Mai Khoi Q. Do

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.

Implication of anti-inflammatory macrophages in regenerative moto-neuritogenesis: Promotion of myoblast migration and neural chemorepellent semaphorin 3A expression in injured muscle

Regenerative mechanisms that regulate intramuscular motor innervation are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed a heretofore unexplored role of resident myogenic stem cell (satellite cell)-derived myoblasts as a key presenter of a secreted neural chemorepellent semaphorin 3A (Sema3A); hepatocyte growth factor (HGF) triggered its expression exclusively at the early-differentiation phase. In order to verify this concept, the present study was designed to clarify a paracrine source of HGF release. In vitro experiments demonstrated that activated anti-inflammatory macrophages (CD206-positive M2) produce HGF and thereby promote myoblast chemoattraction and Sema3A expression. Media from pro-inflammatory macrophage cultures (M1) did not show any significant effect. M2 also enhanced the expression of myoblast-differentiation markers in culture, and infiltrated predominantly at the early-differentiation phase (3-5 days post-injury); M2 were confirmed to produce HGF as monitored by in vivo/ex vivo immunocytochemistry of CD11b/CD206/HGF-positive cells and by HGF in situ hybridization of cardiotoxin- or crush-injured tibialis anterior muscle, respectively. These studies advance our understanding of the stage-specific activation of Sema3A expression signaling. Findings, therefore, encourage the idea that M2 contribute to spatiotemporal up-regulation of extracellular Sema3A concentrations by producing HGF that, in turn, stimulates a burst of Sema3A secretion by myoblasts that are recruited to site of injury. This model may ensure a coordinated delay in re-attachment of motoneuron terminals onto damaged fibers early in muscle regeneration, and thus synchronize the recovery of muscle-fiber integrity and the early resolution of inflammation after injury.

Time‐coordinated prevalence of extracellular HGF, FGF2 and TGF‐β3 in crush‐injured skeletal muscle

Successful regeneration and remodeling of neuromuscular junctions are critical for restoring functional capacities and properties of skeletal muscle after damage, and axon-guidance molecules may be involved in the signaling that regulates such restoration. Recently, we found that early-differentiated satellite cells up-regulate a secreted neural chemorepellent Sema3A upon in vivo muscle-crush injury. The study also revealed that Sema3A expression is up-regulated in primary satellite-cell cultures in response to hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF2) and is prevented by transforming growth factor (TGF)-β2, 3. In order to verify the physiological significance of this regulation in vitro, the present study was designed to estimate the time-course of extracellular HGF, FGF2 and TGF-β3 concentrations after crush-injury of Gastrocnemius muscle in the rat lower hind-limb, using a combination of a non-homogenization/non-spin extraction of extracellular wound fluids and enhanced chemiluminescence-Western blotting analyses. Results clearly demonstrated that active HGF and FGF2 are prevalent in 2-8 days post-crush, whereas active TGF-β3 increases after 12 days, providing a better understanding of the time-coordinated levels of HGF, FGF2 and TGF-β3 that drive regulation of Sema3A expression during regenerative intramuscular moto-neuritogenesis.

Comparative analysis of semaphorin 3A in soleus and EDL muscle satellite cells in vitro toward understanding its role in modulating myogenin expression.

Resident myogenic stem cells, satellite cells, up-regulate a secreted multi-functional modulator, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle-crush injury and treatment with hepatocyte growth factor (HGF) or basic fibroblast growth factor (FGF2). Here, we add evidence that the Sema3A expression and secretion induced by the growth factors is significantly higher in primary cultures from adult rat soleus than from the fast-twitch extensor digitorum longus (EDL) muscle. The higher Sema3A response, revealed by quantitative PCR and Western blotting of cell lysates and conditioned media, may account for the higher myogenin expression of soleus muscle satellite cells early in differentiation since addition of recombinant Sema3A stimulates myogenin expression in cultures. These experiments also showed that mRNA expression of plexin A2, which together with neuropilins, constitutes Sema3A composite-receptors, was higher in satellite cells from soleus than EDL with no difference in plexin A1 and A3 and neuropilin-1 and 2 levels. These comparative studies, therefore, highlight a possible Sema3A-plexin A2-myogenin signaling axis that may ensure promoting early differentiation by soleus muscle satellite cells.

Satellite cells produce neural chemorepellent semaphorin 3A upon muscle injury

Regenerative mechanisms that regulate intramuscular motor innervation. including configuration of the neuromuscular connections are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed a heretofore unexplored role of satellite cells as a key source of a secreted neural chemorepellent semaphorin 3A (Sema3A) expression. In order to verify this concept, there is still a critical need to provide direct evidence to show the up-regulation of Sema3A protein in satellite cells in vivo upon muscle injury. The present study employed a Sema3A/MyoD double-immunohistochemical staining for cryo-sections prepared from cardiotoxin injected gastrocnemius muscle of adult mouse lower hind-limb. Results clearly demonstrated that Sema3A expression was up-regulated in myogenic differentiation-positive satellite cells at 4-12 days post-injury period, the time that corresponds to the cell differentiation phase characterized by increasing myogenin messenger RNA expression. This direct proof encourages a possible implication of satellite cells in the spatiotemporal regulation of extracellular Sema3A concentrations, which potentially ensures coordinating a delay in neurite sprouting and re-attachment of motoneuron terminals onto damaged muscle fibers early in muscle regeneration in synchrony with recovery of muscle-fiber integrity.

