serum-free medium in response to multiplication stimulating activity/rat insulin-like growth factor II (MSA). Cells from 3-month-old rats exhibited a shorter lag phase of growth than cells from 12- or 24-month-old animals. Dose-response curves for MSA with each of the three age groups did not differ in the concentrations of MSA required for a half-maximal response or in the magnitudes of the response. Hormone-binding data using [125I]MSA, however, revealed the highest numbers of MSA-binding sites with lowest affinities in the 3-month-old rat muscle cells; cells from the 24-month-old rats were intermediate and cells from the 12-month-old rats had the highest affinity and lowest number of binding sites. The lower affinity and increased number of binding sites in the young rat cells may be due to greater numbers of IGF type I receptors in muscle from young growing rats. © 1987.
• Dodson, M. V., Allen, R. E., Shimizu, N., Shimizu, Y., & Hossner, K. L. (1987). Interaction of ovine somatomedin and multiplication stimulating activity/rat insulin-like growth factor II with cultured skeletal muscle satellite cells. Acta Endocrinologica, 116(3), 425-432.
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  PMID: 2961189;Abstract: The interactions of 125I-multiplication stimulating activity (MSA) and 125I-ovine somatomedin with receptors on skeletal muscle satellite cells are described. Specific binding of 125I-MSA/rIGF-II was inhibited by MSA/rIGF-II and oSm but not by insulin. Binding of 125I-oSm was inhibited by MSA/rIGF-II, oSm and insulin. In addition, 24-h pre-incubation of satellite cells with insulin increased the amount of 125I-MSA/rIGF-II bound, but insulin concentrations below 550 μg/l had no effect on the subsequent binding of 125I-oSm. Pre-incubation of cultures with oSm or MSA/rIGF-II decreased the subsequent binding of 125I-oSm and 125I-MSA/rIGF-II. These preliminary experiments suggest that oSm is similar to IGF-I in its binding characteristics and that primary cultures of skeletal muscle satellite cells possess type I and type II IGF receptors.
• Allen, R. E., Dodson, M. V., Boxhorn, L. K., Davis, S. L., & Hossner, K. L. (1986). Satellite cell proliferation in response to pituitary hormones.. Journal of animal science, 62(6), 1596-1601.
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  PMID: 3733558;Abstract: Proliferation of rat skeletal muscle satellite cells was studied in vitro, and their ability to respond to a variety of protein hormones was examined, including: growth hormone, prolactin, luteinizing hormone, thyrotropin and fibroblast growth factor. All experiments were conducted in serum-free medium to avoid complicating interactions with serum constituents such as other hormones or binding proteins. Dose-response curves were constructed for each protein and encompassed the physiological range plus concentrations two to three orders of magnitude greater than the physiological range. Of the proteins examined, the only one shown to have the ability to directly stimulate satellite cell proliferation was fibroblast growth factor. None of the anterior pituitary protein hormones had the ability to interact directly with satellite cells to stimulate proliferation in vitro. Therefore, satellite cells seem to be selective in their response to protein hormones, and the classes identified thus far are hormones in the insulin-like growth factor family and fibroblast growth factor. These two classes of protein hormones are quite different and would not be expected to act through a common pathway. Consequently, we have proposed a dual regulatory system that may allow for local as well as systemic stimulation of satellite cells.
• Allen, R. E., Dodson, M. V., Luiten, L. S., & Boxhorn, L. K. (1985). A serum-free medium that supports the growth of cultured skeletal muscle satellite cells. In Vitro, 21(11), 636-640.
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  PMID: 3905759;Abstract: A serum-free medium has been devised that supports the proliferation and differentiation of primary cultures of rat skeletal muscle satellite cells for up to 4 d. The medium consists of a mixture of Dulbecco's modified Eagle's medium and MCDB-104 plus insulin, dexamethasone, pituitary fibroblast growth factor. Deutsch fetuin, and linoleic acid. In addition to promoting the formation of myotubes from satellite cells, a decrease in fibroblast contamination of these cultures was observed when cultures grown in serum-free medium were compared to cultures grown in serum-containing medium.
• Allen, R. E., Luiten, L. S., & Dodson, M. V. (1985). Effect of insulin and linoleic acid on satellite cell differentiation.. Journal of animal science, 60(6), 1571-1579.
