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Tomaz da Silva M, Joshi AS, Castillo MB, Koike TE, Roy A, Gunaratne PH, Kumar A. Fn14 promotes myoblast fusion during regenerative myogenesis. Life Sci Alliance 2023; 6:e202302312. [PMID: 37813488 PMCID: PMC10561765 DOI: 10.26508/lsa.202302312] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Revised: 09/22/2023] [Accepted: 09/26/2023] [Indexed: 10/12/2023] Open
Abstract
Skeletal muscle regeneration involves coordinated activation of an array of signaling pathways. Fibroblast growth factor-inducible 14 (Fn14) is a bona fide receptor for the TWEAK cytokine. Levels of Fn14 are increased in the skeletal muscle of mice after injury. However, the cell-autonomous role of Fn14 in muscle regeneration remains unknown. Here, we demonstrate that global deletion of the Fn14 receptor in mice attenuates muscle regeneration. Conditional ablation of Fn14 in myoblasts but not in differentiated myofibers of mice inhibits skeletal muscle regeneration. Fn14 promotes myoblast fusion without affecting the levels of myogenic regulatory factors in the regenerating muscle. Fn14 deletion in myoblasts hastens initial differentiation but impairs their fusion. The overexpression of Fn14 in myoblasts results in the formation of myotubes having an increased diameter after induction of differentiation. Ablation of Fn14 also reduces the levels of various components of canonical Wnt and calcium signaling both in vitro and in vivo. Forced activation of Wnt signaling rescues fusion defects in Fn14-deficient myoblast cultures. Collectively, our results demonstrate that Fn14-mediated signaling positively regulates myoblast fusion and skeletal muscle regeneration.
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Affiliation(s)
- Meiricris Tomaz da Silva
- Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX, USA
| | - Aniket S Joshi
- Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX, USA
| | - Micah B Castillo
- Department of Biology and Biochemistry, University of Houston, Houston, TX, USA
| | - Tatiana E Koike
- Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX, USA
| | - Anirban Roy
- Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX, USA
| | - Preethi H Gunaratne
- Department of Biology and Biochemistry, University of Houston, Houston, TX, USA
| | - Ashok Kumar
- Department of Pharmacological and Pharmaceutical Sciences, University of Houston College of Pharmacy, Houston, TX, USA
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Duan Y, Li T, Zhang G, Wu P, Chen L, Ding H, Wang J, Sun W. Transcriptome sequencing to explore the effect of miR-214 on chicken primary myoblasts. Anim Biotechnol 2023; 34:1727-1736. [PMID: 35262452 DOI: 10.1080/10495398.2022.2044840] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022]
Abstract
MicroRNAs are involved in a series of biological processes, such as proliferation, differentiation and apoptosis of primary myoblasts. The research group found that miR-214 is highly expressed in chicken primary myoblasts (CPMs), so we used miR-214 as a starting point to explore the biological function of miR-214 in skeletal muscle growth and development. In this experiment, CPMs were cultured in vitro; miR-214 was overexpressed in CPMs; and cell samples were collected for subsequent transcriptome sequencing (RNA-seq). After miR-214 overexpression, we identified 97 differentially expressed genes (DEGs), of which 21 DEGs were up-regulated and 76 DEGs were down-regulated. After bioinformatics analysis, these DEGs were found to be significantly enriched in myofibrils, muscle system processes, myofibril assembly and other biological processes related to muscle development. The significantly enriched KEGGs include focal adhesion and type II diabetes mellitus. The protein network of DEGs was drawn by STRING and Cytoscape software, and 5 DEGs were randomly selected to verify the sequencing results by real-time fluorescence quantification. CAV3 is not only an important node protein in the protein network but also a member of the focal adhesion signaling pathway. It is speculated that miR-214 may regulate muscle development through the focal adhesion signaling pathway.
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Affiliation(s)
- Yanjun Duan
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou, PR China
| | - Tingting Li
- College of Animal Science and Technology, Yangzhou University, Yangzhou, PR China
| | - Genxi Zhang
- College of Animal Science and Technology, Yangzhou University, Yangzhou, PR China
| | - Pengfei Wu
- College of Animal Science and Technology, Yangzhou University, Yangzhou, PR China
| | - Lan Chen
- College of Veterinary Medicine, Institute of Comparative Medicine, Yangzhou University, Yangzhou, PR China
| | - Hao Ding
- College of Animal Science and Technology, Yangzhou University, Yangzhou, PR China
| | - Jinyu Wang
- College of Animal Science and Technology, Yangzhou University, Yangzhou, PR China
| | - Wei Sun
- College of Animal Science and Technology, Yangzhou University, Yangzhou, PR China
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Liu LL, Chen B, Chen SL, Liu WJ. A Genome-Wide Association Study of the Chest Circumference Trait in Xinjiang Donkeys Based on Whole-Genome Sequencing Technology. Genes (Basel) 2023; 14:genes14051081. [PMID: 37239441 DOI: 10.3390/genes14051081] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2023] [Revised: 05/07/2023] [Accepted: 05/09/2023] [Indexed: 05/28/2023] Open
Abstract
Animal genotyping by means of genome-wide association studies is important for connecting phenotypes of interest with their underlying genetics in livestock. However, the use of whole genome sequencing to investigate chest circumference (CC) in donkeys has rarely been reported. We aimed to use the genome-wide association study approach to detect significant single nucleotide polymorphisms (SNPs) and key genes associated with chest circumference traits in Xinjiang donkeys. We assessed 112 Xinjiang donkeys in this study. The chest circumference of each was measured 2 h before milking. We re-sequenced blood samples from the Xinjiang donkeys, and genome-wide association study analyses were performed using a mixed model with the PLINK, GEMMA, and REGENIE programs. We tested 38 donkeys for candidate SNPs for genome-wide association study using three software programs. Additionally, 18 SNP markers reached genome-wide significance (p < 1.61 × 10-9). On the basis of these, 41 genes were identified. Previously proposed candidate genes for CC traits were supported by this study, including NFATC2 (Nuclear Factor of Activated T Cells 2), PROP1 (PROP Paired-Like Homeobox 1), UBB (Ubiquitin B), and HAND2 (Heart and Neural Crest Derivatives Expressed 2). These promising candidates provide a valuable resource for validating potential meat production genes and will facilitate the development of high-yielding Xinjiang donkey breeds through marker-assisted selection or gene editing.
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Affiliation(s)
- Ling-Ling Liu
- Department of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China
| | - Bin Chen
- Department of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China
| | - Sheng-Lei Chen
- Department of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China
| | - Wu-Jun Liu
- Department of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China
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Su Y, Gao X, Wang Y, Li X, Zhang W, Zhao J. Astragalus polysaccharide promotes sheep satellite cell differentiation by regulating miR-133a through the MAPK/ERK signaling pathway. Int J Biol Macromol 2023; 239:124351. [PMID: 37023880 DOI: 10.1016/j.ijbiomac.2023.124351] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Revised: 03/21/2023] [Accepted: 04/03/2023] [Indexed: 04/08/2023]
Abstract
Astragalus polysaccharide (APS) possesses extensive biological activities, pharmacological effects, and anti-fatigue function. MiR-133a is a specifically expressed miRNA in skeletal muscle that participates in the regulation of myoblast proliferation and differentiation. However, little is known about the role of APS in the development of sheep skeletal muscle. In this study, we aimed to investigate the underlying mechanism of APS and miR-133a on the differentiation of sheep skeletal muscle satellite cells (SMSCs) and the regulatory relationship between APS and miR-133a. The results suggested that APS plays a positive regulatory role in the proliferation and differentiation of sheep SMSCs. Moreover, miR-133a significantly promotes SMSC differentiation and the activity of the MAPK/ERK signaling pathway. Importantly, we found that APS function requires the mediation of miR-133a in the differentiation of sheep SMSCs. Taken together, our results indicate that APS accelerates SMSC differentiation by regulating miR-133a via the MAPK/ERK signaling pathway in sheep.
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Affiliation(s)
- Yuan Su
- College of Animal Sciences, Shanxi Agricultural University, Taigu 030801, PR China
| | - Xuyang Gao
- College of Animal Sciences, Shanxi Agricultural University, Taigu 030801, PR China
| | - Yu Wang
- College of Animal Sciences, Shanxi Agricultural University, Taigu 030801, PR China
| | - Xuying Li
- College of Animal Sciences, Shanxi Agricultural University, Taigu 030801, PR China
| | - Weipeng Zhang
- College of Animal Sciences, Shanxi Agricultural University, Taigu 030801, PR China
| | - Junxing Zhao
- College of Animal Sciences, Shanxi Agricultural University, Taigu 030801, PR China.
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Hou D, Qin P, Niu X, Li T, Chen B, Wei C, Jing Z, Han R, Li H, Liu X, Tian Y, Li D, Li Z, Cai H, Kang X. Genome-wide identification evolution and expression of vestigial-like gene family in chicken. Anim Biotechnol 2022; 33:1602-1612. [PMID: 34032551 DOI: 10.1080/10495398.2021.1920425] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Vestigial-like (Vgll) genes are widespread in vertebrates and play an important role in muscle development. In this study, we used bioinformatics methods to systematically identify the chicken VGLL family in the whole genome and investigated its evolutionary history and gene structure features. Tissue expression spectra combined with real-time PCR data were used to analyze the organizational expression pattern of the genes. Based on the maximum likelihood method, a phylogenetic tree of the VGLL family was constructed, and 94 VGLL genes were identified in 24 breeds, among which four VGLL family genes were identified in the chicken genome. Ten motifs were detected in the VGLL genes, and the analysis of introns combined with gene structure revealed that the family was conserved during evolution. Tissue expression analysis suggested that the expression profiles of the VGLL family genes in 16 tissues differed between LU Shi and AA broilers. In addition, a single gene (VGLL2) showed increased expression in chickens at embryonic days 10-16 and was involved in the growth and development of skeletal muscle in chickens in the embryonic stage. In summary, VGLL genes are involved in chicken muscle growth and development, which provides useful information for subsequent functional studies of VGLL genes.
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Affiliation(s)
- Dan Hou
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Panpan Qin
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Xinran Niu
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Tong Li
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Bingjie Chen
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Chengjie Wei
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Zhenzhu Jing
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Ruili Han
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Hong Li
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Xiaojun Liu
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Yadong Tian
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Donghua Li
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Zhuanjian Li
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Hanfang Cai
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
| | - Xiangtao Kang
- Henan Innovative Engineering Research Center of Poultry Germplasm Resource, College of Animal Science and Technology, Henan Agricultural University, Zhengzhou, China
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Lim JY, Kim E, Douglas CM, Wirianto M, Han C, Ono K, Kim SY, Ji JH, Tran CK, Chen Z, Esser KA, Yoo SH. The circadian E3 ligase FBXL21 regulates myoblast differentiation and sarcomere architecture via MYOZ1 ubiquitination and NFAT signaling. PLoS Genet 2022; 18:e1010574. [PMID: 36574402 PMCID: PMC9829178 DOI: 10.1371/journal.pgen.1010574] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2022] [Revised: 01/09/2023] [Accepted: 12/14/2022] [Indexed: 12/28/2022] Open
Abstract
Numerous molecular and physiological processes in the skeletal muscle undergo circadian time-dependent oscillations in accordance with daily activity/rest cycles. The circadian regulatory mechanisms underlying these cyclic processes, especially at the post-transcriptional level, are not well defined. Previously, we reported that the circadian E3 ligase FBXL21 mediates rhythmic degradation of the sarcomere protein TCAP in conjunction with GSK-3β, and Psttm mice harboring an Fbxl21 hypomorph allele show reduced muscle fiber diameter and impaired muscle function. To further elucidate the regulatory function of FBXL21 in skeletal muscle, we investigated another sarcomere protein, Myozenin1 (MYOZ1), that we identified as an FBXL21-binding protein from yeast 2-hybrid screening. We show that FBXL21 binding to MYOZ1 led to ubiquitination-mediated proteasomal degradation. GSK-3β co-expression and inhibition were found to accelerate and decelerate FBXL21-mediated MYOZ1 degradation, respectively. Previously, MYOZ1 has been shown to inhibit calcineurin/NFAT signaling important for muscle differentiation. In accordance, Fbxl21 KO and MyoZ1 KO in C2C12 cells impaired and enhanced myogenic differentiation respectively compared with control C2C12 cells, concomitant with distinct effects on NFAT nuclear localization and NFAT target gene expression. Importantly, in Psttm mice, both the levels and diurnal rhythm of NFAT2 nuclear localization were significantly diminished relative to wild-type mice, and circadian expression of NFAT target genes associated with muscle differentiation was also markedly dampened. Furthermore, Psttm mice exhibited significant disruption of sarcomere structure with a considerable excess of MYOZ1 accumulation in the Z-line. Taken together, our study illustrates a pivotal role of FBXL21 in sarcomere structure and muscle differentiation by regulating MYOZ1 degradation and NFAT2 signaling.
