Hideaki Ohtsubo

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.

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.

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.

APOBEC2 negatively regulates myoblast differentiation in muscle regeneration

Recently we found that the deficiency of APOBEC2, a member of apoB mRNA editing enzyme, catalytic polypeptide-like family, leads to a diminished muscle mass and increased myofiber with centrally-located nuclei known as dystrophic phenotypes. APOBEC2 expression is predominant in skeletal and cardiac muscles and elevated exclusively at the early-differentiation phase of wild-type (WT) myoblast cultures; however the physiological significance is still un-known. Here we show that APOBEC2 is a key negative regulator of myoblast differentiation in muscle regeneration. APOBEC2-knockout (A2KO) mice myoblast cultures displayed a normal morphology of primary myotubes along with earlier increase in fusion index and higher expression levels of myosin heavy chain (MyHC), myogenin and its cooperating factor MEF2C than WT myoblasts. Similar response was observable in APOBEC2-knockdown cultures of WT myoblasts that were transfected with the specific siRNA at the differentiation phase (not proliferation phase). Importantly, cardiotoxin-injured A2KO gastrocnemius muscle provided in vivo evidence by showing larger up-regulation of neonatal MyHC and myogenin and hence earlier regeneration of myofiber structures with diminished cross-sectional areas and minimal Feret diameters. Therefore, the findings highlight a promising role for APOBEC2 in normal progression of regenerative myogenesis at the early-differentiation phase upon muscle injury.

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

Transmembrane proteoglycans syndecan‐2, 4, receptor candidates for the impact of HGF and FGF2 on semaphorin 3A expression in early‐differentiated myoblasts

Regenerative mechanisms that regulate intramuscular motor innervation are thought to reside in the spatiotemporal expression of axon‐guidance molecules. Our previous studies proposed an 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) and basic fibroblast growth factor (FGF2) triggered its expression exclusively at the early differentiation phase. In order to advance this concept, the present study described that transmembrane heparan/chondroitin sulfate proteoglycans syndecan‐2, 4 may be the plausible receptor candidates for HGF and FGF2 to signal Sema3A expression. Results showed that mRNA expression of syndecan‐2, 4 was abundant (two magnitudes higher than syndecan‐1, 3) in early‐differentiated myoblasts and their in vitro knockdown diminished the HGF/FGF2‐induced expression of Sema3A down to a baseline level. Pretreatment with heparitinase and chondroitinase ABC decreased the HGF and FGF2 responses, respectively, in non–knockdown cultures, supporting a possible model that HGF and FGF2 may bind to heparan and chondroitin sulfate chains of syndecan‐2, 4 to signal Sema3A expression. The findings, therefore, extend our understanding that HGF/FGF2‐syndecan‐2, 4 association may stimulate a burst of Sema3A secretion by myoblasts recruited to the site of muscle injury; this would ensure a coordinated delay in the attachment of motoneuron terminals onto fibers early in muscle regeneration, and thus synchronize the recovery of muscle fiber integrity and the early resolution of inflammation after injury with reinnervation toward functional recovery.

Data supporting possible implication of APOBEC2 in self-renewal functions of myogenic stem satellite cells: Toward understanding the negative regulation of myoblast differentiation

This paper provides in vitro phenotypical data to show that APOBEC2, a member of apoB mRNA editing enzyme, catalytic polypeptide-like family, may implicate in self-renewal functions of myogenic stem satellite cells, namely in the re-establishment of quiescent status after activation and proliferation of myoblasts in single-myofiber culture.

Apobec2 deficiency causes mitochondrial defects and mitophagy in skeletal muscle

Apobec2 is a member of the activation‐induced deaminase/apolipoprotein B mRNA editing enzyme catalytic polypeptide cytidine deaminase family expressed in differentiated skeletal and cardiac muscle. We previously reported that Apobec2 deficiency in mice leads to a shift in muscle fiber type, myopathy, and diminished muscle mass. However, the mechanisms of myopathy caused by Apobec2 deficiency and its physiologic functions are unclear. Here we show that, although Apobec2 localizes to the sarcomeric Z‐lines in mouse tissue and cultured myotubes, the sarcomeric structure is not affected in Apobec2‐deficient muscle. In contrast, electron microscopy reveals enlarged mitochondria and mitochondria engulfed by autophagic vacuoles, suggesting that Apobec2 deficiency causes mitochondrial defects leading to increased mitophagy in skeletal muscle. Indeed, Apobec2 deficiency results in increased reactive oxygen species generation and depolarized mitochondria, leading to mitophagy as a defensive response. Furthermore, the exercise capacity of Apobec2−/−mice is impaired, implying Apobec2 deficiency results in ongoing muscle dysfunction. The presence of rimmed vacuoles in myofibers from 10‐mo‐old mice suggests that the chronic muscle damage impairs normal autophagy. We conclude that Apobec2 deficiency causes mitochondrial defects that increase muscle mitophagy, leading to myopathy and atrophy. Our findings demonstrate that Apobec2 is required for mitochondrial homeostasis to maintain normal skeletal muscle function.—Sato, Y., Ohtsubo, H., Nihei, N., Kaneko, T., Sato, Y., Adachi, S.‐I., Kondo, S., Nakamura, M., Mizunoya, W., Iida, H., Tatsumi, R., Rada, C., Yoshizawa, F. Apobec2 deficiency causes mitochondrial defects and mitophagy in skeletal muscle. FASEB J. 32,1428‐1439 (2018). www.fasebj.org

Fast-to-slow shift of muscle fiber-type composition by dietary apple polyphenols in rats: Impact of the low-dose supplementation.

Our previous studies demonstrated that an 8-week intake of 5% (w/w) apple polyphenol (APP) in the diet improves muscle endurance of young-adult rats. In order to identify a lower limit of the dietary contribution of APP to the effect, the experiments were designed for lower-dose supplementation (8-week feeding of 0.5% APP in AIN-93G diet) to 12-week-old male Sprague-Dawley rats. Results clearly showed that the 0.5% APP diet significantly up-regulates slower myosin-heavy-chain (MyHC) isoform ratios (IIx and IIa relative to total MyHC) and myoglobin expression in lower hind-limb muscles examined (P < 0.05). There was a trend to increased fatigue resistance detected from measurements of relative isometric plantar-flexion force torque generated by a stimulus train delivered to the tibial nerve (F(98, 1372) = 1.246, P = 0.0574). Importantly, there was no significant difference in the animal body-phenotypes or locomotor activity shown as total moving distance in light and dark periods. Therefore, the present study encourages the notion that even low APP-intake may increase the proportions of fatigue-resistant myofibers, and has promise as a strategy for modifying performance in human sports and improving function in age-related muscle atrophy.