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  PMID: 3894312;Abstract: Differentiation of rat skeletal muscle satellite cells was studied in vitro. Linoleic acid and insulin, two unrelated compounds that reportedly stimulate differentiation of other types of myogenic cells, were used to examine the regulation of differentiation in satellite cell cultures. As in cultures of chick embryo muscle cells, linoleic acid stimulated fusion but only at low serum concentrations or in defined medium without fibroblast growth factor (FGF). The effects of insulin on differentiation were quite variable, however; at very low cell densities no stimulatory effect was observed. In intermediate and, to a lesser extent, high density satellite cell cultures, the addition of insulin at concentrations between .01 and 1.0 microM stimulated satellite cell fusion. Whenever increases in fusion were observed, however, a parallel increase in cell number was also found. A closer examination of the relationship between differentiation and the presence or absence of mitogenic agents in the medium suggested that a mitogenic signal and the resultant proliferation of cells prevented differentiation. Subsequent experiments indicated that fusion could be induced by lower serum concentration or by removal of FGF, as long as linoleic acid was present in the medium. Therefore, proliferation and differentiation appear to be antagonistic processes in cultured satellite cells. If the rate of proliferation is depressed, either by mitogen removal or by increasing cell density, differentiation is favored. Differentiation can, therefore, be regulated and applied to in vitro studies of satellite cell activity.
• Dodson, M. V., Allen, R. E., & Hossner, K. L. (1985). Ovine somatomedin, multiplication-stimulating activity, and insulin promote skeletal muscle satellite cell proliferation in vitro. Endocrinology, 117(6), 2357-2363.
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  PMID: 3905359;Abstract: Primary cultures of skeletal muscle satellite cells, the postnatal myogenic precursor cells, were induced to proliferate by exposure to physiological levels of somatomedins (Sms)/insulin-like growth factors (IGFs) and pharmacological levels of insulin. These polypeptides were included in medium containing horse serum as well as serum-free defined medium. Dexamethasone inclusion in the serum-containing medium facilitated the ovine Sm (oSm; P < 0.05) and the multiplication-stimulating activity/rat IGF-II (MSA/rIGF-II; P < 0.25) responses, but not the insulin proliferative response. In addition, data from defined medium studies indicate that satellite cells are more sensitive to both IGF moieties than insulin and that the proliferations induced by half-maximal concentrations of oSm and insulin were similar (P < 0.05), but both were different from the proliferation induced by MSA/rIGF-II (P < 0.05). In the presence of insulin concentrations that promote maximum proliferation, the addition of oSm did not produce an additive effect, whereas the addition of MSA/rIGF-II did produce a significant increase in satellite cell proliferation above that induced by insulin. MSA/rIGF-II may, therefore, be stimulating proliferation of satellite cells through a receptor system different from that serving insulin and oSm. Collectively, these data support the hypothesis that Sms/IGFs play an important role in the control of postnatal muscle growth by providing a link between these hormones and one of the significant target cells involved in this process.
• Knudson, B. K., Hogberg, M. G., Merkel, R. A., Allen, R. E., & Magee, W. T. (1985). Developmental comparisons of boars and barrows: I. Growth rate, carcass and muscle characteristics.. Journal of animal science, 61(4), 789-796.
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  PMID: 4066537;Abstract: The rate of gain, carcass measurements and three muscles were evaluated in 65 crossbred boars representing 13 litters that were allotted at 4 wk of age to slaughter weight and treatment groups as follows: 1) 105 kg, castrated; 2) 105 kg, intact; 3) 118 kg, intact; 4) 132 kg, intact and 5) 145 kg, intact. One barrow and four boars within a litter constituted a replicate and each replicate was penned separately. The growth rate of all boars to 105 kg constituted one group and was compared with the growth rate of barrows to 105 kg live body weight. Average daily gain from 4 wk until 105 kg did not differ significantly between boars and barrows. Growth rate of the boars continued at an increasing rate until they reached 87.3 kg live weight, while maximum daily gain of barrows occurred at 76.3 kg live weight or 11 kg less than that of boars. At 105 kg, boars had 31.3% less 10th rib backfat thickness and 2.9% greater carcass length than barrows, but longissimus muscle area did not differ. Barrows had greater backfat thickness at 105 kg than 145-kg boars. As live weight increased from 105 to 145 kg, carcass length, 10th rib backfat thickness and longissimus area of boars increased (P less than .01) linearly. Fat-free muscle weights of the brachialis (BR), semitendinosus (ST) and longissimus (L) did not differ between boars and barrows at 105 kg. Boars at 105 kg had 1.3 and 1.7% more moisture in the BR and ST, respectively, than barrows. Percentage protein, total intramuscular fat and fiber diameter in the BR, ST and L muscles did not differ between boars and barrows at 105 kg or with increasing live weight in boars. Total RNA increased linearly (P less than .05) in the BR and ST as boars increased in live weight from 105 to 145 kg.