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Affiliation(s)
- Ji Ye Lim
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Eunju Kim
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Collin M. Douglas
- Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida, United States of America
| | - Marvin Wirianto
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Chorong Han
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Kaori Ono
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Sun Young Kim
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Justin H. Ji
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Celia K. Tran
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Zheng Chen
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
| | - Karyn A. Esser
- Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, Florida, United States of America
| | - Seung-Hee Yoo
- Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, Houston, Texas, United States of America
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Lee J, Park J, Choe H, Shim K. Insect peptide CopA3 promotes proliferation and PAX7 and MYOD expression in porcine muscle satellite cells. JOURNAL OF ANIMAL SCIENCE AND TECHNOLOGY 2022; 64:1132-1143. [PMID: 36812017 PMCID: PMC9890342 DOI: 10.5187/jast.2022.e81] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/28/2022] [Revised: 09/27/2022] [Accepted: 10/04/2022] [Indexed: 12/14/2022]
Abstract
Insects are a valuable natural source that can produce a variety of bioactive compounds due to their increasing species diversity. CopA3 is an antimicrobial peptide derived from Copris tripartitus (i.e., the dung beetle). It is known to increase the proliferation of colonic epithelial and neuronal stem cells by regulating cell cycle. This research hypothesized that CopA3 can promote the proliferation of porcine muscle satellite cells (MSCs). The effects of CopA3 on porcine MSCs, which are important for muscle growth and regeneration, remain unclear. Here, we investigated the effects of CopA3 on porcine MSCs. According to viability results, we designed four groups: control (without CopA3) and three treatment groups (treated with 5,10, and 25 μg/mL of CopA3). At a CopA3 concentration of 5 μg/mL and 10 μg/mL, the proliferation of MSCs increased more than that observed in the control group. Furthermore, compared to that in the control, CopA3 treatment increased the S phase but decreased the G0/G1 phase ratio. Additionally, early and late apoptotic cells were found to be decreased in the 5 μg/mL group. The expressions of the myogenesis-related transcription factor PAX7 and MYOD proteins were significantly upregulated in the 5 μg/mL and 10 μg/mL groups, whereas the MYOG protein remained undetected in all group. This study suggested that CopA3 promotes muscle cell proliferation by regulating the cell cycle of MSCs and can regulate the activity of MSCs by increasing the expressions of PAX7 and MYOD.
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Affiliation(s)
- Jeongeun Lee
- Department of Agricultural Convergence
Technology, Jeonbuk National University, Jeonju 54896,
Korea
| | - Jinryoung Park
- Department of Stem Cell and Regenerative
Biotechnology, Konkuk University, Seoul 06591, Korea,3D Tissue Culture Research Center, Konkuk
University, Seoul 06591, Korea
| | - Hosung Choe
- Department of Animal Biotechnology,
Jeonbuk National University, Jeonju 54896, Korea
| | - Kwanseob Shim
- Department of Agricultural Convergence
Technology, Jeonbuk National University, Jeonju 54896,
Korea,Department of Animal Biotechnology,
Jeonbuk National University, Jeonju 54896, Korea,Corresponding author: Kwanseob Shim,
Department of Agricultural Convergence Technology, Jeonbuk National University,
Jeonju 54896, Korea. Tel: +82-63-270-2609, E-mail:
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Wang H, Wang X, Li M, Sun H, Chen Q, Yan D, Dong X, Pan Y, Lu S. Genome-Wide Association Study of Growth Traits in a Four-Way Crossbred Pig Population. Genes (Basel) 2022; 13:1990. [PMID: 36360227 PMCID: PMC9689869 DOI: 10.3390/genes13111990] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2022] [Revised: 10/28/2022] [Accepted: 10/28/2022] [Indexed: 04/29/2025] Open
Abstract
Growth traits are crucial economic traits in the commercial pig industry and have a substantial impact on pig production. However, the genetic mechanism of growth traits is not very clear. In this study, we performed a genome-wide association study (GWAS) based on the specific-locus amplified fragment sequencing (SLAF-seq) to analyze ten growth traits on 223 four-way intercross pigs. A total of 227,921 highly consistent single nucleotide polymorphisms (SNPs) uniformly dispersed throughout the entire genome were used to conduct GWAS. A total of 53 SNPs were identified for ten growth traits using the mixed linear model (MLM), of which 18 SNPs were located in previously reported quantitative trait loci (QTL) regions. Two novel QTLs on SSC4 and SSC7 were related to average daily gain from 30 to 60 kg (ADG30-60) and body length (BL), respectively. Furthermore, 13 candidate genes (ATP5O, GHRHR, TRIM55, EIF2AK1, PLEKHA1, BRAP, COL11A2, HMGA1, NHLRC1, SGSM1, NFATC2, MAML1, and PSD3) were found to be associated with growth traits in pigs. The GWAS findings will enhance our comprehension of the genetic architecture of growth traits. We suggested that these detected SNPs and corresponding candidate genes might provide a biological foundation for improving the growth and production performance of pigs in swine breeding.
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Affiliation(s)
- Huiyu Wang
- Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
- Faculty of Animal Science, Xichang University, Xichang 615000, China
| | - Xiaoyi Wang
- Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
| | - Mingli Li
- Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
| | - Hao Sun
- Faculty of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
| | - Qiang Chen
- Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
| | - Dawei Yan
- Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
| | - Xinxing Dong
- Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
| | - Yuchun Pan
- Faculty of Animal Science, Zhejiang University, Hangzhou 310058, China
| | - Shaoxiong Lu
- Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, China
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Kurosaka M, Ogura Y, Sato S, Kohda K, Funabashi T. Transcription factor signal transducer and activator of transcription 6 (STAT6) is an inhibitory factor for adult myogenesis. Skelet Muscle 2021; 11:14. [PMID: 34051858 PMCID: PMC8164270 DOI: 10.1186/s13395-021-00271-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Accepted: 05/18/2021] [Indexed: 01/25/2023] Open
Abstract
Background The signal transducer and activator of transcription 6 (STAT6) transcription factor plays a vitally important role in immune cells, where it is activated mainly by interleukin-4 (IL-4). Because IL-4 is an essential cytokine for myotube formation, STAT6 might also be involved in myogenesis as part of IL-4 signaling. This study was conducted to elucidate the role of STAT6 in adult myogenesis in vitro and in vivo. Methods Myoblasts were isolated from male mice and were differentiated on a culture dish to evaluate the change in STAT6 during myotube formation. Then, the effects of STAT6 overexpression and inhibition on proliferation, differentiation, and fusion in those cells were studied. Additionally, to elucidate the myogenic role of STAT6 in vivo, muscle regeneration after injury was evaluated in STAT6 knockout mice. Results IL-4 can increase STAT6 phosphorylation, but STAT6 phosphorylation decreased during myotube formation in culture. STAT6 overexpression decreased, but STAT6 knockdown increased the differentiation index and the fusion index. Results indicate that STAT6 inhibited myogenin protein expression. Results of in vivo experiments show that STAT6 knockout mice exhibited better regeneration than wild-type mice 5 days after cardiotoxin-induced injury. It is particularly interesting that results obtained using cells from STAT6 knockout mice suggest that this STAT6 inhibitory action for myogenesis was not mediated by IL-4 but might instead be associated with p38 mitogen-activated protein kinase phosphorylation. However, STAT6 was not involved in the proliferation of myogenic cells in vitro and in vivo. Conclusion Results suggest that STAT6 functions as an inhibitor of adult myogenesis. Moreover, results suggest that the IL-4-STAT6 signaling axis is unlikely to be responsible for myotube formation. Supplementary Information The online version contains supplementary material available at 10.1186/s13395-021-00271-8.
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Affiliation(s)
- Mitsutoshi Kurosaka
- Department of Physiology, St. Marianna University School of Medicine, Kawasaki, Kanagawa, 216-8511, Japan
| | - Yuji Ogura
- Department of Physiology, St. Marianna University School of Medicine, Kawasaki, Kanagawa, 216-8511, Japan.
| | - Shuichi Sato
- School of Kinesiology, The University of Louisiana at Lafayette, Lafayette, LA, USA.,New Iberia Research Center, The University of Louisiana at Lafayette, New Iberia, LA, USA
| | - Kazuhisa Kohda
- Department of Physiology, St. Marianna University School of Medicine, Kawasaki, Kanagawa, 216-8511, Japan
| | - Toshiya Funabashi
- Department of Physiology, St. Marianna University School of Medicine, Kawasaki, Kanagawa, 216-8511, Japan
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Duan Y, Wu Y, Yin X, Li T, Chen F, Wu P, Zhang S, Wang J, Zhang G. MicroRNA-214 Inhibits Chicken Myoblasts Proliferation, Promotes Their Differentiation, and Targets the TRMT61A Gene. Genes (Basel) 2020; 11:genes11121400. [PMID: 33255823 PMCID: PMC7760887 DOI: 10.3390/genes11121400] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Revised: 11/18/2020] [Accepted: 11/24/2020] [Indexed: 12/29/2022] Open
Abstract
The proliferation and differentiation of myoblasts is an important process of skeletal muscle development. In this process, microRNAs (miRNAs) play an important role in the proliferation and differentiation of chicken primary myoblasts (CPMs). Our previous study found that miR-214 and the tRNA methyltransferase 61A (TRMT61A) gene were differentially expressed in different stages of proliferation and differentiation. Therefore, this study aimed to explore the effect of miR-214 on the proliferation and differentiation of CPMs and the functional relationship between miR-214 and TRMT61A. In this study, we detected the effect of miR-214 on the proliferation of CPMs by qPCR, flow cytometry, CCK-8, and EdU after the overexpression and interference of miR-214. qPCR, Western blotting, and indirect immunofluorescence were used to detect the effect of miR-214 on the differentiation of the CPMs. The expression patterns of miR-214 and TRMT61A were observed at different time points of differentiation induced by the CPMs. The results show that miR-214 inhibited the proliferation of the CPMs and promoted the differentiation of the CPMs. The Dual-Luciferase Reporter assay and the expression pattern of miR-214 and TRMT61A suggested that they had a negative regulatory target relationship. This study revealed the function and regulatory mechanism of miR-214 in the proliferation and differentiation of CPMs.