Preliminary time-course study of antiinflammatory macrophage infiltration in crush-injured skeletal muscle

Muscle damage induces massive macrophage infiltration of the injury site, in which activated pro-inflammatory and anti-inflammatory phenotypes (currently classified as M1 and M2, respectively) have been documented as distinct functional populations predominant at different times after the conventional acute injury by intramuscular injection of snake venoms (cardiotoxin, notexin) or chemicals (bupivacaine hydrochloride, barium chloride). The present study employed a muscle-crush injury model that may better reflect the physiologic damage and repair processes initiated by contusing a gastrocnemius muscle in the lower hind-limb of adult mice with hemostat forceps, and examined the time-course invasion of M1 and M2 macrophages during muscle regeneration by immunocytochemistry of CD197 and CD206 marker proteins. CD197-positive M1 macrophages were observed exclusively at 1-4 days after crush followed by the alternative prevalence of CD206-positive M2 at 7 days of myogenic differentiation, characterized by increasing levels of myogenin messenger RNA expression. Preliminary PCR analysis showed that M2 may produce hepatocyte growth factor (HGF) in culture, providing additional benefit to understanding that M2 populations actively promote regenerative myogenesis (muscle fiber repair) and moto-neuritogenesis (re-attachment of motoneuron terminals onto damaged fibers) through their time-specific infiltration and release of growth factor at the injury site early in muscle regeneration.

Improvement of Endurance Based on Muscle Fiber-Type Composition by Treatment with Dietary Apple Polyphenols in Rats

A recent study demonstrated a positive effect of apple polyphenol (APP) intake on muscle endurance of young-adult animals. While an enhancement of lipid metabolism may be responsible, in part, for the improvement, the contributing mechanisms still need clarification. Here we show that an 8-week intake of 5% (w/w) APP in the diet, up-regulates two features related to fiber type: the ratio of myosin heavy chain (MyHC) type IIx/IIb and myoglobin protein expression in plantaris muscle of 9-week-old male Fischer F344 rats compared to pair-fed controls (P < 0.05). Results were demonstrated by our SDS-PAGE system specialized for MyHC isoform separation and western blotting of whole muscles. Animal-growth profiles (food intake, body-weight gain, and internal-organ weights) did not differ between the control and 5% APP-fed animals (n = 9/group). Findings may account for the increase in fatigue resistance of lower hind limb muscles, as evidenced by a slower decline in the maximum isometric planter-flexion torque generated by a 100-s train of electrical stimulation of the tibial nerve. Additionally, the fatigue resistance was lower after 8 weeks of a 0.5% APP diet than after 5% APP, supporting an APP-dose dependency of the shift in fiber-type composition. Therefore, the present study highlights a promising contribution of dietary APP intake to increasing endurance based on fiber-type composition in rat muscle. Results may help in developing a novel strategy for application in animal sciences, and human sports and age-related health sciences.

Supplementary immunocytochemistry of hepatocyte growth factor production in activated macrophages early in muscle regeneration.

Regenerative intramuscular motor-innervation is thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies showed that resident myogenic stem cells, satellite cells, up-regulate a secreted neural-chemorepellent semaphorin 3A (Sema3A) during the early-differentiation period, in response to hepatocyte growth factor (HGF) elevated in injured muscle. However, a paracrine source of the HGF release is still unknown. Very recently, we proposed a possible contribution of anti-inflammatory macrophages (CD206-positive M2) by showing that M2 cells infiltrate predominantly at the early-differentiation phase (3-5 days post-injury) and produce/secrete large amounts of HGF. However, in understanding this concept there still remains a critical need to examine if phagocytotic pro-inflammatory macrophages (CD86-positive M1), another activated-phenotype still present at the early-differentiation phase concerned, produce HGF upon muscle injury. The current immunocytochemical study demonstrated that the HGF expression is negative for M1 prepared from cardiotoxin-injured Tibialis anterior muscle at day 5, in contrast to the intense fluorescent-signal of M2 served as a positive control. This supplementary result advances our understanding of a spatiotemporal burst of HGF secretion from M2 populations (not M1) to impact Sema3A expression, which ensures a coordinated delay in attachment of motoneuron terminals onto damaged and generating fibers during the early phase of muscle regeneration.

Slow-Myofiber Commitment by Semaphorin 3A Secreted from Myogenic Stem Cells

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, cell‐membrane receptor (neuropilin2‐plexinA3) → myogenin‐myocyte enhancer factor 2D → 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‐neuropilin1/plexinA1, A2 may enhance slow‐fiber formation by activating signals that inhibit fast‐myosin expression. Importantly, satellite cell‐specific Sema3A conditional‐knockout adult mice (Pax7CreERT2‐Sema3Afl°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

In vitro measurement of post-natal changes in proliferating satellite cell frequency during rat muscle growth.

Satellite cells, resident myogenic stem cells found in postnatal skeletal muscle, are most abundant during early postnatal development and sharply decline in frequency thereafter to adult levels in mice and rats. Therefore, postnatal changes in satellite cell mitotic activities are important aspects for further understanding a muscle growth strategy. In large meat-production animals, however, the traditional in vivo proliferation assay may be less realistic because it requires intra-peritoneal (ip) injection of huge dosage of mutagenic nucleosides, (3)H-labeled thymidine or bromodeoxyuridine (BrdU), at each age-time of sacrifice. We report in the present pilot study using rats that in vivo proliferation activity of satellite cells can be evaluated by an in vitro BrdU-incorporation assay in early cultures. Briefly, satellite cells were prepared from upper hind-limb and back muscles and maintained for 24 h with imposing by BrdU addition for the last 2 h, followed by the regular immunocytochemistry for determining BrdU-incorporated cell percentage. This in vitro assay demonstrated a rapid decrease in proliferating satellite cell frequency to the adult level during about 3-month period after birth, and yielded a high correlation to the measurements by the in vivo BrdU ip-injection method during the postnatal period examined from day-2 to month-11. The in vitro proliferation assay may be further adaptable for large domestic animals by the combination with a muscle biopsy technique that enables age-interval sampling from the same growing animals.