Publications by authors named "So-Ichiro Fukada"

76 Publications

Myofiber androgen receptor increases muscle strength mediated by a skeletal muscle splicing variant of Mylk4.

iScience 2021 Apr 13;24(4):102303. Epub 2021 Mar 13.

Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan.

Androgens have a robust effect on skeletal muscles to increase muscle mass and strength. The molecular mechanism of androgen/androgen receptor (AR) action on muscle strength is still not well known, especially for the regulation of sarcomeric genes. In this study, we generated androgen-induced hypertrophic model mice, myofiber-specific androgen receptor knockout (cARKO) mice supplemented with dihydrotestosterone (DHT). DHT treatment increased grip strength in control mice but not in cARKO mice. Transcriptome analysis by RNA-seq, using skeletal muscles obtained from control and cARKO mice treated with or without DHT, identified a fast-type muscle-specific novel splicing variant of as a target of AR in skeletal muscles. knockout mice exhibited decreased maximum isometric torque of plantar flexion and passive stiffness of myofibers due to reduced phosphorylation of Myomesin 1 protein. This study suggests that androgen-induced skeletal muscle strength is mediated with Mylk4 and Myomesin 1 axis.
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http://dx.doi.org/10.1016/j.isci.2021.102303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041868PMC
April 2021

DNA maintenance methylation enzyme Dnmt1 in satellite cells is essential for muscle regeneration.

Biochem Biophys Res Commun 2021 01 10;534:79-85. Epub 2020 Dec 10.

Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University, Shitsukawa, Toon Ehime, 791-0295, Japan; Department of Pathophysiology, Ehime University Graduate School of Medicine, Shitsukawa, Toon Ehime, 791-0295, Japan; Division of Laboratory Animal Research, Advanced Research Support Center, Ehime University, Shitsukawa, Toon Ehime, 791-0295, Japan. Electronic address:

Epigenetic transcriptional regulation is essential for the differentiation of various types of cells, including skeletal muscle cells. DNA methyltransferase 1 (Dnmt1) is responsible for maintenance of DNA methylation patterns via cell division. Here, we investigated the relationship between Dnmt1 and skeletal muscle regeneration. We found that Dnmt1 is upregulated in muscles during regeneration. To assess the role of Dnmt1 in satellite cells during regeneration, we performed conditional knockout (cKO) of Dnmt1 specifically in skeletal muscle satellite cells using Pax7 mice and Dnmt1 flox mice. Muscle weight and the cross-sectional area after injury were significantly lower in Dnmt1 cKO mice than in control mice. RNA sequencing analysis revealed upregulation of genes involved in cell adhesion and apoptosis in satellite cells from cKO mice. Moreover, satellite cells cultured from cKO mice exhibited a reduced number of cells. These results suggest that Dnmt1 is an essential factor for muscle regeneration and is involved in positive regulation of satellite cell number.
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http://dx.doi.org/10.1016/j.bbrc.2020.11.116DOI Listing
January 2021

Dlk1 regulates quiescence in calcitonin receptor-mutant muscle stem cells.

Stem Cells 2021 03 8;39(3):306-317. Epub 2020 Dec 8.

Project for Muscle Stem Cell Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan.

Muscle stem cells, also called muscle satellite cells (MuSCs), are responsible for skeletal muscle regeneration and are sustained in an undifferentiated and quiescent state under steady conditions. The calcitonin receptor (CalcR)-protein kinase A (PKA)-Yes-associated protein 1 (Yap1) axis is one pathway that maintains quiescence in MuSCs. Although CalcR signaling in MuSCs has been identified, the critical CalcR signaling targets are incompletely understood. Here, we show the relevance between the ectopic expression of delta-like non-canonical Notch ligand 1 (Dlk1) and the impaired quiescent state in CalcR-conditional knockout (cKO) MuSCs. Dlk1 expression was rarely detected in both quiescent and proliferating MuSCs in control mice, whereas Dlk1 expression was remarkably increased in CalcR-cKO MuSCs at both the mRNA and protein levels. It is noteworthy that all Ki67 non-quiescent CalcR-cKO MuSCs express Dlk1, and non-quiescent CalcR-cKO MuSCs are enriched in the Dlk1 fraction by cell sorting. Using mutant mice, we demonstrated that PKA-activation or Yap1-depletion suppressed Dlk1 expression in CalcR-cKO MuSCs, which suggests that the CalcR-PKA-Yap1 axis inhibits the expression of Dlk1 in quiescent MuSCs. Moreover, the loss of Dlk1 rescued the quiescent state in CalcR-cKO MuSCs, which indicates that the ectopic expression of Dlk1 disturbs quiescence in CalcR-cKO. Collectively, our results suggest that ectopically expressed Dlk1 is responsible for the impaired quiescence in CalcR-cKO MuSCs.
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http://dx.doi.org/10.1002/stem.3312DOI Listing
March 2021

Mesenchymal Bmp3b expression maintains skeletal muscle integrity and decreases in age-related sarcopenia.

J Clin Invest 2021 Jan;131(1)

Division for Therapies against Intractable Diseases, Institute for Comprehensive Medical Science (ICMS), Fujita Health University, Toyoake, Japan.