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Chen M, Zhang S, Xu Z, Gao J, Mishra SK, Zhu Q, Zhao X, Wang Y, Yin H, Fan X, Zeng B, Yang M, Yang D, Ni Q, Li Y, Zhang M, Li D. MiRNA Profiling in Pectoral Muscle Throughout Pre- to Post-Natal Stages of Chicken Development. Front Genet 2020; 11:570. [PMID: 32655617 PMCID: PMC7324647 DOI: 10.3389/fgene.2020.00570] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2020] [Accepted: 05/11/2020] [Indexed: 02/05/2023] Open
Abstract
MicroRNA (miRNA) is known to be an important regulator of muscle growth and development. The regulation of microRNA on the skeletal muscle phenotype of animals is mainly achieved by regulating the proliferation and differentiation of myoblasts. In this study, we sequenced a total of 60 samples from 15 developing stages of the pectoral muscle and five other tissues at 300 days of Tibetan chicken. We characterized the expression patterns of miRNAs across muscle developmental stages, and found that the chicken growth and development stage was divided into early-embryonic and late-embryonic as well as postnatal stages. We identified 81 and 21 DE-miRNAs by comparing the miRNA profiles of pectoral muscle of three broad periods and different tissues, respectively; and 271 miRNAs showed time-course patterns. Their potential targets were predicted and used for functional enrichment to understand their regulatory functions. Significantly, GgmiRNA-454 is a time-dependent and tissue-differential expression miRNA. In order to elucidate the role of gga-miRNA-454 in the differentiation of myoblasts, we cultured chicken myoblasts in vitro. The results show that although gga-miRNA-454-3p initiates increase and thereafter decrease during the chicken myoblasts differentiation, it had no effect on primary myoblasts proliferation. Furthermore, we confirm that gga-miRNA-454 inhibits myoblast differentiation by targeting the myotube-associated protein SBF2.
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Affiliation(s)
- Min Chen
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China.,Department of Science and Technology, Chengdu Medical College, Chengdu, China.,Institute of Biopharming, West China Hospital, Sichuan University, Chengdu, China
| | - Shaolan Zhang
- Department of Science and Technology, Chengdu Medical College, Chengdu, China
| | - Zhongxian Xu
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Jian Gao
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Shailendra Kumar Mishra
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Qing Zhu
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Xiaoling Zhao
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Yan Wang
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Huadong Yin
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Xiaolan Fan
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Bo Zeng
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Mingyao Yang
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Deying Yang
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Qingyong Ni
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Yan Li
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Mingwang Zhang
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
| | - Diyan Li
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricltural University, Wenjiang, China
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12
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Der Vartanian A, Chabanais J, Carrion C, Maftah A, Germot A. Downregulation of POFUT1 Impairs Secondary Myogenic Fusion Through a Reduced NFATc2/IL-4 Signaling Pathway. Int J Mol Sci 2019; 20:ijms20184396. [PMID: 31500188 PMCID: PMC6770550 DOI: 10.3390/ijms20184396] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2019] [Revised: 08/26/2019] [Accepted: 09/05/2019] [Indexed: 12/25/2022] Open
Abstract
Past work has shown that the protein O-fucosyltransferase 1 (POFUT1) is involved in mammal myogenic differentiation program. Pofut1 knockdown (Po –) in murine C2C12 cells leads to numerous elongated and thin myotubes, suggesting significant defects in secondary fusion. Among the few pathways involved in this process, NFATc2/IL-4 is described as the major one. To unravel the impact of POFUT1 on secondary fusion, we used wild-type (WT) C2C12 and Po – cell lines to follow Myf6, Nfatc2, Il-4 and Il-4rα expressions during a 120 h myogenic differentiation time course. Secreted IL-4 was quantified by ELISA. IL-4Rα expression and its labeling on myogenic cell types were investigated by Western blot and immunofluorescence, respectively. Phenotypic observations of cells treated with IL-4Rα blocking antibody were performed. In Po –, we found a decrease in nuclei number per myotube and a downexpression of Myf6. The observed downregulation of Nfatc2 is correlated to a diminution of secreted IL-4 and to the low level of IL-4Rα for reserve cells. Neutralization of IL-4Rα on WT C2C12 promotes myonuclear accretion defects, similarly to those identified in Po –. Thus, POFUT1 could be a new controller of myotube growth during myogenesis, especially through NFATc2/IL-4 signaling pathway.
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Affiliation(s)
- Audrey Der Vartanian
- PEIRENE, EA 7500, Glycosylation et différenciation cellulaire, Université de Limoges, F-87000 Limoges, France
- present address: INSERM, IMRB U955-E10, Faculté de Médecine, Université Paris Est Créteil, F-94000 Créteil, France
| | - Julien Chabanais
- PEIRENE, EA 7500, Glycosylation et différenciation cellulaire, Université de Limoges, F-87000 Limoges, France
| | - Claire Carrion
- UMR CNRS 7276, Contrôle de la Réponse Immune et des Lymphoproliférations, Université de Limoges, F-87000 Limoges, France
| | - Abderrahman Maftah
- PEIRENE, EA 7500, Glycosylation et différenciation cellulaire, Université de Limoges, F-87000 Limoges, France
| | - Agnès Germot
- PEIRENE, EA 7500, Glycosylation et différenciation cellulaire, Université de Limoges, F-87000 Limoges, France
- Correspondence: ; Tel.: +33-(0)5-55-45-76-57
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13
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Huang Y, Wu S, Zhang J, Wen H, Zhang M, He F. Methylation status and expression patterns of myomaker gene play important roles in postnatal development in the Japanese flounder (Paralichthys olivaceus). Gen Comp Endocrinol 2019; 280:104-114. [PMID: 31002826 DOI: 10.1016/j.ygcen.2019.04.017] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/10/2018] [Revised: 02/28/2019] [Accepted: 04/16/2019] [Indexed: 01/01/2023]
Abstract
Myomaker is a membrane protein that plays a crucial role in the fusion of myoblasts during muscle growth. DNA methylation, a significant factor, regulates gene expression. The aim of this study was to examine the methylation and mRNA expression patterns of the myomaker gene during 8 different postnatal developmental stages in the Japanese flounder (L: 7 days post hatch (dph); M1: 21 dph; M2: 28 dph; M3: 35 dph; J1: 90 dph; J2: 180 dph; A1: 24 months; A2: 36 months). Muscle tissue samples were taken from Japanese flounder at different postnatal development stages to measure the extent of DNA methylation and gene expression. Methylation level in the promoter and exon 1 of myomaker was measured using bisulfite sequencing, and the relative expression of myomaker during each developmental stage was measured by quantitative PCR. The relative expression levels of myomaker were up-regulated from stages L to M2, M3 to J2, and methylation of myomaker was negatively correlated with mRNA expression. Furthermore, the CpG site located at -26 bp in the promoter was the lowest methylated region in all developmental stages. These results offer a basis for understanding the mechanism by which myomaker regulates muscle formation during postnatal development.
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Affiliation(s)
- Yajuan Huang
- Key Laboratory of Mariculture (Ocean University of China), Ministry of Education, Qingdao 266003, China
| | - Shuxian Wu
- Key Laboratory of Mariculture (Ocean University of China), Ministry of Education, Qingdao 266003, China
| | - Jingru Zhang
- Key Laboratory of Mariculture (Ocean University of China), Ministry of Education, Qingdao 266003, China
| | - Haishen Wen
- Key Laboratory of Mariculture (Ocean University of China), Ministry of Education, Qingdao 266003, China
| | - Meizhao Zhang
- Key Laboratory of Mariculture (Ocean University of China), Ministry of Education, Qingdao 266003, China
| | - Feng He
- Key Laboratory of Mariculture (Ocean University of China), Ministry of Education, Qingdao 266003, China.
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Wang L, Zhu Y, Liu X, Chao Z, Wang Y, Zhong T, Guo J, Zhan S, Li L, Zhang H. Glycogen synthase kinase 3β (GSK3β) regulates the expression of MyHC2a in goat skeletal muscle satellite cells (SMSCs). Anim Sci J 2019; 90:1042-1049. [PMID: 31237073 DOI: 10.1111/asj.13253] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Revised: 05/12/2019] [Accepted: 05/27/2019] [Indexed: 12/16/2022]
Abstract
Glycogen synthase kinase beta (GSK3β) plays an important role in skeletal muscle growth, regeneration, and repair. However, the mechanism of GSK3β regulating MyHC2a expression is currently not clear. In this study, GSK3β inhibition promoted skeletal muscle satellite cells (SMSCs) differentiation and increased expression of MyoD, MyoG, MyHC1, and MyHC2a genes. Then we cloned approximately 1.1 kb of goat MyHC2a gene promoter. The deletion fragment (-514/+55) of MyHC2a promoter exhibited the highest level of promoter activity, and a NFATc2 element in this region was responsible for MyHC2a promoter activity. Treatment of SB216713 significantly decreased the transcriptional activity of the fragment (-514/+55). Furthermore, GSK3β inhibition had no effect on the luciferase activity of MyHC2a promoter after mutating the NFATc2-binding site. These results demonstrated that GSK3β inhibition promoted SMSCs differentiation and regulated the MyHC2a gene expression through NFATc2 in goat-differentiated SMSCs.
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Affiliation(s)
- Linjie Wang
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Yuehua Zhu
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Xin Liu
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Zhe Chao
- Institute of Animal Science and Veterinary Medicine, Hainan Academy of Agricultural Sciences, Haikou, P.R. China
| | - Yan Wang
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Tao Zhong
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Jiazhong Guo
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Siyuan Zhan
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Li Li
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
| | - Hongping Zhang
- Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, P.R. China
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15
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Babačić H, Mehta A, Merkel O, Schoser B. CRISPR-cas gene-editing as plausible treatment of neuromuscular and nucleotide-repeat-expansion diseases: A systematic review. PLoS One 2019; 14:e0212198. [PMID: 30794581 PMCID: PMC6386526 DOI: 10.1371/journal.pone.0212198] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2018] [Accepted: 01/29/2019] [Indexed: 12/26/2022] Open
Abstract
INTRODUCTION The system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (cas) is a new technology that allows easier manipulation of the genome. Its potential to edit genes opened a new door in treatment development for incurable neurological monogenic diseases (NMGDs). The aim of this systematic review was to summarise the findings on the current development of CRISPR-cas for therapeutic purposes in the most frequent NMGDs and provide critical assessment. METHODS AND DATA ACQUISITION We searched the MEDLINE and EMBASE databases, looking for original studies on the use of CRISPR-cas to edit pathogenic variants in models of the most frequent NMGDs, until end of 2017. We included all the studies that met the following criteria: 1. Peer-reviewed study report with explicitly described experimental designs; 2. In vitro, ex vivo, or in vivo study using human or other animal biological systems (including cells, tissues, organs, organisms); 3. focusing on CRISPR as the gene-editing method of choice; and 5. featured at least one NMGD. RESULTS We obtained 404 papers from MEDLINE and 513 from EMBASE. After removing the duplicates, we screened 490 papers by title and abstract and assessed them for eligibility. After reading 50 full-text papers, we finally selected 42 for the review. DISCUSSION Here we give a systematic summary on the preclinical development of CRISPR-cas for therapeutic purposes in NMGDs. Furthermore, we address the clinical interpretability of the findings, giving a comprehensive overview of the current state of the art. Duchenne's muscular dystrophy (DMD) paves the way forward, with 26 out of 42 studies reporting different strategies on DMD gene editing in different models of the disease. Most of the strategies aimed for permanent exon skipping by deletion with CRISPR-cas. Successful silencing of the mHTT gene with CRISPR-cas led to successful reversal of the neurotoxic effects in the striatum of mouse models of Huntington's disease. Many other strategies have been explored, including epigenetic regulation of gene expression, in cellular and animal models of: myotonic dystrophy, Fraxile X syndrome, ataxias, and other less frequent dystrophies. Still, before even considering the clinical application of CRISPR-cas, three major bottlenecks need to be addressed: efficacy, safety, and delivery of the systems. This requires a collaborative approach in the research community, while having ethical considerations in mind.
