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. 2017 Dec;31(12):5196-5207.
doi: 10.1096/fj.201700329R. Epub 2017 Aug 3.

A dynamic ribosomal biogenesis response is not required for IGF-1-mediated hypertrophy of human primary myotubes

Hannah Crossland  1   2 , James A Timmons  1 , Philip J Atherton  3
Affiliations

A dynamic ribosomal biogenesis response is not required for IGF-1-mediated hypertrophy of human primary myotubes

Hannah Crossland et al. FASEB J. 2017 Dec.

Abstract

Increased ribosomal DNA transcription has been proposed to limit muscle protein synthesis, making ribosome biogenesis central to skeletal muscle hypertrophy. We examined the relationship between ribosomal RNA (rRNA) production and IGF-1-mediated myotube hypertrophy in vitro Primary skeletal myotubes were treated with IGF-1 (50 ng/ml) with or without 0.5 µM CX-5461 (CX), an inhibitor of RNA polymerase I. Myotube diameter, total protein, and RNA and DNA levels were measured along with markers of RNA polymerase I regulatory factors and regulators of protein synthesis. CX treatment reduced 45S pre-rRNA expression (-64 ± 5% vs. IGF-1; P < 0.001) and total RNA content (-16 ± 2% vs. IGF-1; P < 0.001) in IGF-1-treated myotubes. IGF-1-mediated increases in myotube diameter (1.27 ± 0.09-fold, P < 0.05 vs. control) and total protein (+20 ± 2%; P < 0.001 vs. control) were not prevented by CX treatment. Suppression of rRNA synthesis during IGF-1 treatment did not prevent early increases in AKT (+203 ± 39% vs. CX; P < 0.001) and p70 S6K1 (269 ± 41% vs. CX; P < 0.001) phosphorylation. Despite robust inhibition of the dynamic ribosomal biogenesis response to IGF-1, myotube diameter and protein accretion were sustained. Thus, while ribosome biogenesis represents a potential site for the regulation of skeletal muscle protein synthesis and muscle mass, it does not appear to be a prerequisite for IGF-1-induced myotube hypertrophy in vitro.-Crossland, H., Timmons, J. A., Atherton, P. J. A dynamic ribosomal biogenesis response is not required for IGF-1-mediated hypertrophy of human primary myotubes.

Keywords: RNA; mTOR; skeletal muscle.

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Figures

Figure 1.
Figure 1.
Images of human myotubes (A), myotube diameter measurements (B), nuclei per myotube (C), and desmin staining of myotubes (D) after 24 h of IGF-1 with or without CX treatment. Light microscope images were taken from differentiated myotubes after 24 h of 50 ng/ml IGF-1 treatment with or without 0.5 µM CX. Human primary myotubes were stained by immunofluorescence for desmin after incubation for 24 h with IGF-1 and/or CX. Number of nuclei per myotube was calculated for each group.
Figure 2.
Figure 2.
Dose–time course of CX treatment and total protein, RNA, and DNA content in human primary myotubes after IGF-1 treatment with or without CX. A, B) Expression of 45S rRNA (A) and c-Myc (B) mRNA was measured in human primary myotubes after 2, 4, and 24 h of CX treatment (0.25 or 0.5 µM). C–E) Total protein (C), RNA (D), and DNA (E) were measured in human primary myotubes after 24 h of IGF-1 (50 ng/ml) treatment with or without 0.5 µM CX. F–H) From these values, the protein/DNA (F), RNA/DNA (G) and RNA/protein (H) ratios were calculated. Data are expressed as means ± sem from 3 independent experiments (n = 4–6 replicates per treatment). *P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated control; ###P < 0.001 vs. CX, ††P < 0.01, †††P < 0.001 vs. IGF-1.
Figure 3.
Figure 3.
Expression of rRNA and Pol I regulatory factors after IGF-1 treatment with or without CX. Expression of 45S (A), 5.8S (B), 28S (C), c-Myc (D), UBF (E), TIF1A (F), and TAF1A (G) after 4 and 24 h of IGF-1 treatment (50 ng/ml) with or without 0.5 µM CX in human primary myotubes. Data are normalized to 18S expression and are expressed as means ± sem from 3 independent experiments (n = 4 replicates per treatment). *P < 0.05, **P < 0.01; ***P < 0.001 vs. untreated control; ##P < 0.01, ###P < 0.001 vs. CX; †††P < 0.001 vs. IGF-1.
Figure 4.
Figure 4.
mRNA expression of RNA Pol I subunits and ribosomal protein genes after IGF-1 treatment with or without CX. Expression of POLR1A (A), POLR1B (B), POLR1C (C), POLR1E (D), RPL13A (E), RPL32 (F), RPS5 (G), and RPS19 (H) mRNA after 4 and 24 h of IGF-1 treatment (50 ng/ml) with or without 0.5 µM CX in human primary myotubes. Data are normalized to 18S expression and expressed as means ± sem from 3 independent experiments (n = 4 replicates per treatment). *P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. CX; †P < 0.05, †††P < 0.001 vs. IGF-1.
Figure 5.
Figure 5.
Protein expression and phosphorylation of selected mTOR signaling components in human primary myotubes after IGF-1 treatment with or without CX. A, B) Representative blots from Western blot analysis of selected signaling targets taken from differentiated myotubes after 4 h of IGF-1 treatment with or without CX. C–F) Relative changes in phosphorylated (phospho)-total mTOR (C), phospho-total p70 S6K1 (D), phospho-total AKT (E), and phospho-total 4E-BP1 (F) after IGF-1 treatment with or without CX. RAU, relative arbitrary units. Data are presented as means ± sem from 3 independent experiments (n = 3 per treatment). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; #P < 0.05, ###P < 0.001 vs. CX.
Figure 6.
Figure 6.
Protein expression and phosphorylation of selected regulators of ribosome biogenesis in human primary myotubes after IGF-1 treatment with or without CX. A) Representative blots from Western blot analysis of selected signaling targets taken from differentiated myotubes after 4 h of IGF-1 treatment with or without CX. B–E) Relative changes in total c-Myc (B), phosphorylated Rb Ser780 (C), total TIF1A (D), and phospho-total UBF (E) after IGF-1 treatment with or without CX. RAU, relative arbitrary units. Data are presented as means ± sem from 3 independent experiments (n = 3 per treatment). *P < 0.05, ***P < 0.001 vs. control; #P < 0.05, ###P < 0.001 vs. CX.
Figure 7.
Figure 7.
Protein expression and phosphorylation of selected regulators of protein breakdown in human primary myotubes after IGF-1 treatment with or without CX. A, B) Representative blots from Western blot analysis of selected signaling targets taken from differentiated myotubes after 4 h of IGF-1 treatment with or without CX. C–F) Relative changes in phosphorylated FOXO3 Ser253 (C), total MuRF1 (D), LC3II/LC3I ratio (E), and ubiquitinated proteins (F) after IGF-1 treatment with or without CX. RAU, relative arbitrary units. Data are presented as means ± sem from 3 independent experiments (n = 3 per treatment). *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. CX.
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