Age-related sarcopenia constitutes an important health problem associated with adverse outcomes. Sarcopenia is closely associated with fat infiltration in muscle, which is attributable to interstitial mesenchymal progenitors. Mesenchymal progenitors are nonmyogenic in nature but are required for homeostatic muscle maintenance. However, the underlying mechanism of mesenchymal progenitor-dependent muscle maintenance is not clear, nor is the precise role of mesenchymal progenitors in sarcopenia. Here, we show that mice genetically engineered to specifically deplete mesenchymal progenitors exhibited phenotypes markedly similar to sarcopenia, including muscle weakness, myofiber atrophy, alterations of fiber types, and denervation at neuromuscular junctions. Through searching for genes responsible for mesenchymal progenitor-dependent muscle maintenance, we found that Bmp3b is specifically expressed in mesenchymal progenitors, whereas its expression level is significantly decreased during aging or adipogenic differentiation. The functional importance of BMP3B in maintaining myofiber mass as well as muscle-nerve interaction was demonstrated using knockout mice and cultured cells treated with BMP3B. Furthermore, the administration of recombinant BMP3B in aged mice reversed their sarcopenic phenotypes. These results reveal previously unrecognized mechanisms by which the mesenchymal progenitors ensure muscle integrity and suggest that age-related changes in mesenchymal progenitors have a considerable impact on the development of sarcopenia.
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http://dx.doi.org/10.1172/JCI139617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773381PMC
January 2021

Author Correction: Adiponectin promotes muscle regeneration through binding to T-cadherin.

Sci Rep 2020 Jul 17;10(1):12219. Epub 2020 Jul 17.

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-66545-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367247PMC
July 2020

Role of damage and management in muscle hypertrophy: Different behaviors of muscle stem cells in regeneration and hypertrophy.

Biochim Biophys Acta Mol Cell Res 2020 09 14;1867(9):118742. Epub 2020 May 14.

Institut National de la Santé et de la Recherche Médicale U1016, Institut Cochin, Paris, France.

Skeletal muscle is a dynamic tissue with two unique abilities; one is its excellent regenerative ability, due to the activity of skeletal muscle-resident stem cells named muscle satellite cells (MuSCs); and the other is the adaptation of myofiber size in response to external stimulation, intrinsic factors, or physical activity, which is known as plasticity. Low physical activity and some disease conditions lead to the reduction of myofiber size, called atrophy, whereas hypertrophy refers to the increase in myofiber size induced by high physical activity or anabolic hormones/drugs. MuSCs are essential for generating new myofibers during regeneration and the increase in new myonuclei during hypertrophy; however, there has been little investigation of the molecular mechanisms underlying MuSC activation, proliferation, and differentiation during hypertrophy compared to those of regeneration. One reason is that 'degenerative damage' to myofibers during muscle injury or upon hypertrophy (especially overloaded muscle) is believed to trigger similar activation/proliferation of MuSCs. However, evidence suggests that degenerative damage of myofibers is not necessary for MuSC activation/proliferation during hypertrophy. When considering MuSC-based therapy for atrophy, including sarcopenia, it will be indispensable to elucidate MuSC behaviors in muscles that exhibit non-degenerative damage, because degenerated myofibers are not present in the atrophied muscles. In this review, we summarize recent findings concerning the relationship between MuSCs and hypertrophy, and discuss what remains to be discovered to inform the development and application of relevant treatments for muscle atrophy.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118742DOI Listing
September 2020

Methods for Accurate Assessment of Myofiber Maturity During Skeletal Muscle Regeneration.

Front Cell Dev Biol 2020 22;8:267. Epub 2020 Apr 22.

Muscle Aging and Regenerative Medicine, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.

Adult skeletal muscle has a remarkable ability to regenerate. Regeneration of mature muscle fibers is dependent on muscle stem cells called satellite cells. Although they are normally in a quiescent state, satellite cells are rapidly activated after injury, and subsequently proliferate and differentiate to make new muscle fibers. Myogenesis is a highly orchestrated biological process and has been extensively studied, and therefore many parameters that can precisely evaluate regenerating events have been established. However, in some cases, it is necessary to evaluate the completion of regeneration rather than ongoing regeneration. In this study, we establish methods for assessing the myofiber maturation during muscle regeneration. By carefully comparing expression patterns of several muscle regeneration-related genes, we found that expression of ( and ), (), and () is gradually increased as muscle regeneration proceeds. In contrast, commonly used regeneration markers such as and are transiently upregulated after muscle injury but their expression decreases as regeneration progresses. Intriguingly, upregulation of , and cannot be achieved in cultured myotubes, indicating that these markers are excellent indicators to assess myofiber maturity. We also show that analyzing re-expression of Myoz1 and dystrophin in individual fiber during regeneration enables accurate assessment of myofiber maturity at the single-myofiber level. Together, our study provides valuable methods that are useful in evaluating muscle regeneration and the efficacy of therapeutic strategies for muscle diseases.
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http://dx.doi.org/10.3389/fcell.2020.00267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188918PMC
April 2020

Calcitonin Receptor Neurons in the Mouse Nucleus Tractus Solitarius Control Energy Balance via the Non-aversive Suppression of Feeding.

Cell Metab 2020 02 16;31(2):301-312.e5. Epub 2020 Jan 16.

Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48105, USA; Division of Endocrinology, Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI 48105, USA; Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48105, USA; Graduate Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48105, USA. Electronic address:

To understand hindbrain pathways involved in the control of food intake, we examined roles for calcitonin receptor (CALCR)-containing neurons in the NTS. Ablation of NTS Calcr abrogated the long-term suppression of food intake, but not aversive responses, by CALCR agonists. Similarly, activating Calcr neurons decreased food intake and body weight but (unlike neighboring Cck cells) failed to promote aversion, revealing that Calcr neurons mediate a non-aversive suppression of food intake. While both Calcr and Cck neurons decreased feeding via projections to the PBN, Cck cells activated aversive CGRP cells while Calcr cells activated distinct non-CGRP PBN cells. Hence, Calcr cells suppress feeding via non-aversive, non-CGRP PBN targets. Additionally, silencing Calcr cells blunted food intake suppression by gut peptides and nutrients, increasing food intake and promoting obesity. Hence, Calcr neurons define a hindbrain system that participates in physiological energy balance and suppresses food intake without activating aversive systems.
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http://dx.doi.org/10.1016/j.cmet.2019.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104375PMC
February 2020

The CalcR-PKA-Yap1 Axis Is Critical for Maintaining Quiescence in Muscle Stem Cells.

Cell Rep 2019 11;29(8):2154-2163.e5

Project for Muscle Stem Cell Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Quiescence is a fundamental property of adult stem cells. Recent evidence indicates that quiescence is not a default state but requires active signaling that prevents accidental or untimely activation of stem cells. The calcitonin receptor (CalcR) is critical for sustaining quiescence in muscle satellite (stem) cells (MuSCs). However, the molecular mechanisms by which CalcR signaling regulates quiescence in MuSCs are enigmatic. Here, we demonstrate that transgenic expression of the catalytic domain of protein kinase A (PKA) restores the quiescence of CalcR-mutant MuSCs and delays MuSC activation. Mechanistically, CalcR-activated PKA phosphorylates Lats1/2, the main effector of Hippo signaling, thereby inhibiting the nuclear accumulation of Yap1, which prevents expression of Hippo-target genes, including cell-cycle-related molecules. Importantly, genetic inactivation of Yap1 in CalcR-mutant MuSCs reinstates quiescence in CalcR-mutant MuSCs, indicating that the CalcR-PKA-Lats1/2-Yap1 axis plays a critical role in sustaining MuSC quiescence.
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http://dx.doi.org/10.1016/j.celrep.2019.10.057DOI Listing
November 2019

Sustained expression of HeyL is critical for the proliferation of muscle stem cells in overloaded muscle.

Elife 2019 09 23;8. Epub 2019 Sep 23.

Project for Muscle Stem Cell Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan.

In overloaded and regenerating muscle, the generation of new myonuclei depends on muscle satellite cells (MuSCs). Because MuSC behaviors in these two environments have not been considered separately, MuSC behaviors in overloaded muscle remain unexamined. Here, we show that most MuSCs in overloaded muscle, unlike MuSCs in regenerating muscle, proliferate in the absence of MyoD expression. Mechanistically, MuSCs in overloaded muscle sustain the expression of , a Notch effector gene, to suppress MyoD expression, which allows effective MuSC proliferation on myofibers and beneath the basal lamina. Although -knockout mice show no impairment in an injury model, in a hypertrophy model, their muscles harbor fewer new MuSC-derived myonuclei due to increased MyoD expression and diminished proliferation, which ultimately causes blunted hypertrophy. Our results show that sustained HeyL expression is critical for MuSC proliferation specifically in overloaded muscle, and thus indicate that the MuSC-proliferation mechanism differs in overloaded and regenerating muscle.
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http://dx.doi.org/10.7554/eLife.48284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6768661PMC
September 2019

Expression and Functional Analyses of Dlk1 in Muscle Stem Cells and Mesenchymal Progenitors during Muscle Regeneration.

Int J Mol Sci 2019 Jul 3;20(13). Epub 2019 Jul 3.

Project for Muscle Stem Cell Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.

Delta like non-canonical Notch ligand 1 () is a paternally expressed gene which is also known as preadipocyte factor 1 (). The accumulation of adipocytes and expression of Dlk1 in regenerating muscle suggests a correlation between fat accumulation and Dlk1 expression in the muscle. Additionally, mice overexpressing Dlk1 show increased muscle weight, while Dlk1-null mice exhibit decreased body weight and muscle mass, indicating that Dlk1 is a critical factor in regulating skeletal muscle mass during development. The muscle regeneration process shares some features with muscle development. However, the role of Dlk1 in regeneration processes remains controversial. Here, we show that mesenchymal progenitors also known as adipocyte progenitors exclusively express Dlk1 during muscle regeneration. Eliminating developmental effects, we used conditional depletion models to examine the specific roles of Dlk1 in muscle stem cells or mesenchymal progenitors. Unexpectedly, deletion of Dlk1 in neither the muscle stem cells nor the mesenchymal progenitors affected the regenerative ability of skeletal muscle. In addition, fat accumulation was not increased by the loss of Dlk1. Collectively, Dlk1 plays essential roles in muscle development, but does not greatly impact regeneration processes and adipogenic differentiation in adult skeletal muscle regeneration.
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http://dx.doi.org/10.3390/ijms20133269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650828PMC
July 2019

Cell-autonomous and redundant roles of Hey1 and HeyL in muscle stem cells: HeyL requires Hes1 to bind diverse DNA sites.

Development 2019 02 20;146(4). Epub 2019 Feb 20.

Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan

The undifferentiated state of muscle stem (satellite) cells (MuSCs) is maintained by the canonical Notch pathway. Although three bHLH transcriptional factors, Hey1, HeyL and Hes1, are considered to be potential effectors of the Notch pathway exerting anti-myogenic effects, neither HeyL nor Hes1 inhibits myogenic differentiation of myogenic cell lines. Furthermore, whether these factors work redundantly or cooperatively is unknown. Here, we showed cell-autonomous functions of Hey1 and HeyL in MuSCs using conditional and genetic null mice. Analysis of cultured MuSCs revealed anti-myogenic activity of both HeyL and Hes1. We found that HeyL forms heterodimeric complexes with Hes1 in living cells. Moreover, our ChIP-seq experiments demonstrated that, compared with HeyL alone, the HeyL-Hes1 heterodimer binds with high affinity to specific sites in the chromatin, including the binding sites of Hey1. Finally, analyses of myogenin promoter activity showed that HeyL and Hes1 act synergistically to suppress myogenic differentiation. Collectively, these results suggest that HeyL and Hey1 function redundantly in MuSCs, and that HeyL requires Hes1 for effective DNA binding and biological activity.
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http://dx.doi.org/10.1242/dev.163618DOI Listing
February 2019

Adiponectin promotes muscle regeneration through binding to T-cadherin.

Sci Rep 2019 01 9;9(1):16. Epub 2019 Jan 9.

Department of Metabolic Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan.

Skeletal muscle has remarkable regenerative potential and its decline with aging is suggested to be one of the important causes of loss of muscle mass and quality of life in elderly adults. Metabolic abnormalities such as obesity were linked with decline of muscle regeneration. On the other hand, plasma levels of adiponectin are decreased in such metabolic conditions. However, plasma levels of adiponectin have been shown to inversely correlate with muscle mass and strength in elderly people especially with chronic heart failure (CHF). Here we have addressed whether adiponectin has some impact on muscle regeneration after cardiotoxin-induced muscle injury in mice. Muscle regeneration was delayed by angiotensin II infusion, mimicking aging and CHF as reported. Adiponectin overexpression in vivo decreased necrotic region and increased regenerating myofibers. Such enhanced regeneration by excess adiponectin was also observed in adiponectin null mice, but not in T-cadherin null mice. Mechanistically, adiponectin accumulated on plasma membrane of myofibers both in mice and human, and intracellularly colocalized with endosomes positive for a multivesicular bodies/exosomes marker CD63 in regenerating myofibers. Purified high-molecular multimeric adiponectin similarly accumulated intracellularly and colocalized with CD63-positive endosomes and enhanced exosome secretion in differentiating C2C12 myotubes but not in undifferentiated myoblasts. Knockdown of T-cadherin in differentiating C2C12 myotubes attenuated both adiponectin-accumulation and adiponectin-mediated exosome production. Collectively, our studies have firstly demonstrated that adiponectin stimulates muscle regeneration through T-cadherin, where intracellular accumulation and exosome-mediated process of adiponectin may have some roles.
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http://dx.doi.org/10.1038/s41598-018-37115-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6327035PMC
January 2019

The Robo4-TRAF7 complex suppresses endothelial hyperpermeability in inflammation.

J Cell Sci 2019 01 2;132(1). Epub 2019 Jan 2.

Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, 565-0781, Japan

Roundabout guidance receptor 4 (Robo4) is an endothelial cell-specific receptor that stabilizes the vasculature in pathological angiogenesis. Although Robo4 has been shown to suppress vascular hyperpermeability induced by vascular endothelial growth factor (VEGF) in angiogenesis, the role of Robo4 in inflammation is poorly understood. In this study, we investigated the role of Robo4 in vascular hyperpermeability during inflammation. Endotoxemia models using mice showed increased mortality and vascular leakage. In endothelial cells, Robo4 suppressed tumor necrosis factor α (TNFα)-induced hyperpermeability by stabilizing VE-cadherin at cell junctions, and deletion assays revealed that the C-terminus of Robo4 was involved in this suppression. Through binding and localization assays, we demonstrated that in endothelial cells, Robo4 binds to TNF receptor-associated factor 7 (TRAF7) through interaction with the C-terminus of Robo4. Gain- and loss-of-function studies of TRAF7 with or without Robo4 expression showed that TRAF7 is required for Robo4-mediated suppression of hyperpermeability. Taken together, our results demonstrate that the Robo4-TRAF7 complex is a novel negative regulator of inflammatory hyperpermeability. We propose this complex as a potential future target for protection against inflammatory diseases.
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http://dx.doi.org/10.1242/jcs.220228DOI Listing
January 2019

Muscle regeneration is disrupted by cancer cachexia without loss of muscle stem cell potential.

PLoS One 2018 9;13(10):e0205467. Epub 2018 Oct 9.

Project for Muscle Stem Cell Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan.

Cancer cachexia is a severe, debilitating condition characterized by progressive body wasting associated with remarkable loss of skeletal muscle weight. It has been reported that cancer cachexia disturbs the regenerative ability of skeletal muscle, but the cellular mechanisms are still unknown. Here, we investigated the skeletal muscle regenerative process in mouse colon-26 (C26) tumor cell-bearing mice as a C26 cancer cachexia model. Although the proliferation and differentiation abilities of muscle stem cells derived from the C26 tumor cell-bearing mice were sustained in vitro, the proliferation and differentiation were severely impaired in the cachexic mice. The numbers of both macrophages and mesenchymal progenitors, which are critical players in muscle regeneration, were reduced in the cancer cachexic mice, indicating that the skeletal muscle regeneration process was disrupted by cancer cachexia. Furthermore, the number of infiltrated neutrophils was also reduced in cancer cachexia mice 24 hours after muscle injury, and the expression of critical chemokines for muscle regeneration was reduced in cancer cachexia model mice compared to control mice. Collectively, although the ability to regeneration of MuSCs was retained, cancer cachexia disturbed skeletal muscle regenerative ability by inhibiting the orchestrated muscle regeneration processes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205467PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177180PMC
March 2019

Angiotensin-converting enzyme 2 deficiency accelerates and angiotensin 1-7 restores age-related muscle weakness in mice.

