R-spondin3 is a myokine that differentiates myoblasts to type I fibres

Figures

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  Figure 1. Rspo3 is specifically expressed in type I fibres. (A) The mRNA expression of the Rspo3 gene was analysed in type I and II fibres (n = 3) using conventional RT-PCR. (B) The mRNA expression level of Rspo3 in type I and II fibres was quantified by quantitative RT-PCR analysis. Values are presented as mean ± SEM (n = 3 mice). **P < 0.01 by Student’s t-test. Uncropped gels can be found in Supplementary Fig. 8.

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  Figure 2. Rspo3 treatment increases MyHC I expression in myotubes. (A) Experimental scheme. Mouse primary myoblasts derived from satellite cells of type I or type II fibres were cultured for 3 d in a differentiation media containing BSA (200 ng/mL; Control) or mouse Rspo3 recombinant protein (200 ng/mL; 6.45 nM). (B) Protein levels of MyHC I and MyHC II in the differentiated myotubes were measured by Western blotting. The expression of these proteins was normalised to that of β-actin. Values are presented as mean ± SEM (n = 6 mice for analysing MyHC I, n = 4–5 mice for analysing MyHC II). **P < 0.01 by Student’s t-test. Uncropped blots can be found in Supplementary Fig. 9.

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  Figure 3. Rspo3 activates the Wnt/β-catenin signalling pathway during myogenic differentiation. (A, B) Mouse primary myoblasts derived from satellite cells of type I and type II fibres were differentiated for 3 d in the absence (BSA; 200 ng/mL) or presence of the mouse Rspo3 recombinant protein (200 ng/mL; 6.45 nM). Cytoplasmic and nucleic fractions were extracted from myotube lysate. Protein levels of β-catenin, lamin A/C (nuclear marker), and α-tubulin (cytosol marker) in myotubes were analysed using Western blotting. Values are presented as mean ± SEM (n = 10–12 mice for analysing cells derived from type I fibres, n = 8 mice for analysing cells derived from type II fibres). *P < 0.05; **P < 0.01 by Student’s t-test. Uncropped blots can be found in Supplementary Fig. 10.

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  Figure 4. Rspo3 does not activate non-canonical Wnt signalling pathways during myogenic differentiation. (A) Mouse primary myoblasts derived from satellite cells of type I and type II fibres were differentiated for 3 d in the absence (BSA; 200 ng/mL) or presence of the mouse Rspo3 recombinant protein (200 ng/mL; 6.45 nM). Protein levels of phospho-JNK1/2/3 and pan-JNK1/2/3 in myotubes were analysed using Western blotting. Values are presented as mean ± SEM (n = 6 mice). (B) Mouse primary myoblasts derived from satellite cells of type I and type II fibres were cultured for 3 d in the differentiation media as described in (A). Protein levels of NFATc1 in myotubes were analysed using Western blotting. Values are presented as mean ± SEM (n = 12 mice for analysing cells derived from type I fibres, n = 10 mice for analysing cells derived from type II fibres). Uncropped blots can be found in Supplementary Fig. 11.

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  Figure 5. The Rspo3-induced MyHC I expression is blocked by a pharmacological inhibitor of the Wnt/β-catenin signalling pathway. (A, B) Mouse primary myoblasts were differentiated in the medium containing the Rspo3 recombinant protein (200 ng/mL; 6.45 nM) with or without the WNT/β-catenin signalling inhibitors, XAV-939 (1.56 µg/mL; 5 µM), for 3 d. Protein levels of β-catenin, MyHC I and MyHC II in myotubes were quantified using Western blotting. Values are presented as mean ± SEM (n = 6–7 mice for analysing cells derived from type II fibres, n = 9–10 mice for analysing cells derived from type I fibres). (A) Myoblasts derived from type II fibre were used. (B) Myoblasts derived from type I fibre were used. *P < 0.05; **P < 0.01 by one-way ANOVA followed by the Tukey post hoc test. Uncropped blots can be found in Supplementary Fig. 12.