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Affiliation(s)
- Haris Babačić
- Friedrich Baur Institute, Department of Neurology, Ludwig-Maximilians-University of Munich, Munich, Germany
- * E-mail: (BS); (HB)
| | - Aditi Mehta
- Faculty of Pharmacy, Ludwig-Maximilians-University of Munich, Munich, Germany
| | - Olivia Merkel
- Faculty of Pharmacy, Ludwig-Maximilians-University of Munich, Munich, Germany
| | - Benedikt Schoser
- Friedrich Baur Institute, Department of Neurology, Ludwig-Maximilians-University of Munich, Munich, Germany
- * E-mail: (BS); (HB)
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16
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Huang L, Chen Y, Huang W, Wu H. Cell pairing and polyethylene glycol (PEG)-mediated cell fusion using two-step centrifugation-assisted single-cell trapping (CAScT). LAB ON A CHIP 2018; 18:1113-1120. [PMID: 29536068 DOI: 10.1039/c7lc01131h] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/18/2023]
Abstract
Here a convenient and effective strategy of two-step centrifugation-assisted single-cell trapping (CAScT) based on an addressable truncated cone-shaped microwell array (TCMA) chip is developed for cell pairing. We describe the operation principles of the method and demonstrate its compatibility with polyethylene glycol (PEG)-mediated cell fusion. Compared with other methods, most of which rely on sophisticated devices and bulky subsidiary equipment, our method is more convenient and exhibits better or comparable performance. Using this method, up to around 6000 heterotypic cell pairs can be formed and addressed within a small area of 1 cm2. The paired cells are then treated with fusogenic PEG for cell fusion. Compared with traditional protocols, cell fusion using this approach is well defined with better control, which leads to an improved yield of heterotypic binucleated hybrids. Furthermore, we demonstrate the capability of our device for long-term cell culture and cell harvesting.
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Affiliation(s)
- Lu Huang
- Shenzhen Research Institute, The Hong Kong University of Science and Technology, Shenzhen, China.
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17
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Cho S, Hong R, Yim P, Yeom M, Lee B, Yang WM, Hong J, Lee HS, Hahm DH. An herbal formula consisting of Schisandra chinensis (Turcz.) Baill, Lycium chinense Mill and Eucommia ulmoides Oliv alleviates disuse muscle atrophy in rats. JOURNAL OF ETHNOPHARMACOLOGY 2018; 213:328-339. [PMID: 29051115 DOI: 10.1016/j.jep.2017.10.008] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/08/2016] [Revised: 09/22/2017] [Accepted: 10/12/2017] [Indexed: 06/07/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE Schisandra chinensis (Turcz.) Baill (SC), Lycium chinense Mill (LC) and Eucommia ulmoides Oliv (EU) are representative tonic herbal medicines that help to strengthen body muscles and bones making them stronger according to the Donguibogam, a tradition medical book of the Joseon Dynasty in Korea. AIM OF THE STUDY To evaluate effects of an herbal formula consisting of SC, LC and EU on muscle atrophy in C2C12 myotubes and in a rat model of immobilization-induced muscle atrophy. MATERIALS AND METHODS Muscle atrophy was developed by cast immobilization of unilateral hindlimb on rats for 3 weeks. Treatments were administered orally 14 times over 3 weeks. After treatments, we compared the change of body weight, muscle weight, grip strength, muscle fiber size, muscle fiber type shift by Grip strength meter, H&E stain and ATPase stain. And western blot was used for evaluating molecular mechanism in muscle atrophy on C2C12 cells. RESULTS When taken individually, SC was the most effective of the three in inhibiting tumor necrosis factor alpha (TNF-α)-induced degeneration of C2C12 myogenesis. The formulation with a mass ratio of 2:1:1 SC: LC: EU (SSLE) was more effective against TNF-α-induced muscle atrophy than was a 1:1:1 SC: LC: EU (SLE) formula or any of the single herbal extracts. In a rat model of disuse muscle atrophy, the SSLE formula significantly inhibited reductions in muscle weight, grip strength and muscle fiber size induced by hindlimb immobilization, in a dose-dependent manner. The formula also inhibited immobilization-induced shifting of the muscle fiber type in soleus muscle. Treatment with SSLE inhibited TNF-α-induced expression of the atrogenes atrogin-1 and muscle RING-finger protein 1 in C2C12 cells. The SSLE formula also increased myoblast differentiation markers (myoD and myogenin) and activation of the Akt and mammalian target of rapamycin (mTOR) signaling pathway. CONCLUSION These findings suggest that the SSLE formula prevents muscle atrophy through inhibition of the ubiquitin-proteasome system as well as upregulation of myoblast differentiation and muscle protein synthesis in C2C12 cells. Taken together, we conclude that the SSLE formula is invaluable for the development of therapeutic medicines to prevent disuse muscle atrophy and its accompanying muscle weakness.
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Affiliation(s)
- Seongguk Cho
- Department of Science in Korean Medicine, Graduate School, Kyung Hee University, 02447 Seoul, Republic of Korea
| | - Riwon Hong
- Department of Science in Korean Medicine, Graduate School, Kyung Hee University, 02447 Seoul, Republic of Korea
| | - Poorm Yim
- Department of Science in Korean Medicine, Graduate School, Kyung Hee University, 02447 Seoul, Republic of Korea
| | - Mijung Yeom
- Acupuncture and Meridian Science Research Center, College of Korean Medicine, Kyung Hee University, 02447 Seoul, Republic of Korea
| | - Bombi Lee
- Acupuncture and Meridian Science Research Center, College of Korean Medicine, Kyung Hee University, 02447 Seoul, Republic of Korea
| | - Woong Mo Yang
- Department of Science in Korean Medicine, Graduate School, Kyung Hee University, 02447 Seoul, Republic of Korea
| | - Jongki Hong
- Colleges of Pharmacy, Kyung Hee University, 02447 Seoul, Republic of Korea
| | - Hyang Sook Lee
- Department of Science in Korean Medicine, Graduate School, Kyung Hee University, 02447 Seoul, Republic of Korea; Acupuncture and Meridian Science Research Center, College of Korean Medicine, Kyung Hee University, 02447 Seoul, Republic of Korea
| | - Dae-Hyun Hahm
- Acupuncture and Meridian Science Research Center, College of Korean Medicine, Kyung Hee University, 02447 Seoul, Republic of Korea; Department of Physiology, School of Medicine, Kyung Hee University, 02447 Seoul, Republic of Korea.
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18
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Glucocorticoids Improve Myogenic Differentiation In Vitro by Suppressing the Synthesis of Versican, a Transitional Matrix Protein Overexpressed in Dystrophic Skeletal Muscles. Int J Mol Sci 2017; 18:ijms18122629. [PMID: 29211034 PMCID: PMC5751232 DOI: 10.3390/ijms18122629] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2017] [Revised: 11/24/2017] [Accepted: 11/27/2017] [Indexed: 12/17/2022] Open
Abstract
In Duchenne muscular dystrophy (DMD), a dysregulated extracellular matrix (ECM) directly exacerbates pathology. Glucocorticoids are beneficial therapeutics in DMD, and have pleiotropic effects on the composition and processing of ECM proteins in other biological contexts. The synthesis and remodelling of a transitional versican-rich matrix is necessary for myogenesis; whether glucocorticoids modulate this transitional matrix is not known. Here, versican expression and processing were examined in hindlimb and diaphragm muscles from mdx dystrophin-deficient mice and C57BL/10 wild type mice. V0/V1 versican (Vcan) mRNA transcripts and protein levels were upregulated in dystrophic compared to wild type muscles, especially in the more severely affected mdx diaphragm. Processed versican (versikine) was detected in wild type and dystrophic muscles, and immunoreactivity was highly associated with newly regenerated myofibres. Glucocorticoids enhanced C2C12 myoblast fusion by modulating the expression of genes regulating transitional matrix synthesis and processing. Specifically, Tgfβ1, Vcan and hyaluronan synthase-2 (Has2) mRNA transcripts were decreased by 50% and Adamts1 mRNA transcripts were increased three-fold by glucocorticoid treatment. The addition of exogenous versican impaired myoblast fusion, whilst glucocorticoids alleviated this inhibition in fusion. In dystrophic mdx muscles, versican upregulation correlated with pathology. We propose that versican is a novel and relevant target gene in DMD, given its suppression by glucocorticoids and that in excess it impairs myoblast fusion, a process key for muscle regeneration.
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Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat Biomed Eng 2017; 1:889-901. [PMID: 29805845 PMCID: PMC5968829 DOI: 10.1038/s41551-017-0137-2] [Citation(s) in RCA: 522] [Impact Index Per Article: 65.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
CRISPR/Cas9-based therapeutics, especially those that can correct gene mutations via homology directed repair (HDR), have the potential to revolutionize the treatment of genetic diseases. However, HDR-based therapeutics are challenging to develop because they require simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types, and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.
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20
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Kurosaka M, Ogura Y, Funabashi T, Akema T. Involvement of Transient Receptor Potential Cation Channel Vanilloid 1 (TRPV1) in Myoblast Fusion. J Cell Physiol 2016; 231:2275-85. [PMID: 26892397 DOI: 10.1002/jcp.25345] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2015] [Accepted: 02/16/2016] [Indexed: 12/15/2022]
Abstract
The mechanisms that underlie the complex process of muscle regeneration after injury remain unknown. Transient receptor potential cation channel vanilloid 1 (TRPV1) is expressed in several cell types, including skeletal muscle, and is activated by high temperature and by certain molecules secreted during tissue inflammation. Severe inflammation and local temperature perturbations are induced during muscle regeneration, which suggests that TRPV1 might be activated and involved in the process. The aim of this study, was to clarify the role of TRPV1 in the myogenic potential of satellite cells responsible for muscle regeneration. We found that mRNA and protein levels of TRPV1 increased during regeneration after cardiotoxin (CTX)-induced muscle injury in mice. Using isolated mouse satellite cells (i.e., myoblasts), we observed that activation of TRPV1 by its agonist capsaicin (CAP) augmented myogenin protein levels. Whereas CAP did not alter myoblast proliferation, it facilitated myoblast fusion (evaluated using myonucleii number per myotube and fusion index). In contrast, suppression of TRPV1 by siRNA impaired myoblast fusion. Using mice, we also demonstrated that intramuscular injection of CAP facilitated muscle repair after CTX-induced muscle injury. Moreover, we showed that these roles of TRPV1 might be mediated by interleukin-4 and calcium signaling during myoblast fusion. Collectively, these results suggest that TRPV1 underlies normal myogenesis through promotion of myoblast fusion. J. Cell. Physiol. 231: 2275-2285, 2016. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Mitsutoshi Kurosaka
- Department of Physiology, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan
| | - Yuji Ogura
- Department of Physiology, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan
| | - Toshiya Funabashi
- Department of Physiology, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan
| | - Tatsuo Akema
- Department of Physiology, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan
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21
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Structure-function analysis of myomaker domains required for myoblast fusion. Proc Natl Acad Sci U S A 2016; 113:2116-21. [PMID: 26858401 DOI: 10.1073/pnas.1600101113] [Citation(s) in RCA: 57] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
During skeletal muscle development, myoblasts fuse to form multinucleated myofibers. Myomaker [Transmembrane protein 8c (TMEM8c)] is a muscle-specific protein that is essential for myoblast fusion and sufficient to promote fusion of fibroblasts with muscle cells; however, the structure and biochemical properties of this membrane protein have not been explored. Here, we used CRISPR/Cas9 mutagenesis to disrupt myomaker expression in the C2C12 muscle cell line, which resulted in complete blockade to fusion. To define the functional domains of myomaker required to direct fusion, we established a heterologous cell-cell fusion system, in which fibroblasts expressing mutant versions of myomaker were mixed with WT myoblasts. Our data indicate that the majority of myomaker is embedded in the plasma membrane with seven membrane-spanning regions and a required intracellular C-terminal tail. We show that myomaker function is conserved in other mammalian orthologs; however, related family members (TMEM8a and TMEM8b) do not exhibit fusogenic activity. These findings represent an important step toward deciphering the cellular components and mechanisms that control myoblast fusion and muscle formation.
