Mechanical stretch induces activation of skeletal muscle satellite cells in vitro.

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.     

Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor

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 N^G-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. muscle regeneration     

Low-pH preparation of skeletal muscle satellite cells can be used to study activation in vitro

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.     

Calcium influx through a possible coupling of cation channels impacts skeletal muscle satellite cell activation in response to mechanical stretch

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.     

Activation of muscle satellite cells in single-fiber cultures.

Satellite stem cell activation is the process by which quiescent precursor cells resident on muscle fibers are recruited to cycle and move. Two processes are reported to affect satellite cell activation. In vivo, nitric oxide (NO) produced by NO synthase in fibers (NOS-Imu) promotes activation. In cell cultures, hepatocyte growth factor (HGF) is the major activating factor isolated from crushed muscle extract (CME). In this study we hypothesized that distinct and possibly related events were mediated by NO and HGF during activation. Intact fibers were cultured in the presence of bromodeoxyuridine (BrdU) to label DNA synthesis over 48 h. Experiments were designed to test the effects of CME, HGF, a NOS substrate L-arginine, and the NOS inhibitor L-NAME on activation, determined as the number of BrdU-positive satellite cells per fiber. Activation was increased significantly by CME, HGF, and L-arginine. L-Arginine increased activation in a dose-response manner. CME-induced activation was reduced significantly by NOS inhibition. Exposure to marcaine (10 min) caused reversible membrane damage without hypercontraction, as shown by characterizing the sarcolemmal integrity. The resulting decrease in satellite cell activation could be overcome by exogenous HGF. Results support the hypothesis that NO is involved in recruiting to cycle those satellite cells resident on fibers. Separate assessments of resident and free muscle cells showed that HGF and NO also participate in mobilizing satellite cells. Since HGF counteracted NOS inhibition and marcaine-induced membrane damage, data suggest that NO may mediate early steps in activation and precede HGF-mediated events.     

Matrix metalloproteinase-2 mediates stretch-induced activation of skeletal muscle satellite cells in a nitric oxide-dependent manner

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 microM sodium nitroprusside a NO donor or mechanical cyclic stretch for 2h 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 microM) to the stretch culture. The 52-kDa species was also generated by treatment of the recombinant MMP2 protein with 1 microM 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.     

C-Met expression and mechanical activation of satellite cells on cultured muscle fibers.

Single-fiber cultures can be used to model satellite cell activation in vivo. Although technical deficiencies previously prevented study of stretch-induced events, here we describe a method developed to study satellite cell gene expression by in situ hybridization (ISH) using protocol modifications for fiber adhesion and fixation. The hypothesis that mechanical stretching activates satellite cells was tested. Fiber cultures were established from normal flexor digitorum brevis muscles and plated on FlexCell dishes with a layer of Vitrogen. After 2 hr of stretch in the presence of BrdU, satellite cells on fibers attached to Vitrogen were activated above control levels. In the absence of activating treatments or mechanical stretch, ISH studies showed 0-6 c-Met+ satellite cells per fiber. Time course experiments demonstrated stable quiescence in the absence of stretch and significant peaks in activation after 30 min and 2 hr of stretch. Frequency distributions for unstretched fiber cultures showed a significantly greater number of quiescent c-Met+ satellite cells than were activated by stretching, suggesting that typical activation stimuli did not trigger cycling in the entire c-Met+ population of satellite cells. These methods have a strong potential to further dissect the nature of stretch-induced activation and gene expression among characterized populations of individual quiescent and activated satellite cells.     

Satellite cells are increasingly refractory to activation by nitric oxide and stretch in aged mouse-muscle cultures

Age-related muscle atrophy or sarcopenia results in progressive loss of muscle function and satellite cells in aging muscle are increasingly refractory to activation that could mitigate atrophy. We know that nitric oxide release triggered by mechanical stretch of skeletal muscle, initiates satellite cell activation in vitro in single fiber, single cell and whole-muscle cultures, and in vivo in animals. This study examined muscle cell activation using tritiated-thymidine incorporation into the DNA of muscle cells in cultured muscles from female mice between 6 weeks and 18 months-of-age. Experiments examined age-related changes in activation by mechanical stretch and/or NO treatments (with the substrate of nitric oxide synthase (l-arginine), a nitric oxide donor (isosorbide dinitrate) and/or nitric oxide synthase inhibition). Activation without stretch was highest at 8 months. Stretching muscles by 10% more than doubled activation in muscles at 6 weeks of age and only a 20% stretch similarly activated cells in cultured 6-month-old muscles. Only treatment with ISDN in combination with a 20% stretch activated cell proliferation in muscles from 8-month-old mice. A nitric-oxide donor drug rescued muscle satellite cells in adult, 8-month-old mice from being refractory to mechanical stretch, apparently by overcoming an ineffective release of nitric oxide during stretch. Results suggest that treatment with nitric oxide has the potential to enhance the effectiveness of exercise in preventing the onset of age-related muscle atrophy in adult muscle.     