J Cachexia Sarcopenia Muscle 2018 10 11;9(5):975-986. Epub 2018 Sep 11.

Department of Geriatric and General Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

Background: A pharmacologic strategy for age-related muscle weakness is desired to improve mortality and disability in the elderly. Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin II into angiotensin 1-7, a peptide known to protect against acute and chronic skeletal muscle injury in rodents. Since physiological aging induces muscle weakness via mechanisms distinct from other muscle disorders, the role of ACE2-angiotensin 1-7 in age-related muscle weakness remains undetermined. Here, we investigated whether deletion of ACE2 alters the development of muscle weakness by aging and whether angiotensin 1-7 reverses muscle weakness in older mice.

Methods: After periodic measurement of grip strength and running distance in male ACE2KO and wild-type mice until 24 months of age, we infused angiotensin 1-7 or vehicle for 4 weeks, and measured grip strength, and excised tissues. Tissues were also excised from younger (3-month-old) and middle-aged (15-month-old) mice. Microarray analysis of RNA was performed using tibialis anterior (TA) muscles from middle-aged mice, and some genes were further tested using RT-PCR.

Results: Grip strength of ACE2KO mice was reduced at 6 months and was persistently lower than that of wild-type mice (p < 0.01 at 6, 12, 18, and 24-month-old). Running distance of ACE2KO mice was shorter than that of wild-type mice only at 24 months of age [371 ± 26 vs. 479 ± 24 (m), p < 0.01]. Angiotensin 1-7 improved grip strength in both types of older mice, with larger effects observed in ACE2KO mice (% increase, 3.8 ± 1.5 and 13.3 ± 3.1 in wild type and ACE2KO mice, respectively). Older, but not middle-aged ACE2KO mice had higher oxygen consumption assessed by a metabolic cage than age-matched wild-type mice. Angiotensin 1-7 infusion modestly increased oxygen consumption in older mice. There was no difference in a wheel-running activity or glucose tolerance between ACE2KO and wild-type mice and between mice with vehicle and angiotensin 1-7 infusion. Analysis of TA muscles revealed that p16INK4a, a senescence-associated gene, and central nuclei of myofibers increased in middle-aged, but not younger ACE2KO mice. p16INK4a and central nuclei increased in TA muscles of older wild-type mice, but the differences between ACE2KO and wild-type mice remained significant (p < 0.01). Angiotensin 1-7 did not alter the expression of p16INK4a or central nuclei in TA muscles of both types of mice. Muscle ACE2 expression of wild-type mice was the lowest at middle age (2.6 times lower than younger age, p < 0.05).

Conclusions: Deletion of ACE2 induced the early manifestation of muscle weakness with signatures of muscle senescence. Angiotensin 1-7 improved muscle function in older mice, supporting future application of the peptide or its analogues in the treatment of muscle weakness in the elderly population.
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http://dx.doi.org/10.1002/jcsm.12334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204583PMC
October 2018

"Of Mice and Measures": A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic.

J Neuromuscul Dis 2018 ;5(4):407-417

Department of Human Genetics, Leiden University Medical Center, the Netherlands.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value. The overall aim is to establish guidance on how to best use the D2/mdx mouse model in preclinical studies.
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http://dx.doi.org/10.3233/JND-180324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218134PMC
January 2019

Implication of basal lamina dependency in survival of Nrf2-null muscle stem cells via an antioxidative-independent mechanism.

J Cell Physiol 2019 02 2;234(2):1689-1698. Epub 2018 Aug 2.

Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator for the induction of antioxidative genes and plays roles in diverse cellular functions. The roles of Nrf2 in muscle regeneration have been investigated, and both important and unimportant roles of Nrf2 for muscle regeneration have been reported. Here, using aged Nrf2-null and Nrf2-dystrophic double-null mice, we showed nonsignificant phenotypes in the muscle regeneration ability of Nrf2-null mice. In contrast with these results, strikingly, almost all Nrf2-null muscle stem cells (MuSCs) isolated by fluorescence-activated cell sorting died in vitro of apoptosis and were not rescued by antioxidative reagents. Although their proliferation was still impaired, the Nrf2-null MuSCs attached to myofibers activated and divided normally, at least in the first round. To elucidate these discrepancies of MuSCs behaviors, we focused on the basal lamina, because both in vivo and single myofiber culture allow MuSCs within the basal lamina to become activated. In a basal lamina-disrupted model, Nrf2-null mice exhibited remarkable regeneration defects without increased levels of reactive oxidative species in MuSCs, suggesting that the existence of the basal lamina affects the survival of Nrf2-null MuSCs. Taken together, these results suggest that the basal lamina compensates for the loss of Nrf2, independent of the antioxidative roles of Nrf2. In addition, experimental conditions might explain the discrepant results of Nrf2-null regenerative ability.
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http://dx.doi.org/10.1002/jcp.27040DOI Listing
February 2019

Reciprocal signalling by Notch-Collagen V-CALCR retains muscle stem cells in their niche.

