Validation of positional candidates Rps6ka6 and Pou3f4 for a locus associated with skeletal muscle mass variability

Konstantinos  Lekkos; Afra A  Bhuiyan; Abdullah Mohammed Albloshi; Paige M  Brooks; Thomas M  Coate; Arimantas Lionikas

doi:10.1093/g3journal/jkae046

Validation of positional candidates Rps6ka6 and Pou3f4 for a locus associated with skeletal muscle mass variability

Konstantinos Lekkos, Afra A Bhuiyan, Abdullah Mohammed Albloshi, Paige M Brooks, Thomas M Coate, Arimantas Lionikas^* (Corresponding Author)

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

Genetic variability significantly contributes to individual differences in skeletal muscle mass; however, the specific genes involved in that process remain elusive. In this study, we examined the role of positional candidates, Rps6ka6 and Pou3f4, of a chromosome X locus, implicated in muscle mass variability in CFW laboratory mice. Histology of hindlimb muscles was studied in CFW male mice carrying the muscle “increasing” allele C (n = 15) or “decreasing” allele T (n = 15) at the peak marker of the locus, rs31308852, and in the Pou3f4y/− and their wild-type male littermates. To study the role of the Rps6ka6 gene, we deleted exon 7 (Rps6ka6-ΔE7) using clustered regularly interspaced palindromic repeats-Cas9 based method in H2Kb myogenic cells creating a severely truncated RSK4 protein. We then tested whether that mutation affected myoblast proliferation, migration, and/or differentiation. The extensor digitorum longus muscle was 7% larger (P < 0.0001) due to 10% more muscle fibers (P = 0.0176) in the carriers of the “increasing” compared with the “decreasing” CFW allele. The number of fibers was reduced by 15% (P = 0.0268) in the slow-twitch soleus but not in the fast-twitch extensor digitorum longus (P = 0.2947) of Pou3f4y/− mice. The proliferation and migration did not differ between the Rps6ka6-ΔE7 and wild-type H2Kb myoblasts. However, indices of differentiation (myosin expression, P < 0.0001; size of myosin-expressing cells, P < 0.0001; and fusion index, P = 0.0013) were significantly reduced in Rps6ka6-ΔE7 cells. This study suggests that the effect of the X chromosome locus on muscle fiber numbers in the fast-twitch extensor digitorum longus is mediated by the Rps6ka6 gene, whereas the Pou3f4 gene affects fiber number in slow-twitch soleus.

Original language	English
Article number	jkae046
Journal	G3: Genes, Genomes, Genetics Mission
Volume	14
Issue number	5
Early online date	5 Apr 2024
DOIs	https://doi.org/10.1093/g3journal/jkae046
Publication status	Published - 7 May 2024

Bibliographical note

Open Acess via the OUP Agreement
Acknowledgments
Authors are grateful for Mr James Archibald for assistance with histological analysis of muscle samples and for the Microscopy and Histology Core Facility at the University of Aberdeen for the help with imaging.
Funding
A.L. was supported by awards AR052879 and AR056280 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, award 249156 from the FP7-PEOPLE-2009-RG programme, award CGA/18/05 from the Chief Scientist Office, award 21/019 from the NHS Grampian, and award 204815/Z/16/Z from the Wellcome Trust. T.M.C. was supported by NIH R01 DC016595. P.M.B. was supported by NIH R01 DC016595, NIH F31 DC019824, and TL1TR001431. Research reported in this article was supported by the National Center for Advancing Translational Sciences under award number TL1TR001431. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Data Availability Statement

The H2Kb cell clones are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.

Supplemental material available at G3 online.