• Knudson, B. K., Hogberg, M. G., Merkel, R. A., Allen, R. E., & Magee, W. T. (1985). Developmental comparisons of boars and barrows: II. Body composition and bone development.. Journal of animal science, 61(4), 797-801.
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  PMID: 4066538;Abstract: Differences in total carcass bone, muscle and fat, and linear measurements of the tibia and radius were evaluated in barrows at 105 kg and boars at 105, 118, 132 and 145 kg live body weight. The carcasses of five replicates were physically separated into skin, bone and soft tissues, and the linear measurements of the tibia and radius were obtained on seven replicates. At live weight of 105 kg, boars did not differ significantly in fat-free muscle, but they had 33.2% less fat, 11% greater bone weight and 14% greater skin weight than barrows. At 145 kg, boars had total carcass fat weight comparable with 105-kg barrows. Fat-free muscle, bone and skin weight of boars increased at linear rates of .41, .083 and .104 kg/kg of body weight increase from 105 to 145 kg, respectively. At 105 kg, density and length of the tibia and radius did not differ between boars and barrows. The tibia of boars were heavier than those of barrows at 105 kg, resulting in a greater ratio of tibia weight to length (indirect measure of bone thickness). As boars increased in live weight from 105 to 145 kg, total weight and length of the tibia and radius increased linearly. The ratio of weight to length of the tibia and radius increased during this 40-kg weight gain, indicating that weight of both bones increased at a greater rate than length. These results indicate that boars and barrows have the same weight of total carcass fat when boars are 40 kg heavier than the barrows. The greater bone weight of boar carcasses compared with barrows is due to greater bone thickness.
• Purchas, R. W., Romsos, D. R., Allen, R. E., & Merkel, R. A. (1985). Muscle growth and satellite cell proliferative activity in obese (OB/OB) mice.. Journal of animal science, 60(3), 644-651.
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  PMID: 3988641;Abstract: Muscle growth of male obese (ob/ob) and lean mice at 2, 3, 5 and 8 wk were analyzed on the basis of weights of gastrocnemius, plantaris and soleus muscles from each hind leg. The carcasses (prepared by removing skin, viscera, head, feet and tail) were analyzed for fat content so that the effect of phenotype on the relationship between muscle weight and fat-free carcass weight could be assessed. For each age group the obese mice had less muscle relative to fat-free carcass weight than lean mice, with the difference being significant at 3 wk (P less than .05) and 8 wk (P less than .025). The proliferative activity of muscle satellite cells in 2- and 3-wk-old obese and lean mice was measured on isolated muscle fibers by autoradiography. Muscle fiber diameter and number of nuclei/unit length were unaffected by phenotype, but the proportion of muscle nuclei showing proliferative activity was lower (P less than .01) in obese than in lean mice at 2 wk (1.05 vs 1.93%, respectively) and 3 wk of age (.23 vs .59%, respectively). These results are consistent with the suggestion that muscle growth is limited by satellite-cell proliferative activity, although direct evidence for a cause and effect relationship is not provided.
• Allen, R. E., Dodson, M. V., & Luiten, L. S. (1984). Regulation of skeletal muscle satellite cell proliferation by bovine pituitary fibroblast growth factor. Experimental Cell Research, 152(1), 154-160.
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  PMID: 6714317;Abstract: Satellite cells in skeletal muscle have been implicated in muscle growth processes and regeneration. However, very little is known about the regulation of their proliferation and differentiation. The effect of fibroblast growth factor (FGF) on the proliferation of myogenic cells from adult rat skeletal muscle, presumably satellite cells, has been examined, and FGF has been found to be a potent mitogen for these cells. The mitogenic properties of serum were also documented and studied in conjunction with FGF. Even under conditions of maximal stimulation by serum, the addition of FGF caused a substantial increase in proliferation of satellite cells. The additive nature of the FGF and serum-stimulatory activity suggests that FGF-like molecules are not the active agents in serum and that more than one pathway may be involved in stimulating satellite cell proliferation. © 1984.
