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Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis
BACKGROUND: Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin (MSTN) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an i...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9727643/ https://www.ncbi.nlm.nih.gov/pubmed/36504704 http://dx.doi.org/10.1016/j.bbrep.2022.101391 |
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author | Rooney, Mary F. Neto, Nuno G.B. Monaghan, Michael G. Hill, Emmeline W. Porter, Richard K. |
author_facet | Rooney, Mary F. Neto, Nuno G.B. Monaghan, Michael G. Hill, Emmeline W. Porter, Richard K. |
author_sort | Rooney, Mary F. |
collection | PubMed |
description | BACKGROUND: Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin (MSTN) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an in vitro system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with MSTN genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C > T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse gluteus medius, were conditionally immortalised and evaluated to determine whether cell phenotype and metabolic function were comparable to functional characteristics previously reported for ex vivo skeletal muscle isolated from Thoroughbred horses with each genotype. RESULTS: Primary myoblasts conditionally immortalised with the temperature sensitive SV40TtsA58 lentivirus vector successfully proliferated and could revert to their primary cell phenotype and differentiate into multinucleated myotubes. Skeletal muscle fibre type, MSTN gene expression, mitochondrial abundance, and mitochondrial function of the three MSTN genotype cell lines, were consistent with equivalent characterisation of ex vivo skeletal muscle samples with these genotypes. Furthermore, addition of coenzyme Q(10) (CoQ(10)) to the cell lines improved mitochondrial function, an observation consistent with ex vivo skeletal muscle samples with these genotypes following supplementation with CoQ(10) in the diet. CONCLUSIONS: The observation that the phenotypic characteristics and metabolic function of the cells lines are equivalent to ex vivo skeletal muscle indicates that this in vitro system will enable efficient and cost-effective analyses of equine skeletal muscle for a range of different applications including understanding metabolic function, testing of nutritional supplements, drug test development and gene doping test development. In the multi-billion-euro international Thoroughbred horse industry research advances in the biological function of skeletal muscle are likely to have considerable impact. Furthermore, this novel genotype-specific system may be adapted and applied to human biomedicine to improve understanding of the effects of myostatin in human physiology and medicine. |
format | Online Article Text |
id | pubmed-9727643 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-97276432022-12-08 Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis Rooney, Mary F. Neto, Nuno G.B. Monaghan, Michael G. Hill, Emmeline W. Porter, Richard K. Biochem Biophys Rep Research Article BACKGROUND: Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin (MSTN) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an in vitro system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with MSTN genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C > T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse gluteus medius, were conditionally immortalised and evaluated to determine whether cell phenotype and metabolic function were comparable to functional characteristics previously reported for ex vivo skeletal muscle isolated from Thoroughbred horses with each genotype. RESULTS: Primary myoblasts conditionally immortalised with the temperature sensitive SV40TtsA58 lentivirus vector successfully proliferated and could revert to their primary cell phenotype and differentiate into multinucleated myotubes. Skeletal muscle fibre type, MSTN gene expression, mitochondrial abundance, and mitochondrial function of the three MSTN genotype cell lines, were consistent with equivalent characterisation of ex vivo skeletal muscle samples with these genotypes. Furthermore, addition of coenzyme Q(10) (CoQ(10)) to the cell lines improved mitochondrial function, an observation consistent with ex vivo skeletal muscle samples with these genotypes following supplementation with CoQ(10) in the diet. CONCLUSIONS: The observation that the phenotypic characteristics and metabolic function of the cells lines are equivalent to ex vivo skeletal muscle indicates that this in vitro system will enable efficient and cost-effective analyses of equine skeletal muscle for a range of different applications including understanding metabolic function, testing of nutritional supplements, drug test development and gene doping test development. In the multi-billion-euro international Thoroughbred horse industry research advances in the biological function of skeletal muscle are likely to have considerable impact. Furthermore, this novel genotype-specific system may be adapted and applied to human biomedicine to improve understanding of the effects of myostatin in human physiology and medicine. Elsevier 2022-12-05 /pmc/articles/PMC9727643/ /pubmed/36504704 http://dx.doi.org/10.1016/j.bbrep.2022.101391 Text en © 2022 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Article Rooney, Mary F. Neto, Nuno G.B. Monaghan, Michael G. Hill, Emmeline W. Porter, Richard K. Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis |
title | Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis |
title_full | Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis |
title_fullStr | Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis |
title_full_unstemmed | Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis |
title_short | Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis |
title_sort | conditionally immortalised equine skeletal muscle cell lines for in vitro analysis |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9727643/ https://www.ncbi.nlm.nih.gov/pubmed/36504704 http://dx.doi.org/10.1016/j.bbrep.2022.101391 |
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