Autocrine Phosphorylation of p70S6k in Response to Acute Stretch in Myotubes

Phosphorylation of 70-KDa S6 kinase (p70^S6k) is correlated with in vivo skeletal muscle hypertrophy. Experiments tested whether mechanical stretch is sufficient to increase p70^S6k phosphorylation in skeletal myotubes. Immediately following stretch, there was a small increase in p70^S6k phosphorylation (63.2 ± 8.5%) with maximal phosphorylation at 3 h (129.5 ± 22.2%) and it remained elevated through 24 h (46.0 ± 17.2%). To test whether an autocrine mechanism is involved, unstretched myotubes were incubated with medium from the stretch group for 10 min. Conditioned medium resulted in the phosphorylation of p70^S6k in unstretched myotubes (92.8 ± 28.9%) to levels comparable to the 3-h stretch group. These data indicate that p70^S6k is phosphorylated in stretched myotubes via a mechanism that most likely involves an autocrine signaling pathway.     

Satellite cell activation on fibers: modeling events in vivo--an invited review

Knowledge of the events underlying satellite cell activation and the counterpart maintenance of quiescence is essential for planning therapies that will promote the growth and regeneration of skeletal muscle in healthy, disease and aging. By modeling those events of satellite cell activation in studies of single muscle fibers or muscles in culture, the roles of mechanical stretching and nitric oxide are becoming understood. Recent studies demonstrated that stretch-induced activation is very rapid and exhibits some features of satellite cell heterogeneity. As well, gene expression studies showed that expression of the c-met receptor gene rises rapidly after stretching muscles in culture compared to those without stretch. This change in gene expression during activation, and the maintenance of quiescence in both normal and dystrophic muscles are dependent on NO, as they are blocked by inhibition of nitric oxide synthase (NOS). Mechanical, contractile activity is the defining feature of muscle function. Therefore, ongoing studies of stretch effects in satellite cell activation and quiescence in quiescent fiber and muscle cultures provides appropriate models by which to explore the regulatory steps in muscle in vivo under many conditions related to disease, repair, rehabilitation, growth and the prevention or treatment of atrophy.     

The dynamics of the nitric oxide release-transient from stretched muscle cells

Potent nitric oxide (NO) signals are described for many forms of cell-cell communication. Although NO plays a significant role in skeletal muscle metabolism and contractility and in precursor activation during muscle formation and stretching, there is no direct evidence of stretch-induced NO release from muscle. Differentiated muscle cell cultures from normal and dystrophic mdx mice were preloaded with the NO-specific dye DAF-2 (diaminofluorescein-2) before stretching. NO release was detected by video-microscopy. NO was released rapidly from wild-type (WT) cells after stretch and intensity declined rapidly to a plateau. Mdx cells showed much less NO release. Direct observations of the time-course of stretch-induced NO release in WT cells is congruent with the hypothesis of NO-mediated stretch activation of satellite cells in normal skeletal muscle. Distinct differences in the time-course between normal and dystrophic cells indicate visualization methods for NO release will be a sensitive measure of NOS-1 restoration following diverse treatment approaches to muscular dystrophy.     

Evidence that nitric oxide-induced synthesis of cGMP occurs in a paracrine but not an autocrine fashion and that the site of its release can be regulated: studies in dorsal root ganglia in vivo and in vitro.

