Skeletal muscle satellite cells play an important role in muscle regeneration. Previous work has ... more 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.
It has previously been shown that mechanical stretch induces activation of cultured quiescent sat... more 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.
Recently, we found that resident myogenic stem satellite cells upregulate a multi-functional secr... more Recently, we found that resident myogenic stem satellite cells upregulate a multi-functional secreted protein, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle injury; however, its physiological significance is still unknown. Here we show that Sema3A impacts slow-twitch fiber generation through a signaling pathway, cellmembrane receptor (neuropilin2-plexinA3) fi myogenin-myocyte enhancer factor 2D fi slow myosin heavy chain. This novel axis was found by small interfering RNA-transfection experiments in myoblast cultures, which also revealed an additional element that Sema3A-neuropi-lin1/plexinA1, A2 may enhance slow-fiber formation by activating signals that inhibit fastmyosin expression. Importantly, satellite cell-specific Sema3A conditional-knockout adult mice (Pax7CreER T2-Sema3A fl 8 x activated by tamoxifen-i.p. injection) provided direct in vivo evidence for the Sema3A-driven program, by showing that slow-fiber generation and muscle endurance were diminished after repair from cardiotoxin-injury of gastrocnemius muscle. Overall, the findings highlight an active role for satellite cell-secreted Sema3A ligand as a key "commitment factor" for the slow-fiber population during muscle regeneration. Results extend our understanding of the myogenic stem-cell strategy that regulates fiber-type differentiation and is responsible for skeletal muscle contractility, energy metabolism, fatigue resistance, and its susceptibility to aging and disease. STEM CELLS 2017;35:1815-1834 SIGNIFICANCE STATEMENT This study demonstrates that the semaphorin 3A ligand secreted from the early-differentiated myogenic stem satellite cells, is a key "commitment factor" as it establishes the slow-fiber population during muscle regeneration through signaling from a cell-membrane receptor (neuropi-lin2-plexinA3) ! myogenin ! slow myosin. The pathway appears to work independently of innervation status or transcriptional circuitry centered on PGC1a/PPARd elements, and also operates very early, before these well-known systems are established in regenerating muscle and contribute to fiber-type maturation and maintenance. We anticipate that slow-fiber generation can be enhanced with agonists of the receptor neuropilin2-plexinA3 (including natural agonists in food ingredients). This work on stem-cell biology is fundamental to muscle function, and has major implications for developing novel strategies in health sciences for promoting slow-fiber expression in humans to combat sarcopenia, promote muscle endurance and potentially slow the progression of amyotrophic lateral sclerosis (ALS), and importantly for strategies to sustain food security through meat-animal production.
Application of mechanical stretch to cultured adult rat muscle satellite cells results in release... more 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 N-nitrol-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.
We have shown that hepatocyte growth factor/scatter factor can stimulate activation and early div... more 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.
Mechanical stretch induces activation of cultured quiescent satellite cells and the activation re... more Mechanical stretch induces activation of cultured quiescent satellite cells and the activation response is owing to rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c-met receptor. We provide new evidence that the stretch activation is dependent on nitric oxide (NO) production. Stretch activation could be abolished by the addition of N G-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NO synthesis, but not by N G-nitro-D-arginine methyl ester hydrochloride, a less active enantiomer of L-NAME. Adding HGF to the L-NAME culture restored the activation response, indicating that L-NAME does not directly inhibit satellite cell activation, but acts upstream from the HGF release. In addition, immunoblots of satellite cell lysate revealed the presence of nitric oxide synthase. These experiments suggest that NO is involved in linking mechanical perturbation of satellite cells to chemical signaling responsible for HGF release from its sequestration in vitro.
Satellite cells, resident myogenic stem cells found between the basement membrane and the sarcole... more 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.
The International Journal of Biochemistry & Cell Biology, 2006
When skeletal muscle is stretched or injured, satellite cells are activated to enter the cell cyc... more 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. [
When skeletal muscle is stretched or injured, satellite cells are activated to proliferate, and t... more 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. Muscle Nerve, 2004
When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the... more 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, NO x 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.
