Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis.
Abstract: Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the () gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with 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 , were conditionally immortalised and evaluated to determine whether cell phenotype and metabolic function were comparable to functional characteristics previously reported for skeletal muscle isolated from Thoroughbred horses with each genotype. Unassigned: 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, gene expression, mitochondrial abundance, and mitochondrial function of the three genotype cell lines, were consistent with equivalent characterisation of skeletal muscle samples with these genotypes. Furthermore, addition of coenzyme Q (CoQ) to the cell lines improved mitochondrial function, an observation consistent with skeletal muscle samples with these genotypes following supplementation with CoQ in the diet. Unassigned: The observation that the phenotypic characteristics and metabolic function of the cells lines are equivalent to skeletal muscle indicates that this 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.
© 2022 The Authors.
Publication Date: 2022-12-05 PubMed ID: 36504704PubMed Central: PMC9727643DOI: 10.1016/j.bbrep.2022.101391Google Scholar: Lookup
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Summary
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The research article discusses the creation of three new equine skeletal muscle cell lines to aid in functional experiments related to horse racing performance. The cell lines represent different genotypes of the myostatin gene, and were found to maintain characteristics similar to muscle tissue from Thoroughbred horses of the same genotypes.
Introduction
- The study focuses on a genetic factor greatly influencing the performance of Thoroughbred racehorses – a major quantitative trait locus at the myostatin (MSTN) gene. The gene’s specific variation determines a horse’s suitability for specific race distances.
- An ‘in vitro’ model system was needed to conduct functional experiments, which led to the development of three novel equine skeletal muscle cell lines.
- These cell lines reflect the phenotypic variation associated with the MSTN genotype (CC/II, CT/IN, and TT/NN).
Methods and Findings
- Primary equine skeletal muscle myoblasts, which are muscle cells in an early stage of differentiation, were successfully isolated from Thoroughbred horses and conditionally immortalised using the SV40TtsA58 lentivirus vector. This allowed the cells to proliferate in a controlled environment.
- The immortalised myoblasts were able to revert to their primary cell phenotype and differentiate into multinucleated myotubes, which are precursors to muscle fibers.
- Evaluation of the cell lines revealed consistency on several key points, such as muscle fiber type, MSTN gene expression, mitochondrial abundance, and function, with MSTN skeletal muscle samples isolated from Thoroughbred horses of the equivalent genotypes.
- The addition of coenzyme Q to the cell lines improved mitochondrial function, mirroring similar improvements observed in MSTN skeletal muscle samples from horses of the same genotypes when coenzyme Q was added to their diet.
Implication and Applications
- As these cell lines mirror the traits of MSTN skeletal muscle in Thoroughbred horses, they can be employed in cost-effective analyses to improve understanding of equine skeletal muscle function, test nutritional supplements, develop drug tests, and support gene doping test development.
- Considering the high-stakes nature of the Thoroughbred horse industry, research advances in the biological function of skeletal muscle could be incredibly significant.
- On a broader scale, this novel genotype-specific system could potentially be adapted to human biomedicine, enhancing understanding of the effects of myostatin on human physiology and medicine.
Cite This Article
APA
Rooney MF, Neto NGB, Monaghan MG, Hill EW, Porter RK.
(2022).
Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis.
Biochem Biophys Rep, 33, 101391.
https://doi.org/10.1016/j.bbrep.2022.101391 Publication
Researcher Affiliations
- School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, 152-160, Pearse Street, Dublin 2, Ireland.
- Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, 152-160, Pearse Street, Dublin 2, Ireland.
- Department of Mechanical, Manufacturing and Biomedical Engineering, Trinity College Dublin, Dublin 2, Ireland.
- Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, 152-160, Pearse Street, Dublin 2, Ireland.
- Department of Mechanical, Manufacturing and Biomedical Engineering, Trinity College Dublin, Dublin 2, Ireland.
- Plusvital Ltd., The Highline, Pottery Road, Dun Laoghaire, Co. Dublin, Ireland.
- School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland.
- School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute (TBSI), Trinity College Dublin, 152-160, Pearse Street, Dublin 2, Ireland.
Conflict of Interest Statement
EWH is a shareholder in and Chief Scientific Officer of Plusvital Ltd., an equine nutrition and genetic testing company. Plusvital Ltd. has been granted a licence for commercial use of the data that is contained within multiple granted patents and patent applications including (patent reference numbers): EP2352850, JP5667057, US8771943, AU2009290452, NZ591711, US9249470 and US2016215335. EWH is named on these patents. MFR, NGBN, MGM and RKP declare that they have no competing interests. Other than EWH, Plusvital Ltd. played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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