Biomechanical effects on microRNA expression in skeletal muscle differentiation

Caroline Rhim; William E. Kraus; George A. Truskey; Caroline Rhim; William E. Kraus; George A. Truskey

doi:10.3934/bioeng.2020014

AIMS Bioengineering

2020, Volume 7, Issue 3: 147-164. doi: 10.3934/bioeng.2020014

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Biomechanical effects on microRNA expression in skeletal muscle differentiation

1.
Departments of Biomedical Engineering, Duke University, 101 Science Drive, Durham, NC 27708 USA
2.
Duke Molecular Physiology Institute, Duke University School of Medicine, 300 N Duke St, Durham, NC, 27701, USA

Received: 28 April 2020 Accepted: 15 June 2020 Published: 19 June 2020

We examined whether mechanical stretch affected expression of muscle-specific microRNAs (miRNAs) that regulate proliferation (miR-133a) or differentiation (miR-1, -206). Real-time quantitative RT-PCR was used to assess miRNA regulation in the murine myoblast cell line C2C12 exposed to stretch regimens that promote either proliferation (high stretch: 17%, 1 Hz) or differentiation (moderate stretch: 10%, 0.5 Hz) after adding media that promotes differentiation. Controls consisted of myoblasts cultured under static conditions. While miRNA expression was not affected by high stretch, a significant effect of stretch (P < 0.05) was seen after 4 days with the moderate stretch regimen. All three microRNAs were upregulated by stretch, with the most significant increase for miR-1. Myoblast maturation was enhanced with a moderate stretch regimen, as assessed by a higher percentage of nuclei in straited fibers and an increase in Mef2c gene expression. Correspondingly, HDAC4 protein expression, a direct target of miR-1 and repressor of Mef2, was decreased with the moderate stretch regimen. Over-expression of miR-1 abrogated the effect of stretch on miR-1, miR-133a and miR-206 levels compared to its negative control but did not alter miR-133a or miR-206 levels. Treatment with an antisense mRNA to miR-1 similarly diminished the stretch-mediated response. Results indicate that the differential response of skeletal myoblasts to moderate and high stretch cyclic stretch regimens is due, in part, to muscle specific miRNA expression.
- C2C12 myoblasts,
- mechanotransduction,
- gene regulation
Citation: Caroline Rhim, William E. Kraus, George A. Truskey. Biomechanical effects on microRNA expression in skeletal muscle differentiation[J]. AIMS Bioengineering, 2020, 7(3): 147-164. doi: 10.3934/bioeng.2020014

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Abstract

We examined whether mechanical stretch affected expression of muscle-specific microRNAs (miRNAs) that regulate proliferation (miR-133a) or differentiation (miR-1, -206). Real-time quantitative RT-PCR was used to assess miRNA regulation in the murine myoblast cell line C2C12 exposed to stretch regimens that promote either proliferation (high stretch: 17%, 1 Hz) or differentiation (moderate stretch: 10%, 0.5 Hz) after adding media that promotes differentiation. Controls consisted of myoblasts cultured under static conditions. While miRNA expression was not affected by high stretch, a significant effect of stretch (P < 0.05) was seen after 4 days with the moderate stretch regimen. All three microRNAs were upregulated by stretch, with the most significant increase for miR-1. Myoblast maturation was enhanced with a moderate stretch regimen, as assessed by a higher percentage of nuclei in straited fibers and an increase in Mef2c gene expression. Correspondingly, HDAC4 protein expression, a direct target of miR-1 and repressor of Mef2, was decreased with the moderate stretch regimen. Over-expression of miR-1 abrogated the effect of stretch on miR-1, miR-133a and miR-206 levels compared to its negative control but did not alter miR-133a or miR-206 levels. Treatment with an antisense mRNA to miR-1 similarly diminished the stretch-mediated response. Results indicate that the differential response of skeletal myoblasts to moderate and high stretch cyclic stretch regimens is due, in part, to muscle specific miRNA expression.

Acknowledgments

The authors would like to thank Cindy Cheng and Stephen Morton for their assistance, and Dr. H.S. Kim from the UNC Department of Cell Biology for oligonucleotide synthesis. We would also like to acknowledge funding from the National Institutes of Health (NIH) grant AR55195 and the McChesney Foundation.

Author contributions

C.R. conceived of the research, designed and performed experiments, analyzed results and wrote the manuscript draft. W.E.K. analyzed results and edited the manuscript. G.A.T. Conceived of the research with C.R., obtained funding, analyzed results, and edited the manuscript.

Conflict of interest

All authors declare no conflicts of interest.

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