The International Journal of Biochemistry & Cell Biology, 2008
When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the... more 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 [
American Journal of Physiology-cell Physiology, Jun 1, 2006
Ikeuchi, and Ronald E. Allen. Satellite cell activation in stretched skeletal muscle and the role... more Ikeuchi, and Ronald E. Allen. Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor.
American Journal of Physiology-cell Physiology, Jun 15, 2012
Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite c... more Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch.
The International Journal of Biochemistry & Cell Biology, 2008
When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the... more 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 [
Skeletal muscle regeneration is a multifaceted process requiring the spatial and temporal coordin... more 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% O 2) or hypoxic (1% O 2) conditions. Hypoxic CM recapitulated a MVF angiogenic response identical to HGF deficient satellite cell CM. Hypoxic conditions increased satellite cell HIF-1a 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.
Cultured quiescent satellite cells were subjected to mechanical stretch in a FlexerCell System. I... more 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.
It has previously been shown that mechanical stretch induces activation of cultured quiescent sat... more 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.
ABSTRACTSatellite cells, resident myogenic stem cells found between the basement membrane and the... more ABSTRACTSatellite 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 stimul...
Mechanical stretch induces activation of cultured quiescent satellite cells and the activation re... more Mechanical stretch induces activation of cultured quiescent satellite cells and the activation response is owing to rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c-met receptor. We provide new evidence that the stretch activation is dependent on nitric oxide (NO) production. Stretch activation could be abolished by the addition of N G-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NO synthesis, but not by N G-nitro-D-arginine methyl ester hydrochloride, a less active enantiomer of L-NAME. Adding HGF to the L-NAME culture restored the activation response, indicating that L-NAME does not directly inhibit satellite cell activation, but acts upstream from the HGF release. In addition, immunoblots of satellite cell lysate revealed the presence of nitric oxide synthase. These experiments suggest that NO is involved in linking mechanical perturbation of satellite cells to chemical signaling responsible for HGF release from its sequestration in vitro.
Skeletal muscle satellite cells play an important role in muscle regeneration. Previous work has ... more 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.
It has previously been shown that mechanical stretch induces activation of cultured quiescent sat... more 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.
Recently, we found that resident myogenic stem satellite cells upregulate a multi-functional secr... more Recently, we found that resident myogenic stem satellite cells upregulate a multi-functional secreted protein, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle injury; however, its physiological significance is still unknown. Here we show that Sema3A impacts slow-twitch fiber generation through a signaling pathway, cellmembrane receptor (neuropilin2-plexinA3) fi myogenin-myocyte enhancer factor 2D fi slow myosin heavy chain. This novel axis was found by small interfering RNA-transfection experiments in myoblast cultures, which also revealed an additional element that Sema3A-neuropi-lin1/plexinA1, A2 may enhance slow-fiber formation by activating signals that inhibit fastmyosin expression. Importantly, satellite cell-specific Sema3A conditional-knockout adult mice (Pax7CreER T2-Sema3A fl 8 x activated by tamoxifen-i.p. injection) provided direct in vivo evidence for the Sema3A-driven program, by showing that slow-fiber generation and muscle endurance were diminished after repair from cardiotoxin-injury of gastrocnemius muscle. Overall, the findings highlight an active role for satellite cell-secreted Sema3A ligand as a key "commitment factor" for the slow-fiber population during muscle regeneration. Results extend our understanding of the myogenic stem-cell strategy that regulates fiber-type differentiation and is responsible for skeletal muscle contractility, energy metabolism, fatigue resistance, and its susceptibility to aging and disease. STEM CELLS 2017;35:1815-1834 SIGNIFICANCE STATEMENT This study demonstrates that the semaphorin 3A ligand secreted from the early-differentiated myogenic stem satellite cells, is a key "commitment factor" as it establishes the slow-fiber population during muscle regeneration through signaling from a cell-membrane receptor (neuropi-lin2-plexinA3) ! myogenin ! slow myosin. The pathway appears to work independently of innervation status or transcriptional circuitry centered on PGC1a/PPARd elements, and also operates very early, before these well-known systems are established in regenerating muscle and contribute to fiber-type maturation and maintenance. We anticipate that slow-fiber generation can be enhanced with agonists of the receptor neuropilin2-plexinA3 (including natural agonists in food ingredients). This work on stem-cell biology is fundamental to muscle function, and has major implications for developing novel strategies in health sciences for promoting slow-fiber expression in humans to combat sarcopenia, promote muscle endurance and potentially slow the progression of amyotrophic lateral sclerosis (ALS), and importantly for strategies to sustain food security through meat-animal production.