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Luo W, Li E, Nie Q, Zhang X. Myomaker, Regulated by MYOD, MYOG and miR-140-3p, Promotes Chicken Myoblast Fusion. Int J Mol Sci 2015; 16:26186-201. [PMID: 26540045 PMCID: PMC4661805 DOI: 10.3390/ijms161125946] [Citation(s) in RCA: 87] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2015] [Revised: 10/16/2015] [Accepted: 10/22/2015] [Indexed: 12/21/2022] Open
Abstract
The fusion of myoblasts is an important step during skeletal muscle differentiation. A recent study in mice found that a transmembrane protein called Myomaker, which is specifically expressed in muscle, is critical for myoblast fusion. However, the cellular mechanism of its roles and the regulatory mechanism of its expression remain unclear. Chicken not only plays an important role in meat production but is also an ideal model organism for muscle development research. Here, we report that Myomaker is also essential for chicken myoblast fusion. Forced expression of Myomaker in chicken primary myoblasts promotes myoblast fusion, whereas knockdown of Myomaker by siRNA inhibits myoblast fusion. MYOD and MYOG, which belong to the family of myogenic regulatory factors, can bind to a conserved E-box located proximal to the Myomaker transcription start site and induce Myomaker transcription. Additionally, miR-140-3p can inhibit Myomaker expression and myoblast fusion, at least in part, by binding to the 3ʹ UTR of Myomaker in vitro. These findings confirm the essential roles of Myomaker in avian myoblast fusion and show that MYOD, MYOG and miR-140-3p can regulate Myomaker expression.
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Affiliation(s)
- Wen Luo
- Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
- Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, South China Agricultural University, Guangzhou 510642, China.
- Key Lab of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, South China Agricultural University, Guangzhou 510642, China.
| | - Erxin Li
- Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
- Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, South China Agricultural University, Guangzhou 510642, China.
- Key Lab of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, South China Agricultural University, Guangzhou 510642, China.
| | - Qinghua Nie
- Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
- Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, South China Agricultural University, Guangzhou 510642, China.
- Key Lab of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, South China Agricultural University, Guangzhou 510642, China.
| | - Xiquan Zhang
- Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
- Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, South China Agricultural University, Guangzhou 510642, China.
- Key Lab of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, South China Agricultural University, Guangzhou 510642, China.
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Feeney SJ, McGrath MJ, Sriratana A, Gehrig SM, Lynch GS, D’Arcy CE, Price JT, McLean CA, Tupler R, Mitchell CA. FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1). PLoS One 2015; 10:e0117665. [PMID: 25695429 PMCID: PMC4335040 DOI: 10.1371/journal.pone.0117665] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2014] [Accepted: 12/29/2014] [Indexed: 01/01/2023] Open
Abstract
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.
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Affiliation(s)
- Sandra J. Feeney
- Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia
| | - Meagan J. McGrath
- Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia
| | - Absorn Sriratana
- Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia
| | - Stefan M. Gehrig
- Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Victoria, 3010, Australia
| | - Gordon S. Lynch
- Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Victoria, 3010, Australia
| | - Colleen E. D’Arcy
- Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia
| | - John T. Price
- Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia
- Centre for Chronic Disease Prevention and Management, College of Health and Biomedicine, Victoria University, Melbourne, Victoria, 8001, Australia
| | - Catriona A. McLean
- Department of Anatomical Pathology, Alfred Hospital, Prahran, Victoria, 3004, Australia
- Department of Medicine, Central Clinical School, Monash University, Clayton, VIC, 3800, Australia
| | - Rossella Tupler
- Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, 01655, United States of America
- Dipartimento di Scienze della Vita, Universita di Modena e Reggio Emilia, 41125, Modena, Italy
| | - Christina A. Mitchell
- Department of Biochemistry & Molecular Biology, Monash University, Clayton, Victoria, 3800, Australia
- * E-mail:
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Costa A, Toschi A, Murfuni I, Pelosi L, Sica G, Adamo S, Scicchitano BM. Local overexpression of V1a-vasopressin receptor enhances regeneration in tumor necrosis factor-induced muscle atrophy. BIOMED RESEARCH INTERNATIONAL 2014; 2014:235426. [PMID: 24971321 PMCID: PMC4055243 DOI: 10.1155/2014/235426] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/27/2014] [Revised: 04/22/2014] [Accepted: 04/23/2014] [Indexed: 01/25/2023]
Abstract
Skeletal muscle atrophy occurs during disuse and aging, or as a consequence of chronic diseases such as cancer and diabetes. It is characterized by progressive loss of muscle tissue due to hypotrophic changes, degeneration, and an inability of the regeneration machinery to replace damaged myofibers. Tumor necrosis factor (TNF) is a proinflammatory cytokine known to mediate muscle atrophy in many chronic diseases and to inhibit skeletal muscle regeneration. In this study, we investigated the role of Arg-vasopressin-(AVP-)dependent pathways in muscles in which atrophy was induced by local overexpression of TNF. AVP is a potent myogenesis-promoting factor and is able to enhance skeletal muscle regeneration by stimulating Ca(2+)/calmodulin-dependent kinase and calcineurin signaling. We performed morphological and molecular analyses and demonstrated that local over-expression of the AVP receptor V1a enhances regeneration of atrophic muscle. By upregulating the regeneration/differentiation markers, modulating the inflammatory response, and attenuating fibrogenesis, the stimulation of AVP-dependent pathways creates a favourable environment for efficient and sustained muscle regeneration and repair even in the presence of elevated levels of TNF. This study highlights a novel in vivo role for AVP-dependent pathways, which may represent an interesting strategy to counteract muscle decline in aging or in muscular pathologies.
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Affiliation(s)
- Alessandra Costa
- DAHFMO Unit of Histology and Medical Embryology, Interuniversity Institute of Myology, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy
| | - Angelica Toschi
- DAHFMO Unit of Histology and Medical Embryology, Interuniversity Institute of Myology, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy
| | - Ivana Murfuni
- DAHFMO Unit of Histology and Medical Embryology, Interuniversity Institute of Myology, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy
| | - Laura Pelosi
- DAHFMO Unit of Histology and Medical Embryology, Interuniversity Institute of Myology, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy
| | - Gigliola Sica
- Institute of Histology and Embryology, Catholic University School of Medicine, L.go F. Vito, 1, 00168 Rome, Italy
| | - Sergio Adamo
- DAHFMO Unit of Histology and Medical Embryology, Interuniversity Institute of Myology, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy
| | - Bianca Maria Scicchitano
- DAHFMO Unit of Histology and Medical Embryology, Interuniversity Institute of Myology, Sapienza University of Rome, Via A. Scarpa 16, 00161 Rome, Italy
- Institute of Histology and Embryology, Catholic University School of Medicine, L.go F. Vito, 1, 00168 Rome, Italy
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25
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Wilding BR, McGrath MJ, Bonne G, Mitchell CA. FHL1 mutations that cause clinically distinct human myopathies form protein aggregates and impair myoblast differentiation. J Cell Sci 2014; 127:2269-81. [DOI: 10.1242/jcs.140905] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
FHL1 mutations cause several clinically heterogeneous myopathies including Reducing Body Myopathy (RBM), Scapuloperoneal Myopathy (SPM) and X-Linked Myopathy with Postural Muscle Atrophy (XMPMA). The molecular mechanisms underlying the pathogenesis of FHL1 myopathies are unknown. Protein aggregates designated “Reducing Bodies” (RBs) containing mutant FHL1 are detected in RBM muscle but not several other FHL1 myopathies. Here RBM, SPM and XMPMA FHL1 mutants were expressed in C2C12 cells and showed equivalent protein expression to wild-type FHL1 and formed aggregates positive for the RB stain Menadione-NBT, analogous to RBM muscle aggregates. However HCM and EDMD FHL1 mutants generally exhibited reduced expression. Wild-type FHL1 promotes myoblast differentiation however RBM, SPM and XMPMA mutations impaired differentiation, consistent with loss-of-normal function. Furthermore, SPM and XMPMA mutants retarded myotube formation relative to vector control consistent with a dominant-negative/toxic function. Mutant FHL1 myotube formation was partially rescued by expression of the FHL1-binding partner constitutively-active NFATc1. This is the first study to show FHL1 mutations identified in several clinically distinct myopathies lead to similar protein aggregation and impaired myotube formation suggesting a common pathogenic mechanism despite heterogenous clinical features.
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26
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Turner NJ, Londono R, Dearth CL, Culiat CT, Badylak SF. Human NELL1 protein augments constructive tissue remodeling with biologic scaffolds. Cells Tissues Organs 2013; 198:249-65. [PMID: 24335144 DOI: 10.1159/000356491] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/18/2013] [Indexed: 11/19/2022] Open
Abstract
Biologic scaffolds composed of extracellular matrix (ECM) derived from decellularized tissues effectively reprogram key stages of the mammalian response to injury, altering the wound microenvironment from one that promotes scar tissue formation to one that stimulates constructive and functional tissue remodeling. In contrast, engineered scaffolds, composed of purified ECM components such as collagen, lack the complex ultrastructure and composition of intact ECM and may promote wound healing but lack factors that facilitate constructive and functional tissue remodeling. The objective of the present study was to test the hypothesis that addition of NELL1, a signaling protein that controls cell growth and differentiation, enhances the constructive tissue remodeling of a purified collagen scaffold. An abdominal wall defect model in the rat of 1.5-cm(2) partial thickness was used to compare the constructive remodeling of a bovine type I collagen scaffold to a biologic scaffold derived from small intestinal submucosa (SIS)-ECM with and without augmentation with 17 μg NELL1 protein. Samples were evaluated histologically at 14 days and 4 months. The contractile response of the defect site was also evaluated at 4 months. Addition of NELL1 protein improved the constructive remodeling of collagen scaffolds but not SIS-ECM scaffolds. Results showed an increase in the contractile force of the remodeled skeletal muscle and a fast:slow muscle composition similar to native tissue in the collagen-treated group. The already robust remodeling response to SIS-ECM was not enhanced by NELL1 at the dose tested. These findings suggest that NELL1 protein does contribute to the enhanced constructive remodeling of skeletal muscle.