Keywords

RSK4
myogenesis
muscle fibers
Cell Movement/genetics
Cell Proliferation
Ribosomal Protein S6 Kinases, 90-kDa/genetics
Male
Muscle, Skeletal/metabolism
Genetic Loci
Myoblasts/metabolism
Animals
Alleles
Cell Differentiation/genetics
Mice

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Cite this

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title = "Validation of positional candidates Rps6ka6 and Pou3f4 for a locus associated with skeletal muscle mass variability",

abstract = "Genetic variability significantly contributes to individual differences in skeletal muscle mass; however, the specific genes involved in that process remain elusive. In this study, we examined the role of positional candidates, Rps6ka6 and Pou3f4, of a chromosome X locus, implicated in muscle mass variability in CFW laboratory mice. Histology of hindlimb muscles was studied in CFW male mice carrying the muscle “increasing” allele C (n = 15) or “decreasing” allele T (n = 15) at the peak marker of the locus, rs31308852, and in the Pou3f4y/− and their wild-type male littermates. To study the role of the Rps6ka6 gene, we deleted exon 7 (Rps6ka6-ΔE7) using clustered regularly interspaced palindromic repeats-Cas9 based method in H2Kb myogenic cells creating a severely truncated RSK4 protein. We then tested whether that mutation affected myoblast proliferation, migration, and/or differentiation. The extensor digitorum longus muscle was 7% larger (P < 0.0001) due to 10% more muscle fibers (P = 0.0176) in the carriers of the “increasing” compared with the “decreasing” CFW allele. The number of fibers was reduced by 15% (P = 0.0268) in the slow-twitch soleus but not in the fast-twitch extensor digitorum longus (P = 0.2947) of Pou3f4y/− mice. The proliferation and migration did not differ between the Rps6ka6-ΔE7 and wild-type H2Kb myoblasts. However, indices of differentiation (myosin expression, P < 0.0001; size of myosin-expressing cells, P < 0.0001; and fusion index, P = 0.0013) were significantly reduced in Rps6ka6-ΔE7 cells. This study suggests that the effect of the X chromosome locus on muscle fiber numbers in the fast-twitch extensor digitorum longus is mediated by the Rps6ka6 gene, whereas the Pou3f4 gene affects fiber number in slow-twitch soleus.",

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author = "Konstantinos Lekkos and Bhuiyan, {Afra A} and Albloshi, {Abdullah Mohammed} and Brooks, {Paige M} and Coate, {Thomas M} and Arimantas Lionikas",

note = "Open Acess via the OUP Agreement Acknowledgments Authors are grateful for Mr James Archibald for assistance with histological analysis of muscle samples and for the Microscopy and Histology Core Facility at the University of Aberdeen for the help with imaging. Funding A.L. was supported by awards AR052879 and AR056280 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, award 249156 from the FP7-PEOPLE-2009-RG programme, award CGA/18/05 from the Chief Scientist Office, award 21/019 from the NHS Grampian, and award 204815/Z/16/Z from the Wellcome Trust. T.M.C. was supported by NIH R01 DC016595. P.M.B. was supported by NIH R01 DC016595, NIH F31 DC019824, and TL1TR001431. Research reported in this article was supported by the National Center for Advancing Translational Sciences under award number TL1TR001431. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.",

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doi = "10.1093/g3journal/jkae046",

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AU - Bhuiyan, Afra A

AU - Albloshi, Abdullah Mohammed

AU - Brooks, Paige M

AU - Coate, Thomas M

AU - Lionikas, Arimantas

N1 - Open Acess via the OUP Agreement Acknowledgments Authors are grateful for Mr James Archibald for assistance with histological analysis of muscle samples and for the Microscopy and Histology Core Facility at the University of Aberdeen for the help with imaging. Funding A.L. was supported by awards AR052879 and AR056280 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, award 249156 from the FP7-PEOPLE-2009-RG programme, award CGA/18/05 from the Chief Scientist Office, award 21/019 from the NHS Grampian, and award 204815/Z/16/Z from the Wellcome Trust. T.M.C. was supported by NIH R01 DC016595. P.M.B. was supported by NIH R01 DC016595, NIH F31 DC019824, and TL1TR001431. Research reported in this article was supported by the National Center for Advancing Translational Sciences under award number TL1TR001431. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