Nature 2018 05 23;557(7707):714-718. Epub 2018 May 23.

INSERM IMRB U955-E10, UPEC, ENVA, EFS, Créteil, France.

The cell microenvironment, which is critical for stem cell maintenance, contains both cellular and non-cellular components, including secreted growth factors and the extracellular matrix. Although Notch and other signalling pathways have previously been reported to regulate quiescence of stem cells, the composition and source of molecules that maintain the stem cell niche remain largely unknown. Here we show that adult muscle satellite (stem) cells in mice produce extracellular matrix collagens to maintain quiescence in a cell-autonomous manner. Using chromatin immunoprecipitation followed by sequencing, we identified NOTCH1/RBPJ-bound regulatory elements adjacent to specific collagen genes, the expression of which is deregulated in Notch-mutant mice. Moreover, we show that Collagen V (COLV) produced by satellite cells is a critical component of the quiescent niche, as depletion of COLV by conditional deletion of the Col5a1 gene leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by the Calcitonin receptor, for which COLV acts as a surrogate local ligand. Systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects found in COLV-null satellite cells. This study reveals a Notch-COLV-Calcitonin receptor signalling cascade that maintains satellite cells in a quiescent state in a cell-autonomous fashion, and raises the possibility that similar reciprocal mechanisms act in diverse stem cell populations.
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http://dx.doi.org/10.1038/s41586-018-0144-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5985950PMC
May 2018

A novel long non-coding RNA Myolinc regulates myogenesis through TDP-43 and Filip1.

J Mol Cell Biol 2018 04;10(2):102-117

Institute of Cardiovascular Regeneration, Centre for Molecular Medicine, Goethe University Frankfurt, Frankfurt am Main 60590, Germany.

Myogenesis is a complex process required for skeletal muscle formation during embryonic development and for regeneration and growth of myofibers in adults. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) play key roles in regulating cell fate decision and function in various tissues. However, the role of lncRNAs in the regulation of myogenesis remains poorly understood. In this study, we identified a novel muscle-enriched lncRNA called 'Myolinc (AK142388)', which we functionally characterized in the C2C12 myoblast cell line. Myolinc is predominately localized in the nucleus, and its levels increase upon induction of the differentiation. Knockdown of Myolinc impairs the expression of myogenic regulatory factors and formation of multi-nucleated myotubes in cultured myoblasts. Myolinc also regulates the expression of Filip1 in a cis-manner. Similar to Myolinc, knockdown of Filip1 inhibits myogenic differentiation. Furthermore, Myolinc binds to TAR DNA-binding protein 43 (TDP-43), a DNA/RNA-binding protein that regulates the expression of muscle genes (e.g. Acta1 and MyoD). Knockdown of TDP-43 inhibits myogenic differentiation. We also show that Myolinc-TDP-43 interaction is essential for the binding of TDP-43 to the promoter regions of muscle marker genes. Finally, we show that silencing of Myolinc inhibits skeletal muscle regeneration in adult mice. Altogether, our study identifies a novel lncRNA that controls key regulatory networks of myogenesis.
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http://dx.doi.org/10.1093/jmcb/mjy025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191624PMC
April 2018

[Homeostasis and Disorder of Musculoskeletal System.Molecular mechanism underlying muscle development and regeneration.]

Clin Calcium 2018;28(3):329-333

Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, Japan.

Skeletal muscle composes 30-40% of our body weight and is formed by multinuclear cells called myofibers. The formation of myofiber depends on the dynamic proliferation, differentiation and fusion of the myogenic progenitors during development. In the adult stage, the skeletal muscle exhibits excellent regeneration ability as well, depended on the muscle stem(satellite)cells that generate and repair myofibers. In this review, we would like to introduce ① the mechanisms of myogenic progenitor-dependent myofiber formation in myogenesis, ② the common fusion mechanism for myogenesis and muscle regeneration, and ③ the current status and prospects for clinical application utilizing satellite cells.
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http://dx.doi.org/CliCa1803329333DOI Listing
February 2019

The roles of muscle stem cells in muscle injury, atrophy and hypertrophy.

Authors:
So-Ichiro Fukada

J Biochem 2018 May;163(5):353-358

Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita City, Osaka 565-0871, Japan.

Skeletal muscle is composed of multinuclear cells called myofibers. Muscular dystrophy (a genetic muscle disorder) induces instability in the cell membrane of myofibers and eventually causes myofibre damage. Non-genetic muscle disorders, including sarcopenia, diabetes, bedridden immobility and cancer cachexia, lead to atrophy of myofibres. In contrast, resistance training induces myofibre hypertrophy. Thus, myofibres exhibit a plasticity that is strongly affected by both intrinsic and extrinsic factors. There is no doubt that muscle stem cells (MuSCs, also known as muscle satellite cells) are indispensable for muscle repair/regeneration, but their contributions to atrophy and hypertrophy are still controversial. The present review focuses on the relevance of MuSCs to (i) muscle diseases and (ii) hypertrophy. Further, this review addresses fundamental questions about MuSCs to clarify the onset or progression of these diseases and which might lead to development of a MuSC-based therapy.
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http://dx.doi.org/10.1093/jb/mvy019DOI Listing
May 2018

The Ror1 receptor tyrosine kinase plays a critical role in regulating satellite cell proliferation during regeneration of injured muscle.

J Biol Chem 2017 09 8;292(38):15939-15951. Epub 2017 Aug 8.