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Affiliation(s)
- Neill J Turner
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pa., USA
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27
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Transthyretin is a key regulator of myoblast differentiation. PLoS One 2013; 8:e63627. [PMID: 23717457 PMCID: PMC3661549 DOI: 10.1371/journal.pone.0063627] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2012] [Accepted: 04/04/2013] [Indexed: 12/25/2022] Open
Abstract
Transthyretin (TTR) is a known carrier protein for thyroxine (T4) and retinol-binding protein in the blood that is primarily synthesized in the liver and choroid plexus of the brain. Herein, we report that the TTR gene is expressed in skeletal muscle tissue and up-regulated during myotube formation in C2C12 cells. TTR silencing (TTRkd) significantly reduced myogenin expression and myotube formation, whereas myogenin silencing (MYOGkd) did not have any effect on TTR gene expression. Both TTRkd and MYOGkd led to a decrease in calcium channel related genes including Cav1.1, STIM1 and Orai1. A significant decrease in intracellular T4 uptake during myogenesis was observed in TTRkd cells. Taken together, the results of this study suggest that TTR initiates myoblast differentiation via affecting expression of the genes involved during early stage of myogenesis and the genes related to calcium channel.
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28
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Abstract
Myoblast fusion is a critical process that contributes to the growth of muscle during development and to the regeneration of myofibers upon injury. Myoblasts fuse with each other as well as with multinucleated myotubes to enlarge the myofiber. Initial studies demonstrated that myoblast fusion requires extracellular calcium and changes in cell membrane topography and cytoskeletal organization. More recent studies have identified several cell-surface and intracellular proteins that mediate myoblast fusion. Furthermore, emerging evidence suggests that myoblast fusion is also regulated by the activation of specific cell-signaling pathways that lead to the expression of genes whose products are essential for the fusion process and for modulating the activity of molecules that are involved in cytoskeletal rearrangement. Here, we review the roles of the major signaling pathways in mammalian myoblast fusion.
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Affiliation(s)
- Sajedah M Hindi
- Department of Anatomical Sciences and Neurobiology, School of Medicine, University of Louisville, Louisville, KY 40202, USA
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29
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Markworth JF, Cameron-Smith D. Arachidonic acid supplementation enhances in vitro skeletal muscle cell growth via a COX-2-dependent pathway. Am J Physiol Cell Physiol 2013; 304:C56-67. [DOI: 10.1152/ajpcell.00038.2012] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Arachidonic acid (AA) is the metabolic precursor to a diverse range of downstream bioactive lipid mediators. A positive or negative influence of individual eicosanoid species [e.g., prostaglandins (PGs), leukotrienes, and hydroxyeicosatetraenoic acids] has been implicated in skeletal muscle cell growth and development. The collective role of AA-derived metabolites in physiological states of skeletal muscle growth/atrophy remains unclear. The present study aimed to determine the direct effect of free AA supplementation and subsequent eicosanoid biosynthesis on skeletal myocyte growth in vitro . C2C12 (mouse) skeletal myocytes induced to differentiate with supplemental AA exhibited dose-dependent increases in the size, myonuclear content, and protein accretion of developing myotubes, independent of changes in cell density or the rate/extent of myogenic differentiation. Nonselective (indomethacin) or cyclooxygenase 2 (COX-2)-selective (NS-398) nonsteroidal anti-inflammatory drugs blunted basal myogenesis, an effect that was amplified in the presence of supplemental free AA substrate. The stimulatory effects of AA persisted in preexisting myotubes via a COX-2-dependent (NS-389-sensitive) pathway, specifically implying dependency on downstream PG biosynthesis. AA-stimulated growth was associated with markedly increased secretion of PGF2α and PGE2; however, incubation of myocytes with PG-rich conditioned medium failed to mimic the effects of direct AA supplementation. In vitro AA supplementation stimulates PG release and skeletal muscle cell hypertrophy via a COX-2-dependent pathway.
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Affiliation(s)
- James F. Markworth
- School of Exercise and Nutrition Science, Deakin University, Melbourne, Australia; and
- Liggins Institute, University of Auckland, Auckland, New Zealand
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30
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Enwere EK, Holbrook J, Lejmi-Mrad R, Vineham J, Timusk K, Sivaraj B, Isaac M, Uehling D, Al-awar R, LaCasse E, Korneluk RG. TWEAK and cIAP1 regulate myoblast fusion through the noncanonical NF-κB signaling pathway. Sci Signal 2012; 5:ra75. [PMID: 23074266 DOI: 10.1126/scisignal.2003086] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
The fusion of mononucleated muscle progenitor cells (myoblasts) into multinucleated muscle fibers is a critical aspect of muscle development and regeneration. We identified the noncanonical nuclear factor κB (NF-κB) pathway as a signaling axis that drives the recruitment of myoblasts into new muscle fibers. Loss of cellular inhibitor of apoptosis 1 (cIAP1) protein led to constitutive activation of the noncanonical NF-κB pathway and an increase in the number of nuclei per myotube. Knockdown of essential mediators of NF-κB signaling, such as p100, RelB, inhibitor of κB kinase α, and NF-κB-inducing kinase, attenuated myoblast fusion in wild-type myoblasts. In contrast, the extent of myoblast fusion was increased when the activity of the noncanonical NF-κB pathway was enhanced by increasing the abundance of p52 and RelB or decreasing the abundance of tumor necrosis factor (TNF) receptor-associated factor 3, an inhibitor of this pathway. Low concentrations of the cytokine TNF-like weak inducer of apoptosis (TWEAK), which preferentially activates the noncanonical NF-κB pathway, also increased myoblast fusion, without causing atrophy or impairing myogenesis. These results identify roles for TWEAK, cIAP1, and noncanonical NF-κB signaling in the regulation of myoblast fusion and highlight a role for cytokine signaling during adult skeletal myogenesis.
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Affiliation(s)
- Emeka K Enwere
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada
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31
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Garikipati DK, Rodgers BD. Myostatin inhibits myosatellite cell proliferation and consequently activates differentiation: evidence for endocrine-regulated transcript processing. J Endocrinol 2012; 215:177-87. [PMID: 22872758 DOI: 10.1530/joe-12-0260] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Myostatin is a potent negative regulator of muscle growth in mammals. Despite high structural conservation, functional conservation in nonmammalian species is only assumed. This is particularly true for fish due to the presence of several myostatin paralogs: two in most species and four in salmonids (MSTN-1a, -1b, -2a, and -2b). Rainbow trout are a rich source of primary myosatellite cells as hyperplastic muscle growth occurs even in adult fish. These cells were therefore used to determine myostatin's effects on proliferation whereas our earlier studies reported its effects on quiescent cells. As in mammals, recombinant myostatin suppressed proliferation with no changes in cell morphology. Expression of MSTN-1a was several fold higher than the other paralogs and was autoregulated by myostatin, which also upregulated the expression of key differentiation markers: Myf5, MyoD1, myogenin, and myosin light chain. Thus, myostatin-stimulated cellular growth inhibition activates rather than represses differentiation. IGF-1 stimulated proliferation but had minimal and delayed effects on differentiation and its actions were suppressed by myostatin. However, IGF-1 upregulated MSTN-2a expression and the processing of its transcript, which is normally unprocessed. Myostatin therefore appears to partly mediate IGF-stimulated myosatellite differentiation in rainbow trout. This also occurs in mammals, although the IGF-stimulated processing of MSTN-2a transcripts is highly unique and is indicative of subfunctionalization within the gene family. These studies also suggest that the myokine's actions, including its antagonistic relationship with IGF-1, are conserved and that the salmonid gene family is functionally diverging.
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Affiliation(s)
- Dilip K Garikipati
- Department of Animal Sciences, 124 ASLB, School of Molecular Biosciences, Washington Center for Muscle Biology, Washington State University, Pullman, Washington 99164, USA
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32
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Wang PY, Thissen H, Tsai WB. The roles of RGD and grooved topography in the adhesion, morphology, and differentiation of C2C12 skeletal myoblasts. Biotechnol Bioeng 2012; 109:2104-15. [PMID: 22359221 DOI: 10.1002/bit.24452] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2011] [Revised: 01/16/2012] [Accepted: 01/18/2012] [Indexed: 02/01/2023]
Abstract
Both chemical and topographic cues are crucial for the development of skeletal muscle. In this study, the relative roles of both signals in regard to cell adhesion, morphology, and differentiation of C2C12 skeletal myoblasts were investigated. Grooved polystyrene substrates containing grooves with approximately 900 nm in width with 600 nm ridge spans and 665 nm in depth were conjugated with the cell adhesion peptide arginine-glycine-aspartic acid (RGD). RGD conjugation significantly enhanced the adhesion, growth and differentiation of C2C12 cells. On the other hand, anisotropic topography primarily directed the direction and alignment of myoblasts and myotubes. The results in this study provide information regarding the relative roles of chemical and topographic cues in musculoskeletal myogenesis, and are of interest to applications in muscle tissue engineering.
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Affiliation(s)
- Peng-Yuan Wang
- Department of Chemical Engineering, National Taiwan University, No. 1, Roosevelt Rd., Sec. 4, Taipei 106, Taiwan
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33
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Minetti GC, Feige JN, Rosenstiel A, Bombard F, Meier V, Werner A, Bassilana F, Sailer AW, Kahle P, Lambert C, Glass DJ, Fornaro M. G i2 Signaling Promotes Skeletal Muscle Hypertrophy, Myoblast Differentiation, and Muscle Regeneration. Sci Signal 2011; 4:ra80. [DOI: 10.1126/scisignal.2002038] [Citation(s) in RCA: 60] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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Angione AR, Jiang C, Pan D, Wang YX, Kuang S. PPARδ regulates satellite cell proliferation and skeletal muscle regeneration. Skelet Muscle 2011; 1:33. [PMID: 22040534 PMCID: PMC3223495 DOI: 10.1186/2044-5040-1-33] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2011] [Accepted: 11/01/2011] [Indexed: 01/07/2023] Open
Abstract
Peroxisome proliferator-activated receptors (PPARs) are a class of nuclear receptors that play important roles in development and energy metabolism. Whereas PPARδ has been shown to regulate mitochondrial biosynthesis and slow-muscle fiber types, its function in skeletal muscle progenitors (satellite cells) is unknown. Since constitutive mutation of Pparδ leads to embryonic lethality, we sought to address this question by conditional knockout (cKO) of Pparδ using Myf5-Cre/Pparδflox/flox alleles to ablate PPARδ in myogenic progenitor cells. Although Pparδ-cKO mice were born normally and initially displayed no difference in body weight, muscle size or muscle composition, they later developed metabolic syndrome, which manifested as increased body weight and reduced response to glucose challenge at age nine months. Pparδ-cKO mice had 40% fewer satellite cells than their wild-type littermates, and these satellite cells exhibited reduced growth kinetics and proliferation in vitro. Furthermore, regeneration of Pparδ-cKO muscles was impaired after cardiotoxin-induced injury. Gene expression analysis showed reduced expression of the Forkhead box class O transcription factor 1 (FoxO1) gene in Pparδ-cKO muscles under both quiescent and regenerating conditions, suggesting that PPARδ acts through FoxO1 in regulating muscle progenitor cells. These results support a function of PPARδ in regulating skeletal muscle metabolism and insulin sensitivity, and they establish a novel role of PPARδ in muscle progenitor cells and postnatal muscle regeneration.
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Affiliation(s)
- Alison R Angione
- Department of Animal Sciences, Purdue University, 901 West State Street, West Lafayette, IN 47907, USA.
