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Abstract
Background
The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes.
Methods
After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1).
Results
MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively.
Conclusions
Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo.
Electronic supplementary material
The online version of this article (doi:10.1186/1550-2783-11-38) contains supplementary material, which is available to authorized users.
Copyright comment
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Electronic supplementary material
The online version of this article (doi:10.1186/1550-2783-11-38) contains supplementary material, which is available to authorized users.
Copyright comment
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Acknowledgements
Internal funding from Auburn University’s School of Kinesiology was used to fund this study. The authors thank Dr. David Pascoe and Dr. Andreas Kavazis for intellectual input, as well as Dr. Heidi Kluess for allowing us to use her laboratory’s fluorescent microscope.
Competing interests
Besides SB, and JAR, none of the authors have non-finacial and/or financial competing interests. SB, and JAR are employed by Metabolic Technologies, Inc., but both authors intellectually contributed to study design and data write-up. Therefore, all co-authors agreed that their intellectual input into this project warranted co-authorship.
Authors’ contributions
CDF, BSF, RHA, VJD, SB, JAR, JMW, MDR. This person has made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. CBM, MDR. This person primarily was involved in drafting the manuscript or revising it critically for important intellectual content. CBM, CDF, BSF, RHA, VJD, SB, JAR, JMW, MDR. This person gave final approval of the version to be published. CBM, CDF, BSF, RHA, VJD, SB, JAR, JMW, MDR This person agrees to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.