From the Division of Cell Physiology, Department of Physiology and Cell Biology, and

The Ror family receptor tyrosine kinases, Ror1 and Ror2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and Ror2 during skeletal muscle regeneration. Using an skeletal muscle injury model, we show that expression of Ror1 and Ror2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1β, after injury and that inhibition of TNF-α and IL-1β by neutralizing antibodies suppresses expression of and in injured muscles. Importantly, expression of , but not , was induced primarily in Pax7-positive satellite cells (SCs) after muscle injury, and administration of neutralizing antibodies decreased the proportion of Pax7-positive proliferative SCs after muscle injury. We also found that stimulation of a mouse myogenic cell line, C2C12 cells, with TNF-α or IL-1β induced expression of Ror1 via NF-κB activation and that suppressed expression of Ror1 inhibited their proliferative responses in SCs. Intriguingly, SC-specific depletion of decreased the number of Pax7-positive SCs after muscle injury. Collectively, these findings indicate for the first time that Ror1 has a critical role in regulating SC proliferation during skeletal muscle regeneration. We conclude that Ror1 might be a suitable target in the development of diagnostic and therapeutic approaches to manage muscular disorders.
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http://dx.doi.org/10.1074/jbc.M117.785709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612123PMC
September 2017

Vestigial-like 2 contributes to normal muscle fiber type distribution in mice.

Sci Rep 2017 08 2;7(1):7168. Epub 2017 Aug 2.

Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka, 565-8565, Japan.

Skeletal muscle is composed of heterogeneous populations of myofibers that are classified as slow- and fast-twitch fibers. The muscle fiber-type is regulated in a coordinated fashion by multiple genes, including transcriptional factors and microRNAs (miRNAs). However, players involved in this regulation are not fully elucidated. One of the members of the Vestigial-like factors, Vgll2, is thought to play a pivotal role in TEA domain (TEAD) transcription factor-mediated muscle-specific gene expression because of its restricted expression in skeletal muscles of adult mice. Here, we generated Vgll2 null mice and investigated Vgll2 function in adult skeletal muscles. These mice presented an increased number of fast-twitch type IIb fibers and exhibited a down-regulation of slow type I myosin heavy chain (MyHC) gene, Myh7, which resulted in exercise intolerance. In accordance with the decrease in Myh7, down-regulation of miR-208b, encoded within Myh7 gene and up-regulation of targets of miR-208b, Sox6, Sp3, and Purβ, were observed in Vgll2 deficient mice. Moreover, we detected the physical interaction between Vgll2 and TEAD1/4 in neonatal skeletal muscles. These results suggest that Vgll2 may be both directly and indirectly involved in the programing of slow muscle fibers through the formation of the Vgll2-TEAD complex.
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http://dx.doi.org/10.1038/s41598-017-07149-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540913PMC
August 2017

Gm7325 is MyoD-dependently expressed in activated muscle satellite cells.

Biomed Res 2017 ;38(3):215-219

Graduate School of Pharmaceutical Sciences, Kyoto University.

The Gm7325 gene, bioinformatically identified in the mouse genome, encodes a small protein but has not been characterized until recently. Our gene expression analysis revealed that Gm7325 transcription is remarkably upregulated in injured skeletal muscle tissues. Activated satellite cells and immature myotubes were densely decorated with positive signals for Gm7325 mRNA in in situ hybridization analysis, while no obvious signals were observed in quiescent satellite cells and mature myofibers. In the 5'-flanking regions of mouse Gm7325 and its human homologue, conserved E-box motifs for helix-loop-helix transcription factors are repeatedly arranged around the putative promoter regions. Reporter gene assays suggested that MyoD, a master transcription factor for myogenesis, binds to the conserved E-box motifs to activate Gm7325 expression. Therefore, Gm7325, as a novel MyoD-target gene, is specifically induced in activated satellite cells, and may have an important role in skeletal myogenesis.
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http://dx.doi.org/10.2220/biomedres.38.215DOI Listing
March 2018

Notch ligands regulate the muscle stem-like state ex vivo but are not sufficient for retaining regenerative capacity.

PLoS One 2017 12;12(5):e0177516. Epub 2017 May 12.

Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.

Myogenic stem cells are a promising avenue for the treatment of muscular disorders. Freshly isolated muscle stem cells have a remarkable engraftment ability in vivo, but their cell number is limited. Current conventional culture conditions do not allow muscle stem cells to expand in vitro with their bona fide engraftment efficiency, requiring the improvement of culture procedures for achieving successful cell-therapy for muscle disorders. Here we expanded mouse muscle stem cells and human myoblasts with Notch ligands, DLL1, DLL4, and JAG1 to activate Notch signaling in vitro and to investigate whether these cells could retain their engraftment efficiency. Notch signaling promotes the expansion of Pax7+MyoD- mouse muscle stem-like cells and inhibits differentiation even after passage in vitro. Treatment with Notch ligands induced the Notch target genes and generated PAX7+MYOD- stem-like cells from human myoblasts previously cultured on conventional culture plates. However, cells treated with Notch ligands exhibit a stem cell-like state in culture, yet their regenerative ability was less than that of freshly isolated cells in vivo and was comparable to that of the control. These unexpected findings suggest that artificial maintenance of Notch signaling alone is insufficient for improving regenerative capacity of mouse and human donor-muscle cells and suggest that combinatorial events are critical to achieve muscle stem cell and myoblast engraftment potential.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177516PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428926PMC
September 2017

Adult murine cardiomyocytes exhibit regenerative activity with cell cycle reentry through STAT3 in the healing process of myocarditis.