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35
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Toschi A, Severi A, Coletti D, Catizone A, Musarò A, Molinaro M, Nervi C, Adamo S, Scicchitano BM. Skeletal muscle regeneration in mice is stimulated by local overexpression of V1a-vasopressin receptor. Mol Endocrinol 2011; 25:1661-73. [PMID: 21816902 PMCID: PMC5417231 DOI: 10.1210/me.2011-1049] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2011] [Accepted: 07/05/2011] [Indexed: 01/25/2023] Open
Abstract
Skeletal muscle has a remarkable capacity to regenerate after mechanical or pathological injury. We show that the V1a receptor (V1aR) for vasopressin, a potent myogenic-promoting factor that stimulates differentiation and hypertrophy in vitro, is expressed in mouse skeletal muscle and modulated during regeneration after experimental injury. We used gene delivery by electroporation to overexpress the myc-tagged vasopressin V1aR in specific muscles, thus sensitizing them to circulating vasopressin. The correct localization on the surface of the fibers of the recombinant product was demonstrated by confocal immunofluorescence directed against the myc tag. V1aR overexpression dramatically enhanced regeneration. When compared with mock-transfected controls, V1aR overexpressing muscles exhibited significantly accelerated activation of satellite cells and increased expression of differentiation markers. Downstream of V1aR activation, calcineurin was strongly up-regulated and stimulated the expression of IL-4, a potent mediator of myogenic cell fusion. The central role of calcineurin in mediating V1aR-dependent myogenesis was also demonstrated by using its specific inhibitor, cyclosporine A. This study identifies skeletal muscle as a physiological target of hormones of the vasopressin family and reveals a novel in vivo role for vasopressin-dependent pathways. These findings unveil several steps, along a complex signaling pathway, that may be exploited as potential targets for the therapy of diseases characterized by altered muscle homeostasis and regeneration.
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MESH Headings
- Animals
- Arginine Vasopressin/pharmacology
- Biomarkers/metabolism
- Calcineurin/metabolism
- Cell Differentiation/drug effects
- Desmin/metabolism
- Female
- Gene Expression Regulation/drug effects
- Interleukin-4/genetics
- Interleukin-4/metabolism
- Mice
- Mice, Inbred C57BL
- Muscle, Skeletal/metabolism
- Muscle, Skeletal/pathology
- Muscle, Skeletal/physiology
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Receptors, Vasopressin/genetics
- Receptors, Vasopressin/metabolism
- Regeneration/drug effects
- Regeneration/physiology
- Satellite Cells, Skeletal Muscle/drug effects
- Satellite Cells, Skeletal Muscle/metabolism
- Satellite Cells, Skeletal Muscle/pathology
- Signal Transduction/drug effects
- Transfection
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Affiliation(s)
- Angelica Toschi
- Department of Anatomical, Histological, Forensic, and Orthopaedic Sciences, Sapienza University of Rome, Rome, Italy
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Johnston APW, Bellamy LM, Lisio MD, Parise G. Captopril treatment induces hyperplasia but inhibits myonuclear accretion following severe myotrauma in murine skeletal muscle. Am J Physiol Regul Integr Comp Physiol 2011; 301:R363-9. [DOI: 10.1152/ajpregu.00766.2010] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The role of ANG II in skeletal muscle and satellite cell regulation is largely unknown. Cardiotoxin (CTX) was used to investigate whether muscle injury activates a local ANG II signaling system. Following injury, immunohistochelmistry (IHC) analysis revealed a robust increase in the intensity of angiotensinogen and angiotensin type 1 (AT1) receptor expression. As regeneration proceeded, however, AT1 and angiotensinogen were downregulated. Nuclear accretion and fiber formation were also assessed during muscle regeneration in mice treated with captopril (an angiotensin-converting enzyme inhibitor). When ANG II formation was blocked through the use of captopril, we observed a significantly reduced accretion of nuclei into myofibers (−25%), while tibialis anterior total fiber number was significantly increased +37%. This phenotype appeared to be due to alterations in satellite cell differentiation kinetics; captopril treatment led to sustained mRNA expression of markers associated with quiescence and proliferation (Myf5, Pax7) and simultaneously delayed or inhibited the expression of myogenin. IHC staining supported these findings, revealing that captopril treatment resulted in a strong trend ( P = 0.06) for a decrease in the proportion of myogenin-positive myoblasts. Furthermore, these observations were associated with a delay in muscle fiber maturation; captopril treatment resulted in sustained expression of embryonic myosin heavy chain. Collectively, these findings demonstrate that localized skeletal muscle angiotensin signaling is important to muscle fiber formation, myonuclear accretion, and satellite cell function.
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Affiliation(s)
| | | | | | - Gianni Parise
- Departments of 1Kinesiology and Medical Physics and
- Applied Radiation Sciences, McMaster University, Hamilton, Ontario, Canada
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Oláh T, Fodor J, Ruzsnavszky O, Vincze J, Berbey C, Allard B, Csernoch L. Overexpression of transient receptor potential canonical type 1 (TRPC1) alters both store operated calcium entry and depolarization-evoked calcium signals in C2C12 cells. Cell Calcium 2011; 49:415-25. [DOI: 10.1016/j.ceca.2011.03.012] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2010] [Revised: 03/29/2011] [Accepted: 03/30/2011] [Indexed: 12/25/2022]
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Madaro L, Marrocco V, Fiore P, Aulino P, Smeriglio P, Adamo S, Molinaro M, Bouché M. PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase. Mol Biol Cell 2011; 22:1409-19. [PMID: 21346196 PMCID: PMC3078083 DOI: 10.1091/mbc.e10-10-0821] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Using both in vivo and in vitro protein kinase C (PKC) θ mutant models, we found that PKCθ, the PKC isoform predominantly expressed in skeletal muscle, is required for myoblast fusion and myofiber growth, by regulating focal adhesion kinase activity and, in turn, the expression of the pro-fusion genes caveolin-3 and β1D-integrin. Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKCθ, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKCθ is strongly up-regulated following freeze injury–induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKCθ knockout and muscle-specific PKCθ dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKCθ mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKCθ mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKCθ in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKCθ-null myoblasts. We thus propose that PKCθ signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.
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Affiliation(s)
- Luca Madaro
- Department of Anatomy, Sapienza University of Rome, Rome, Italy
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Molecular mechanisms of myoblast fusion across species. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2011; 713:113-35. [PMID: 21432017 DOI: 10.1007/978-94-007-0763-4_8] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Skeletal muscle development, growth and regeneration depend on the ability of progenitor myoblasts to fuse to one another in a series of ordered steps. Whereas the cellular steps leading to the formation of a multinucleated myofiber are conserved in several model organisms, the molecular regulatory factors may vary. Understanding the common and divergent mechanisms regulating myoblast fusion in Drosophila melanogaster (fruit fly), Danio rerio (zebrafish) and Mus musculus (mouse) provides a better insight into the process of myoblast fusion than any of these models could provide alone. Deciphering the mechanisms of myoblast fusion from simpler to more complex organisms is of fundamental interest to skeletal muscle biology and may provide therapeutic avenues for various diseases that affect muscle.
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40
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Markworth JF, Cameron-Smith D. Prostaglandin F2α stimulates PI3K/ERK/mTOR signaling and skeletal myotube hypertrophy. Am J Physiol Cell Physiol 2010; 300:C671-82. [PMID: 21191105 DOI: 10.1152/ajpcell.00549.2009] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Cyclooxygenase (COX) enzymes mediate the synthesis of proinflammatory prostaglandin (PG) species from cellular arachidonic acid. COX/PGs have been implicated in skeletal muscle growth/regeneration; however, the mechanisms by which PGs influence skeletal muscle adaptation are poorly understood. The present study aimed to investigate PGF(2α) signaling and its role in skeletal myotube hypertrophy. PGF(2α) or the FP receptor agonist fluprostenol increased C2C12 myotube diameter. This effect was abolished by the FP receptor antagonist AL8810 and mammalian target of rapamycin (mTOR) inhibition. PGF(2α) stimulated time- and dose-dependent increases in the phosphorylation of extracellular receptor kinase (ERK)1/2 (Thr202/Tyr204), p70S6 kinase (p70S6K) (Thr389 and Thr421/Ser424), and eukaryotic initiation factor 4G (eIF4G) (Ser1108) without influencing Akt (Ser473). Pretreatment with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 and the ERK inhibitor PD98059 blocked F prostanoid receptor signaling responses, whereas rapamycin blocked heightened p70S6K/eIF4G phosphorylation without influencing ERK1/2 phosphorylation. These data suggest that activation of the F prostanoid receptor is coupled to C2C12 myotube growth and intracellular signaling via a PI3K/ERK/mTOR-dependent pathway.
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Affiliation(s)
- James F Markworth
- School of Exercise and Nutrition Sciences, Deakin Univ., 221 Burwood Highway, Burwood, Victoria 3125, Australia
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41
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Stiber JA, Rosenberg PB. The role of store-operated calcium influx in skeletal muscle signaling. Cell Calcium 2010; 49:341-9. [PMID: 21176846 DOI: 10.1016/j.ceca.2010.11.012] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2010] [Accepted: 11/29/2010] [Indexed: 11/29/2022]
Abstract
In cardiac and skeletal muscle Ca(2+) release from intracellular stores triggers actomyosin cross-bridge formation and the generation of contractile force. In the face of large fluctuations of intracellular calcium ([Ca(2+)](i)) that occur with contractile activity, myocytes are able to sense and respond to changes in workload and patterns of activation through calcium signaling pathways which modulate gene expression and cellular metabolism. Store-operated calcium influx has emerged as a mechanism by which calcium signaling pathways are activated in order to respond to the changing demands of the myocyte. Abnormalities of store-operated calcium influx may contribute to maladaptive muscle remodeling in multiple disease states. The importance of store-operated calcium influx in muscle is confirmed in mice lacking STIM1 which die perinatally and in patients with mutations on STIM1 or Orai1 who exhibit a myopathy exhibited by hypotonia. In this review, we consider the role of store-operated Ca(2+) entry into skeletal muscle as a critical mediator of Ca(2+) dependent gene expression and how alterations in Ca(2+) influx may influence muscle development and disease.
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Affiliation(s)
- Jonathan A Stiber
- Department of Medicine, Duke University Medical Center, Durham, NC 27710, United States
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42
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Mittal A, Bhatnagar S, Kumar A, Paul PK, Kuang S, Kumar A. Genetic ablation of TWEAK augments regeneration and post-injury growth of skeletal muscle in mice. THE AMERICAN JOURNAL OF PATHOLOGY 2010; 177:1732-42. [PMID: 20724600 DOI: 10.2353/ajpath.2010.100335] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
Impairment in the regeneration process is a critical determinant for skeletal muscle wasting in chronic diseases and degenerative muscle disorders. Inflammatory cytokines are known to cause significant muscle wasting, however, their role in myofiber regeneration is less clear. In this study we have investigated the role of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in skeletal muscle regeneration in vivo. Our results show that expression levels of TWEAK and its receptor Fn14 are significantly increased in skeletal muscles of mice after injury. Genetic deletion of TWEAK increased the fiber cross-sectional area and levels of embryonic isoform of myosin heavy chain in regenerating tibial anterior muscle. Conversely, muscle-specific transgenic overexpression of TWEAK reduced the fiber cross-sectional area and levels of the embryonic myosin heavy chain in regenerating muscle. TWEAK induced the expression of several inflammatory molecules and increased interstitial fibrosis in regenerating muscle. Genetic ablation of TWEAK suppressed, whereas overexpression of TWEAK increased, the activation of nuclear factor-kappa B without affecting the activation of Akt or p38 kinase in regenerating myofibers. Primary myoblasts from TWEAK-null mice showed enhanced differentiation in vitro, whereas myoblasts from TWEAK-Tg mice showed reduced differentiation compared with wild-type mice. Collectively, our study suggests that TWEAK negatively regulates muscle regeneration and that TWEAK is a potential therapeutic target to enhance skeletal muscle regeneration in vivo.