Application of mechanical stretch to cultured adult rat muscle satellite cells results in release... more 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 N-nitrol-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.
We have shown that hepatocyte growth factor/scatter factor can stimulate activation and early div... more 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.
Mechanical stretch induces activation of cultured quiescent satellite cells and the activation re... more Mechanical stretch induces activation of cultured quiescent satellite cells and the activation response is owing to rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c-met receptor. We provide new evidence that the stretch activation is dependent on nitric oxide (NO) production. Stretch activation could be abolished by the addition of N G-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NO synthesis, but not by N G-nitro-D-arginine methyl ester hydrochloride, a less active enantiomer of L-NAME. Adding HGF to the L-NAME culture restored the activation response, indicating that L-NAME does not directly inhibit satellite cell activation, but acts upstream from the HGF release. In addition, immunoblots of satellite cell lysate revealed the presence of nitric oxide synthase. These experiments suggest that NO is involved in linking mechanical perturbation of satellite cells to chemical signaling responsible for HGF release from its sequestration in vitro.
Satellite cells, resident myogenic stem cells found between the basement membrane and the sarcole... more 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.
The International Journal of Biochemistry & Cell Biology, 2006
When skeletal muscle is stretched or injured, satellite cells are activated to enter the cell cyc... more 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. [
When skeletal muscle is stretched or injured, satellite cells are activated to proliferate, and t... more 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. Muscle Nerve, 2004
When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the... more 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, NO x 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.
The International Journal of Biochemistry & Cell Biology, 2008
When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the... more 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 [
American Journal of Physiology-cell Physiology, Jun 1, 2006
Ikeuchi, and Ronald E. Allen. Satellite cell activation in stretched skeletal muscle and the role... more Ikeuchi, and Ronald E. Allen. Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor.
American Journal of Physiology-cell Physiology, Jun 15, 2012
Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite c... more Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch.
The International Journal of Biochemistry & Cell Biology, 2008
When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the... more 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 [
Skeletal muscle regeneration is a multifaceted process requiring the spatial and temporal coordin... more 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% O 2) or hypoxic (1% O 2) conditions. Hypoxic CM recapitulated a MVF angiogenic response identical to HGF deficient satellite cell CM. Hypoxic conditions increased satellite cell HIF-1a 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.
Cultured quiescent satellite cells were subjected to mechanical stretch in a FlexerCell System. I... more 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.
It has previously been shown that mechanical stretch induces activation of cultured quiescent sat... more 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.
ABSTRACTSatellite cells, resident myogenic stem cells found between the basement membrane and the... more ABSTRACTSatellite 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 stimul...
Mechanical stretch induces activation of cultured quiescent satellite cells and the activation re... more Mechanical stretch induces activation of cultured quiescent satellite cells and the activation response is owing to rapid release of hepatocyte growth factor (HGF) from its extracellular association with satellite cells and its subsequent presentation to the c-met receptor. We provide new evidence that the stretch activation is dependent on nitric oxide (NO) production. Stretch activation could be abolished by the addition of N G-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NO synthesis, but not by N G-nitro-D-arginine methyl ester hydrochloride, a less active enantiomer of L-NAME. Adding HGF to the L-NAME culture restored the activation response, indicating that L-NAME does not directly inhibit satellite cell activation, but acts upstream from the HGF release. In addition, immunoblots of satellite cell lysate revealed the presence of nitric oxide synthase. These experiments suggest that NO is involved in linking mechanical perturbation of satellite cells to chemical signaling responsible for HGF release from its sequestration in vitro.
Uploads
Papers by Ronald Allen