As nitric oxide is a gas, it cannot be stored and has to be synthesized as required. This suggests that it could be released wherever nitric oxide synthase (NOS) is activated and due to its unstable state will react with appropriate targets at this site of production. In both dissociated dorsal root ganglion (DRG) cultures and in acutely isolated, but intact, DRG, treatment with capsaicin or bradykinin caused cGMP synthesis, which could be blocked by NOS inhibitors. The cGMP was synthesized in cells different from those expressing the neuronal isoform of NOS (nNOS). In dissociated cultures many of the cells stimulated to produce cGMP were neurons, whereas in isolated ganglia they were always satellite glia cells. Surprisingly, the satellite glia cells surrounding the nNOS-containing neurons did not contain cGMP. Following nerve section in adult rats, many axotomized ganglion neurons expressed nNOS. Again in these axotomized ganglia, most cGMP was expressed in the satellite glia surrounding nNOS-negative neurons. However, an nNOS-selective inhibitor reduced the cGMP present in these axotomized ganglia, suggesting that the cGMP synthesized is stimulated by NO (nitrogen monoxide) produced by nNOS. In both dissociated cultures and axotomized ganglia, nNOS-containing processes were observed close to cGMP-positive cells. These observations lead to the suggestion that NO acts in a paracrine fashion when stimulating the synthesis of cGMP and may not be synthesized at all sites containing nNOS.     

A role for nitric oxide in muscle repair: nitric oxide-mediated activation of muscle satellite cells

Muscle satellite cells are quiescent precursors interposed between myofibers and a sheath of external lamina. Although their activation and recruitment to cycle enable muscle repair and adaptation, the activation signal is not known. Evidence is presented that nitric oxide (NO) mediates satellite cell activation, including morphological hypertrophy and decreased adhesion in the fiber-lamina complex. Activation in vivo occurred within 1 min after injury. Cell isolation and histology showed that pharmacological inhibition of nitric oxide synthase (NOS) activity prevented the immediate injury-induced myogenic cell release and delayed the hypertrophy of satellite cells in that muscle. Transient activation of satellite cells in contralateral muscles 10 min later suggested that a circulating factor may interact with NO-mediated signaling. Interestingly, satellite cell activation in muscles of mdx dystrophic mice and NOS-I knockout mice quantitatively resembled NOS-inhibited release of normal cells, in agreement with reports of displaced and reduced NOS expression in dystrophin-deficient mdx muscle and the complete loss of NOS-I expression in knockout mice. Brief NOS inhibition in normal and mdx mice during injury produced subtle alterations in subsequent repair, including apoptosis in myotube nuclei and myotube formation inside laminar sheaths. Longer NOS inhibition delayed and restricted the extent of repair and resulted in fiber branching. A model proposes the hypothesis that NO release mediates satellite cell activation, possibly via shear-induced rapid increases in NOS activity that produce "NO transients."     

Stretch-induced growth in chicken wing muscles: role of soluble growth-promoting factors

The involvement of soluble growth-promoting factors in stretch-induced hypertrophy of the Patagialis muscle (PAT) in the chicken wing was investigated. Soluble extracts were prepared from young chicken PAT muscles made hypertrophic by passive stretch and from unstretched contralateral controls. Extracts were tested for their ability to stimulate cell proliferation and creatine phosphokinase (CPK) activity in primary monolayer cultures of chick embryo muscle cells. Factors were present in muscle extracts which showed a dose-dependent stimulation of cell proliferation and CPK activity in vitro. Passive stretch for 5 days produced a rapid hypertrophy of the PAT which was accompanied by a dramatic increase in the activity of the growth factor(s). Release of stretch resulted in an arrest of growth and an immediate fall in growth factor activity. The difference in growth-stimulating activity between control and stretched PAT extracts could be demonstrated in chicken transferrin-sensitive chick myoblast cultures. Stretch thus induces an increase in a class-specific growth factor, possibly Transferrin, in the PAT. Stretched PAT extracts stimulated: (a) chick myoblast proliferation to a greater extent than an optimum concentration of chick embryo extract, and (b) CPK activity in vitro to a greater extent than excess Transferrin. Both control and stretched PAT extracts supported the growth of rat myoblasts. We conclude that PAT muscle extracts also contain unknown growth factor(s) which are different from Transferrin.     

Fibroblast nemosis induces angiogenic responses of endothelial cells

Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-κB. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.     