PY - 2024/5/7

Y1 - 2024/5/7

N2 - Genetic variability significantly contributes to individual differences in skeletal muscle mass; however, the specific genes involved in that process remain elusive. In this study, we examined the role of positional candidates, Rps6ka6 and Pou3f4, of a chromosome X locus, implicated in muscle mass variability in CFW laboratory mice. Histology of hindlimb muscles was studied in CFW male mice carrying the muscle “increasing” allele C (n = 15) or “decreasing” allele T (n = 15) at the peak marker of the locus, rs31308852, and in the Pou3f4y/− and their wild-type male littermates. To study the role of the Rps6ka6 gene, we deleted exon 7 (Rps6ka6-ΔE7) using clustered regularly interspaced palindromic repeats-Cas9 based method in H2Kb myogenic cells creating a severely truncated RSK4 protein. We then tested whether that mutation affected myoblast proliferation, migration, and/or differentiation. The extensor digitorum longus muscle was 7% larger (P < 0.0001) due to 10% more muscle fibers (P = 0.0176) in the carriers of the “increasing” compared with the “decreasing” CFW allele. The number of fibers was reduced by 15% (P = 0.0268) in the slow-twitch soleus but not in the fast-twitch extensor digitorum longus (P = 0.2947) of Pou3f4y/− mice. The proliferation and migration did not differ between the Rps6ka6-ΔE7 and wild-type H2Kb myoblasts. However, indices of differentiation (myosin expression, P < 0.0001; size of myosin-expressing cells, P < 0.0001; and fusion index, P = 0.0013) were significantly reduced in Rps6ka6-ΔE7 cells. This study suggests that the effect of the X chromosome locus on muscle fiber numbers in the fast-twitch extensor digitorum longus is mediated by the Rps6ka6 gene, whereas the Pou3f4 gene affects fiber number in slow-twitch soleus.

AB - Genetic variability significantly contributes to individual differences in skeletal muscle mass; however, the specific genes involved in that process remain elusive. In this study, we examined the role of positional candidates, Rps6ka6 and Pou3f4, of a chromosome X locus, implicated in muscle mass variability in CFW laboratory mice. Histology of hindlimb muscles was studied in CFW male mice carrying the muscle “increasing” allele C (n = 15) or “decreasing” allele T (n = 15) at the peak marker of the locus, rs31308852, and in the Pou3f4y/− and their wild-type male littermates. To study the role of the Rps6ka6 gene, we deleted exon 7 (Rps6ka6-ΔE7) using clustered regularly interspaced palindromic repeats-Cas9 based method in H2Kb myogenic cells creating a severely truncated RSK4 protein. We then tested whether that mutation affected myoblast proliferation, migration, and/or differentiation. The extensor digitorum longus muscle was 7% larger (P < 0.0001) due to 10% more muscle fibers (P = 0.0176) in the carriers of the “increasing” compared with the “decreasing” CFW allele. The number of fibers was reduced by 15% (P = 0.0268) in the slow-twitch soleus but not in the fast-twitch extensor digitorum longus (P = 0.2947) of Pou3f4y/− mice. The proliferation and migration did not differ between the Rps6ka6-ΔE7 and wild-type H2Kb myoblasts. However, indices of differentiation (myosin expression, P < 0.0001; size of myosin-expressing cells, P < 0.0001; and fusion index, P = 0.0013) were significantly reduced in Rps6ka6-ΔE7 cells. This study suggests that the effect of the X chromosome locus on muscle fiber numbers in the fast-twitch extensor digitorum longus is mediated by the Rps6ka6 gene, whereas the Pou3f4 gene affects fiber number in slow-twitch soleus.

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KW - Ribosomal Protein S6 Kinases, 90-kDa/genetics

KW - Male

KW - Muscle, Skeletal/metabolism

KW - Genetic Loci

KW - Myoblasts/metabolism

KW - Animals

KW - Alleles

KW - Cell Differentiation/genetics

KW - Mice

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DO - 10.1093/g3journal/jkae046

M3 - Article

C2 - 38577978

SN - 2160-1836

VL - 14

JO - G3: Genes, Genomes, Genetics Mission

JF - G3: Genes, Genomes, Genetics Mission

IS - 5

M1 - jkae046

ER -

Validation of positional candidates Rps6ka6 and Pou3f4 for a locus associated with skeletal muscle mass variability

Abstract

Bibliographical note

Data Availability Statement

Keywords

Access to Document

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