• Allen, R. E., Masak, K. C., McAllister, P. K., & Merkel, R. A. (1983). Effect of growth hormone, testosterone and serum concentration on actin synthesis in cultured satellite cells.. Journal of animal science, 56(4), 833-837.
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  PMID: 6853380;Abstract: Because most of the DNA in a mature muscle accumulates during postnatal life and is derived from satellite cells, cellular regulation of protein accumulation in muscle fibers originating from satellite cells is an important aspect of muscle growth control. These experiments were designed to study the modulation of a alpha-actin accumulation in satellite cell-derived myotubes by serum and two anabolic hormones commonly assumed to be involved in muscle growth regulation; growth hormone and testosterone. Satellite cells were cultured from rats ranging in age from 5 d to 1 yr. After fusion into myotubes, various levels of pig serum, 3 to 20%, were added to culture medium, and the amount of alpha-actin per myotube nucleus was determined 4 d later. The quantity increased with increasing percentages of serum in the medium. For an assessment of the extent to which serum stimulatory activity was due to growth hormone or testosterone in the serum, similar experiments were conducted with medium containing 10% pig serum plus various levels of porcine growth hormone or testosterone. Neither of these hormones stimulated actin accumulation at concentrations from 10(-9) to 10(-6) M, a range encompassing physiological levels for each hormone. These experiments do not support the premise that either growth hormone and (or) testosterone are the blood-borne agents that interact directly with muscle to stimulate protein accretion, because neither growth hormone nor testosterone acted directly on muscle cells in vitro to stimulate muscle protein accumulation.
• Allen, R. E., McAllister, P. K., Masak, K. C., & Anderson, G. R. (1982). Influence of age on accumulation of α-actin in satellite-cell-derived myotubes in vitro. Mechanisms of Ageing and Development, 18(1), 89-95.
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  PMID: 7062790;Abstract: Because muscle satellite cells have been implicated in the process of muscle growth and mass regulation, as well as regeneration, alterations in the capacity of satellite cells to differentiate and accumulate muscle specific proteins during aging could play a role in the process of senile muscle atrophy. Skeletal muscle satellite cells were cultured from male rats from the following four age groups: neonatal rats (less than 5 days of age), growing rats (1-3 months of age), adult rats (9-12 months of age) and old rats (more than 24 months of age). A series of experiments was conducted in which cultures were harvested at daily intervals following fusion, and the amount of α-actin per myotube nucleus was determined. Analysis of maximum actin accumulation in myotubes from each experiment within each age group revealed no significant differences among cells derived from growing, adult or old rats; however, myotubes differentiating from neonatal muscle cells were able to accumulate more than three times as much α-actin per myotube nuclei as cells from the other three age groups. This result may reflect fundamental differences between authentic satellite cells and myogenic cells of prenatal origin. Aside from differences between neonatal cells and satellite cells, satellite cells from old muscle do not appear to have a diminished capacity to accumulate muscle-specific proteins following differentiation into muscle fibers. © 1982.
• Allen, R. E. (1980). Disproportionate accumulation of myosin and tropomyosin in cultured muscle cells. European Journal of Cell Biology, 21(3), 247-253.
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  PMID: 7449767;Abstract: Accumulation of two major myofibrillar proteins, myosin and tropomyosin was monitored in differentiating skeletal muscle cultures. The tropomyosin subunit to myosin heavy chain accumulation rate ratio was more than twice the stoichiometric ratio of tropomyosin subunit to myosin heavy chain in nature skeletal muscle myofibrils and crude myofibrils from cultured muscle cells. Electron microscopy revealed normal patterns of myofibril assembly in these muscle cultures. Therefore, the observed disproportionate accumulation of tropomyosin and myosin heavy chain may be reflecting normal cellular conditions for de novo myofibril assembly.
• Allen, R. E., Masak, K. C., & McAllister, P. K. (1980). Staining protein in isoelectric focusing gels with fast green. Analytical Biochemistry, 104(2), 494-498.
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  PMID: 6160787;Abstract: A rapid, simple technique for staining proteins in isoelectric focusing polyacrylamide gels was demonstrated using fast green in 10% acetic acid. Fast green has the distinct advantage of not binding to ampholytes, thus staining only protein. Maximum staining was achieved within 5 min, and bands were visible after 3 to 6 h of destaining. Background stain removal, however, was not complete until 72 h after placing gels in a diffusion destainer. Gel quantitation was demonstrated with actin using fast green and Coomassie brilliant blue R-250. A standard curve prepared with fast green was linear from 0.5 to 8 μg of actin in contrast to Coomassie brilliant blue R-250 which provided linearity from 0.1 to 2.5 μg actin. Application of fast green staining to quantitation of α-actin from cultured muscle satellite cells has been demonstrated. © 1980.