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Affiliation(s)
- Ashwani Mittal
- Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky 40202, USA
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43
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Bower NI, Johnston IA. Discovery and characterization of nutritionally regulated genes associated with muscle growth in Atlantic salmon. Physiol Genomics 2010; 42A:114-30. [PMID: 20663983 DOI: 10.1152/physiolgenomics.00065.2010] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
A genomics approach was used to identify nutritionally regulated genes involved in growth of fast skeletal muscle in Atlantic salmon (Salmo salar L.). Forward and reverse subtractive cDNA libraries were prepared comparing fish with zero growth rates to fish growing rapidly. We produced 7,420 ESTs and assembled them into nonredundant clusters prior to annotation. Contigs representing 40 potentially unrecognized nutritionally responsive candidate genes were identified. Twenty-three of the subtractive library candidates were also differentially regulated by nutritional state in an independent fasting-refeeding experiment and their expression placed in the context of 26 genes with established roles in muscle growth regulation. The expression of these genes was also determined during the maturation of a primary myocyte culture, identifying 13 candidates from the subtractive cDNA libraries with putative roles in the myogenic program. During early stages of refeeding DNAJA4, HSPA1B, HSP90A, and CHAC1 expression increased, indicating activation of unfolded protein response pathways. Four genes were considered inhibitory to myogenesis based on their in vivo and in vitro expression profiles (CEBPD, ASB2, HSP30, novel transcript GE623928). Other genes showed increased expression with feeding and highest in vitro expression during the proliferative phase of the culture (FOXD1, DRG1) or as cells differentiated (SMYD1, RTN1, MID1IP1, HSP90A, novel transcript GE617747). The genes identified were associated with chromatin modification (SMYD1, RTN1), microtubule stabilization (MID1IP1), cell cycle regulation (FOXD1, CEBPD, DRG1), and negative regulation of signaling (ASB2) and may play a role in the stimulation of myogenesis during the transition from a catabolic to anabolic state in skeletal muscle.
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Affiliation(s)
- Neil I Bower
- Scottish Oceans Institute, School of Biology, University of St Andrews, St Andrews, Fife, United Kingdom.
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Pavlath GK. Spatial and functional restriction of regulatory molecules during mammalian myoblast fusion. Exp Cell Res 2010; 316:3067-72. [PMID: 20553712 DOI: 10.1016/j.yexcr.2010.05.025] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2010] [Revised: 05/21/2010] [Accepted: 05/21/2010] [Indexed: 10/19/2022]
Abstract
Myoblast fusion is a highly regulated process that is key for forming skeletal muscle during development and regeneration in mammals. Much remains to be understood about the molecular regulation of myoblast fusion. Some molecules that influence mammalian muscle fusion display specific cellular localization during myogenesis. Such molecules can be localized to the contact region between two fusing cells either in both cells or only in one of the cells. How distinct localization of molecules contributes to fusion is not clear. Further complexity exists as other molecules are functionally restricted to myoblasts at later stages of myogenesis to regulate their fusion with multinucleated myotubes. This review examines these three categories of molecules and discusses how spatial and functional restriction may contribute to the formation of a multinucleated cell. Understanding how and why molecules become restricted in location or function is likely to provide further insights into the mechanisms regulating mammalian muscle fusion.
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Affiliation(s)
- Grace K Pavlath
- Department of Pharmacology, Emory University, 1510 Clifton Road, Atlanta, GA 30322, USA.
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45
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Ten Broek RW, Grefte S, Von den Hoff JW. Regulatory factors and cell populations involved in skeletal muscle regeneration. J Cell Physiol 2010; 224:7-16. [PMID: 20232319 DOI: 10.1002/jcp.22127] [Citation(s) in RCA: 129] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Skeletal muscle regeneration is a complex process, which is not yet completely understood. Satellite cells, the skeletal muscle stem cells, become activated after trauma, proliferate, and migrate to the site of injury. Depending on the severity of the myotrauma, activated satellite cells form new multinucleated myofibers or fuse to damaged myofibers. The specific microenvironment of the satellite cells, the niche, controls their behavior. The niche contains several components that maintain satellite cells quiescence until they are activated. In addition, a great diversity of stimulatory and inhibitory growth factors such as IGF-1 and TGF-beta1 regulate their activity. Donor-derived satellite cells are able to improve muscle regeneration, but their migration through the muscle tissue and across endothelial layers is limited. Less than 1% of their progeny, the myoblasts, survive the first days upon intra-muscular injection. However, a range of other multipotent muscle- and non-muscle-derived stem cells are involved in skeletal muscle regeneration. These stem cells can occupy the satellite cell niche and show great potential for the treatment of skeletal muscle injuries and diseases. The aim of this review is to discuss the niche factors, growth factors, and other stem cells, which are involved in skeletal muscle regeneration. Knowledge about the factors regulating satellite cell activity and skeletal muscle regeneration can be used to improve the treatment of muscle injuries and diseases.
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Affiliation(s)
- Roel W Ten Broek
- Department of Orthodontics and Oral Biology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands
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46
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Abstract
This study shows that forcing c-Flip overexpression in undifferentiated skeletal myogenic cells in vivo results in early aging muscle phenotype. In the transgenic mice, adult muscle histology, histochemistry and biochemistry show strong alterations: reduction of fibers size and muscle mass, mitochondrial abnormalities, increase in protein oxidation and apoptosis markers and reduced AKT/GSK3β phosphorylation. In the infant, higher levels of Pax-7, PCNA, P-ERK and active-caspase-3 were observed, indicating enhanced proliferation and concomitant apoptosis of myogenic precursors. Increased proliferation correlated with NF-κB activation, detected as p65 phosphorylation, and with high levels of embryonic myosin heavy chain. Reduced regenerative potential after muscle damage in the adult and impaired fiber growth associated with reduced NFATc2 activation in the infant were also observed, indicating that the satellite cell pool is prematurely compromised. Altogether, these data show a role for c-Flip in modulating skeletal muscle phenotype by affecting the proliferative potential of undifferentiated cells. This finding indicates a novel additional mechanism through which c-Flip might possibly control tissue remodeling.
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47
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Bower NI, Johnston IA. Paralogs of Atlantic salmon myoblast determination factor genes are distinctly regulated in proliferating and differentiating myogenic cells. Am J Physiol Regul Integr Comp Physiol 2010; 298:R1615-26. [PMID: 20375265 DOI: 10.1152/ajpregu.00114.2010] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
The mRNA expression of myogenic regulatory factors, including myoD1 (myoblast determination factor) gene paralogs, and their regulation by amino acids and insulin-like growth factors were investigated in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon (Salmo salar). The cell cycle and S phase were determined as 28.1 and 13.3 h, respectively, at 18 degrees C. Expression of myoD1b and myoD1c peaked at 8 days of culture in the initial proliferation phase and then declined more than sixfold as cells differentiated and was correlated with PCNA (proliferating cell nuclear antigen) expression (R = 0.88, P < 0.0001; R = 0.70, P < 0.0001). In contrast, myoD1a transcripts increased from 2 to 8 days and remained at elevated levels as myotubes were formed. mRNA levels of myoD1c were, on average, 3.1- and 5.7-fold higher than myoD1a and myoD1b, respectively. Depriving cells of amino acids and serum led to a rapid increase in pax7 and a decrease in myoD1c and PCNA expression, indicating a transition to a quiescent state. In contrast, amino acid replacement in starved cells produced significant increases in myoD1c (at 6 h), PCNA (at 12 h), and myoD1b (at 24 h) and decreases in pax7 expression as cells entered the cell cycle. Our results are consistent with temporally distinct patterns of myoD1c and myoD1b expression at the G(1) and S/G(2) phases of the cell cycle. Treatment of starved cells with insulin-like growth factor I or II did not alter expression of the myoD paralogs. It was concluded that, in vitro, amino acids alone are sufficient to stimulate expression of genes regulating myogenesis in myoblasts involving autocrine/paracrine pathways. The differential responses of myoD paralogs during myotube maturation and amino acid treatments suggest that myoD1b and myoD1c are primarily expressed in proliferating cells and myoD1a in differentiating cells, providing evidence for their subfunctionalization following whole genome and local duplications in the Atlantic salmon lineage.
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Affiliation(s)
- Neil I Bower
- Scottish Oceans Institute, School of Biology, University of St. Andrews, St. Andrews, Fife, United Kingdom.
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48
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Abstract
In Drosophila, as in mammals, myoblast fusion is fundamental for development. This fusion process has two distinct phases that share common ultrastructural features and at least some molecular players between Drosophila and vertebrates. Here, we integrate the latest data on the key molecular players and ultrastructural features found during myoblast fusion into a new working model to explain this fundamental cellular process. At cell-cell contact sites, a protein complex (FuRMAS) serves as a signalling centre and might restrict the area of membrane fusion. The FuRMAS consists of a ring of cell adhesion molecules, signalling proteins, and F-actin. Regulated F-actin branching plays a pivotal role in myoblast fusion with regard to vesicle transport, fusion pore formation, and expansion as well as the integration of the fusion-competent myoblast into the growing myotube. Interestingly, local F-actin accumulation is a typical feature of other transient adhesive structures such as the immunological synapse, podosomes, and invadopodia. Developmental Dynamics 238:1513-1525, 2009. (c) 2009 Wiley-Liss, Inc.
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Affiliation(s)
- Susanne-Filiz Onel
- Philipps-Universität Marburg, Fachbereich Biologie, Entwicklungsbiologie, Marburg, Germany
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49
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Zádor E. dnRas stimulates autocrine-paracrine growth of regenerating muscle via calcineurin-NFAT-IL-4 pathway. Biochem Biophys Res Commun 2008; 375:265-70. [PMID: 18706889 DOI: 10.1016/j.bbrc.2008.08.024] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2008] [Accepted: 08/06/2008] [Indexed: 12/21/2022]
Abstract
Ras and calcineurin are members of two independent pathways in muscle growth but their interaction is not known. This work shows that the transfection of about 1% of the muscle fibers with dominant negative Ras (dnRas) shows a wilder effect; it stimulates the fiber growth in the entire regenerating soleus muscle, including the nontransfected fibers. Co-transfection with the calcineurin inhibitor cain/cabin prevented the growth stimulation. Injection of antibody for interleukin-4 (IL-4) also abolished the growth ameliorating effect. These results suggest that the inactivation of Ras in 1% of the fibers upregulates the calcineurin-NFAT-IL-4 pathway and the secreted IL-4 triggers fiber growth stimulation in the whole regenerating soleus muscle of the rat. The results highlight the importance of the autocrine-paracrine regulation in muscle regeneration and hint to a novel method of gene theraphy of degenerative-regenerative muscle dystrophies.
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Affiliation(s)
- Erno Zádor
- Institute of Biochemistry, Faculty of Medicine, University of Szeged, Szeged, Dóm tér 9, H-6720, Hungary.
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50
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Meadows E, Cho JH, Flynn JM, Klein WH. Myogenin regulates a distinct genetic program in adult muscle stem cells. Dev Biol 2008; 322:406-14. [PMID: 18721801 DOI: 10.1016/j.ydbio.2008.07.024] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2008] [Revised: 07/11/2008] [Accepted: 07/12/2008] [Indexed: 01/28/2023]
Abstract
In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.
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Affiliation(s)
- Eric Meadows
- Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
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