Sci Rep 2017 05 3;7(1):1407. Epub 2017 May 3.

Laboratory of Clinical Science and Biomedicine, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Mammalian cardiomyocytes substantially lose proliferative capacity immediately after birth, limiting adult heart regeneration after injury. However, clinical myocarditis appears to be self-limiting with tissue-reparative properties. Here, we investigated the molecular mechanisms underlying the recovery from myocarditis with regard to cardiomyocyte proliferation using an experimental autoimmune myocarditis (EAM) model. Three weeks after EAM induction (EAM3w), cardiac tissue displayed infiltration of inflammatory cells with cardiomyocyte apoptosis. However, by EAM5w, the myocardial damage was remarkably attenuated, associated with an increase in cardiomyocytes that were positively stained with cell cycle markers at EAM3w. Cardiomyocyte fate mapping study revealed that the proliferating cardiomyocytes primarily derived from pre-existing cardiomyocytes. Signal transducer and activator of transcription 3 (STAT3) was robustly activated in cardiomyocytes during inflammation, accompanied by induction of interleukin-6 family cytokines. Cardiomyocyte-specific ablation of STAT3 gene suppressed the frequency of cycling cardiomyocytes in the recovery period without influencing inflammatory status, resulting in impaired tissue repair and cardiac dysfunction. Finally, microarray analysis revealed that the expression of regeneration-related genes, metallothioneins and clusterin, in cardiomyocytes was decreased by STAT3 gene deletion. These data show that adult mammalian cardiomyocytes restore regenerative capacity with cell cycle reentry through STAT3 as the heart recovers from myocarditis-induced cardiac damage.
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http://dx.doi.org/10.1038/s41598-017-01426-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431117PMC
May 2017

[Molecular mechanism maintaining muscle satellite cells and the roles in sarcopenia.]

Clin Calcium 2017;27(3):339-344

Laboratory of Molecular and Cellular Physiology, Graduate School of Pharmaceutical Sciences, Osaka University, Japan.

Skeletal muscle has its stem cell named satellite cell. The absence of satellite cells does not allow muscle regeneration, it is unquestionable that satellite cell is indispensable for muscle regeneration processes. A certain number of satellite cells appear to be necessary for the successful muscle regeneration, meaning the maintenance of the satellite cells is essential for the functional homeostasis of skeletal muscle. Recent studies have revealed the molecular mechanism underlying satellite cell maintenance in a steady state. A loss of those molecules responsible for the maintenance often results in decreased satellite cell pool and reduced regeneration ability. On the other hand, the contribution of satellite cells to muscle hypertrophy or aged-related atrophy(sarcopenia)is controversial. In this review, we will introduce the molecules that regulate satellite cells homeostasis in the dormant state and then further discuss the recent results on the roles of satellite cell in sarcopenia.
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http://dx.doi.org/CliCa1703339344DOI Listing
October 2017

Modified forelimb grip strength test detects aging-associated physiological decline in skeletal muscle function in male mice.

Sci Rep 2017 02 8;7:42323. Epub 2017 Feb 8.

Department of Geriatric and General Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

The conventional forelimb grip strength test is a widely used method to assess skeletal muscle function in rodents; in this study, we modified this method to improve its variability and consistency. The modified test had lower variability among trials and days than the conventional test in young C57BL6 mice, especially by improving the variabilities in male. The modified test was more sensitive than the conventional test to detect a difference in motor function between female and male mice, or between young and old male mice. When the modified test was performed on male mice during the aging process, reduction of grip strength manifested between 18 and 24 months of age at the group level and at the individual level. The modified test was similar to the conventional test in detecting skeletal muscle dysfunction in young male dystrophic mice. Thus, the modified forelimb grip strength test, with its improved validity and reliability may be an ideal substitute for the conventional method.
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http://dx.doi.org/10.1038/srep42323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5296723PMC
February 2017

Impaired regenerative capacity and lower revertant fibre expansion in dystrophin-deficient mdx muscles on DBA/2 background.

Sci Rep 2016 12 7;6:38371. Epub 2016 Dec 7.

Department of Medical Genetics, University of Alberta Faculty of Medicine and Dentistry, Edmonton, Alberta, T6G2H7, Canada.

Duchenne muscular dystrophy, one of the most common lethal genetic disorders, is caused by mutations in the DMD gene and a lack of dystrophin protein. In most DMD patients and animal models, sporadic dystrophin-positive muscle fibres, called revertant fibres (RFs), are observed in otherwise dystrophin-negative backgrounds. RFs are thought to arise from skeletal muscle precursor cells and clonally expand with age due to the frequent regeneration of necrotic fibres. Here we examined the effects of genetic background on muscle regeneration and RF expansion by comparing dystrophin-deficient mdx mice on the C57BL/6 background (mdx-B6) with those on the DBA/2 background (mdx-DBA), which have a more severe phenotype. Interestingly, mdx-DBA muscles had significantly lower RF expansion than mdx-B6 in all age groups, including 2, 6, 12, and 18 months. The percentage of centrally nucleated fibres was also significantly lower in mdx-DBA mice compared to mdx-B6, indicating that less muscle regeneration occurs in mdx-DBA. Our study aligns with the model that RF expansion reflects the activity of precursor cells in skeletal muscles, and it serves as an index of muscle regeneration capacity.
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http://dx.doi.org/10.1038/srep38371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5141435PMC
December 2016