• Allen, R. E., McAllister, P. K., & Masak, K. C. (1980). Myogenic potential of satellite cells in skeletal muscle of old rats. A brief note. Mechanisms of Ageing and Development, 13(2), 105-109.
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  PMID: 7431999;Abstract: Muscle satellite cells from old (> 30 months) female Long-Evans rats were isolated and grown in cell culture. Satellite cells differentiated in culture to form multinucleated myotubes that had the ability to accumulate muscle-specific proteins. Therefore, satellite cells exist in muscle through senescence and retain their myogenic potential. © 1980.
• Allen, R. E., Merkel, R. A., & Young, R. B. (1979). Cellular aspects of muscle growth: myogenic cell proliferation.. Journal of animal science, 49(1), 115-127.
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  PMID: 500507;
• Allen, R. E., Stromer, M. H., Goll, D. E., & Robson, R. M. (1979). Accumulation of myosin, actin, tropomyosin, and α-actinin in cultured muscle cells. Developmental Biology, 69(2), 655-660.
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  PMID: 437357;Abstract: In an effort to understand the conditions that promote the assembly of myofibrillar proteins in muscle cells, the temporal sequence of accumulation of four myofibrillar proteins, actin, myosin, tropomyosin, and α-actinin, was monitored during the period of de novo assembly of myofibrils in differentiating muscle cells. Isotope dilution experiments indicated that all four proteins were accumulated simultaneously. Therefore, assembly of myofibrils may be occurring in the presence of a full complement of myofibrillar proteins. © 1979.
• Young, R. B., & Allen, R. E. (1979). Transitions in gene activity during development of muscle fibers.. Journal of animal science, 48(4), 837-852.
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  PMID: 383671;
• Suzuki, A., Goll, D. E., Singh, I., Allen, R. E., Robson, R. M., & Stromer, M. H. (1976). Some properties of purified skeletal muscle α actinin. Journal of Biological Chemistry, 251(21), 6860-6870.
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  PMID: 977599;Abstract: Highly purified α actinin can be made by using the low ionic strength extraction procedure and then subjecting the crude α actinin fraction obtained with this extraction procedure to successive chromatography on DEAE cellulose and hydroxyapatite. Hydroxyapatite chromatography specifically removes a protein having a subunit molecular weight of 42,000 on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Hydroxyapatite purified α actinin sediments entirely as a 6.21 S boundary in the analytical ultracentrifuge with no trace of the small 9 to 10 S boundary seen in earlier α actinin preparations purified by DEAE cellulose chromatography. In 100 mM KCl, 20 mM Tris.acetate, pH 7.5, hydroxyapatite purified α actinin has a diffusion coefficient (Do 20,w) of 2.71 x 10 -7 cm 2.s -1, an intrinsic viscosity of 20.6 ml.g -1, a molecular weight of 201,000 ± 4,300 (plus or minus least squares standard error) as determined by sedimentation equilibrium, and a molecular weight of 210,000 as determined by sedimentation diffusion. In 6 M guanidine HCl, hydroxyapatite purified α actinin has a molecular weight of 106,000 ± 6,300 as determined by sedimentation equilibrium and a molecular weight of 100,000 as determined by a calibrated 4% agarose gel permeation column. SDS polyacrylamide gel electrophoresis gives a molecular weight of 96,000 to 100,000 for hydroxyapatite purified α actinin. Rod shaped particles 44 x 390 to 400 Å are seen in electron micrographs of negatively stained α actinin. By assuming 45% hydration and a molecular weight of 206,000, dimensions of approximately 40 x 500Å can be calculated for the α actinin molecule by using either So 20,w, Do 20,w, intrinsic viscosity, or a calibrated 6% agarose gel permeation column. Hydroxyapatite purified α actinin has an α helical content of 74% as measured by circular dichroism at 208 nm.

Presentations

• Allen, R. E. (2011). Satellite Cell Therapy: Cell Delivery. Satellite Cell Therapy: Cell Delivery. Tucson, AZ.
• Allen, R. E. (2011). What Ever Happened to the Old Animal Husbandry Department. Arizona Cattlemen's convention.