Jamming dynamics of stretch-induced surfactant release by alveolar type II cells

Secretion of pulmonary surfactant by alveolar epithelial type II cells is vital for the reduction of interfacial surface tension, thus preventing lung collapse. To study secretion dynamics, rat alveolar epithelial type II cells were cultured on elastic membranes and cyclically stretched. The amounts of phosphatidylcholine, the primary lipid component of surfactant, inside and outside the cells, were measured using radiolabeled choline. During and immediately after stretch, cells secreted less surfactant than unstretched cells; however, stretched cells secreted significantly more surfactant than unstretched cells after an extended lag period. We developed a model based on the hypothesis that stretching leads to jamming of surfactant traffic escaping the cell, similar to vehicular traffic jams. In the model, stretch increases surfactant transport from the interior to the exterior of the cell. This transport is mediated by a surface layer with a finite capacity due to the limited number of fusion pores through which secretion occurs. When the amount of surfactant in the surface layer approaches this capacity, interference among lamellar bodies carrying surfactant reduces the rate of secretion, effectively creating a jam. When the stretch stops, the jam takes an extended time to clear, and subsequently the amount of secreted surfactant increases. We solved the model analytically and show that its dynamics are consistent with experimental observations, implying that surfactant secretion is a fundamentally nonlinear process with memory representing collective behavior at the level of single cells. Our results thus highlight the importance of a jamming dynamics in stretch-induced cellular secretory processes.     

Dynamic fibroblast cytoskeletal response to subcutaneous tissue stretch ex vivo and in vivo

Cytoskeleton-dependent changes in cell shape are well-established factors regulating a wide range of cellular functions including signal transduction, gene expression, and matrix adhesion. Although the importance of mechanical forces on cell shape and function is well established in cultured cells, very little is known about these effects in whole tissues or in vivo. In this study we used ex vivo and in vivo models to investigate the effect of tissue stretch on mouse subcutaneous tissue fibroblast morphology. Tissue stretch ex vivo (average 25% tissue elongation from 10 min to 2 h) caused a significant time-dependent increase in fibroblast cell body perimeter and cross-sectional area (ANOVA, P < 0.01). At 2 h, mean fibroblast cell body cross-sectional area was 201% greater in stretched than in unstretched tissue. Fibroblasts in stretched tissue had larger, "sheetlike" cell bodies with shorter processes. In contrast, fibroblasts in unstretched tissue had a "dendritic" morphology with smaller, more globular cell bodies and longer processes. Tissue stretch in vivo for 30 min had effects that paralleled those ex vivo. Stretch-induced cell body expansion ex vivo was inhibited by colchicine and cytochalasin D. The dynamic, cytoskeleton-dependent responses of fibroblasts to changes in tissue length demonstrated in this study have important implications for our understanding of normal movement and posture, as well as therapies using mechanical stimulation of connective tissue including physical therapy, massage, and acupuncture. mechanotransduction; connective tissue; tensegrity; musculoskeletal manipulations; acupuncture     

Multiple aspects of the phenotype of mammary epithelial cells transformed by expression of activated M-Ras depend on an autocrine mechanism mediated by hepatocyte growth factor/scatter factor

Multiple aspects of the transformed phenotype induced in a murine mammary epithelial cell line scp-2 by expression of activated G22V M-Ras, including maintainance of cell number at low density, anchorage-independent growth, invasion of Matrigel, and secretion of matrix metalloproteinases (MMP) 2 and 9, were dependent on an autocrine mechanism. Conditioned medium from dense cultures of scp-2 cells expressing G22V M-Ras, but not from parental cells, induced activation of Erk and Akt in cells expressing G22V M-Ras, maintained the cell number and promoted anchorage-independent growth of cells expressing G22V M-Ras (although not the parental cells), and induced scattering of MDCK cells. The latter activities were blocked by neutralizing antibodies to hepatocyte growth factor/scatter factor (HGF/SF) and could be mimicked by HGF/SF. Anti-HGF/SF antibodies also inhibited invasion of Matrigel, and the production of MMP-2 and MMP-9, together with urokinase-type plasminogen activator, was secreted by G22V M-Ras scp-2 cells but not by parental cells. Invasion of Matrigel was blocked by an inhibitor of MMPs, BB94, and by the mitogen-activated protein kinase kinase 1/2 kinase inhibitor PD98059 but was only marginally affected by the phosphatidylinositol 3-kinase inhibitor LY294002. Autocrine HGF/SF was thus critical for expression of key features of the phenotype of mammary epithelial cells transformed by expression of activated M-Ras.