doi: 10.1016/j.celrep.2016.02.014. Epub 2016 Feb 25.
Nfix Regulates Temporal Progression of Muscle Regeneration through Modulation of Myostatin Expression
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- PMID:26923583
- PMCID: PMC4793149
- DOI: 10.1016/j.celrep.2016.02.014
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Nfix Regulates Temporal Progression of Muscle Regeneration through Modulation of Myostatin Expression
Giuliana Rossi et al. Cell Rep..
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Abstract
Nfix belongs to a family of four highly conserved proteins that act as transcriptional activators and/or repressors of cellular and viral genes. We previously showed a pivotal role for Nfix in regulating the transcriptional switch from embryonic to fetal myogenesis. Here, we show that Nfix directly represses the Myostatin promoter, thus controlling the proper timing of satellite cell differentiation and muscle regeneration. Nfix-null mice display delayed regeneration after injury, and this deficit is reversed upon in vivo Myostatin silencing. Conditional deletion of Nfix in satellite cells results in a similar delay in regeneration, confirming the functional requirement for Nfix in satellite cells. Moreover, mice lacking Nfix show reduced myofiber cross sectional area and a predominant slow twitching phenotype. These data define a role for Nfix in postnatal skeletal muscle and unveil a mechanism for Myostatin regulation, thus providing insights into the modulation of its complex signaling pathway.
Keywords: Myostatin; Nfix; regeneration; satellite cells; skeletal muscle.
Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
Figures
Nfix Is Expressed by SCs and Its Absence Leads to a Reduced Myofiber Cross Sectional Area and to an Increased Slow MyHC Expression (A) Immunofluorescence analysis of Nfix expression (green) in Pax7+ SCs (red) on aTibialis anterior muscle section of aMlc3f-lacz mouse, where myonuclei are in purple (β-gal). Hoechst was used to stain nuclei (n = 4 mice). The scale bar represents 25 μm. (B) H&E staining onTibialis anterior muscles from WT andNfix-null mice (n = 5 mice). The scale bar represents 100 μm. (C) Graphical representation of the myofiber cross sectional area (CSA) in WT andNfix-null muscles. The plotted values represent the distribution of n = 65 measurements on five random microscope fields (∗∗∗p < 0.001 and two-tailed unpaired t test). The data are presented as mean ± whiskers from min to max. (D) Western blot analysis of slow MyHC expression in WT andNfix-nullSoleus (SOL) and EDL muscles. The β-tubulin was used to normalize. See also Figure S1.
SCs Lacking Nfix Are Characterized by a Delayed Differentiation (A) Immunofluorescence analysis of MyoD (green) and Myogenin (Myog, red) expression on single muscle fibers from WT andNfix-null muscles after 24, 48, 72, and 96 hr in culture. Hoechst was used to stain nuclei (n = 3 WT and 3Nfix-null mice). The scale bar represents 50 μm. (B) Quantification of MyoD+/Myogenin+ and MyoD+/Myogenin− SCs associated with WT andNfix-null myofibers at 24, 48, 72, and 96 hr in culture. The quantification is the result of three independent experiments on three WT and threeNfix-null mice. The data are presented as mean ± SD (not significant, ns;∗∗∗p < 0.001 with n = 11 WT myofibers and n = 16Nfix-null myofibers; and two-tailed unpaired t test). See also Figure S2.
Absence of Nfix Leads to a Marked Impairment of Muscle Regeneration after Injury (A) Immunofluorescence for dMHC (green) on regenerating WT andNfix-nullTibialis anterior muscles 5, 7, 10, 14, and 28 days (d) following CTX injection. The laminin is marked in red and Hoechst was used to stain nuclei (n = 3 mice for each group). The scale bar represents 50 μm. (B) Western blot analysis of Myogenin and Myostatin expression on WT andNfix-null muscles 3, 5, 10, and 28 days (d) following CTX injection. The β-tubulin was used to normalize. (C) Western blot analysis of pSmad3 and Smad3 expression on WT andNfix-null muscles 5 and 7 days (d) following CTX injection. GAPDH was used to normalize. (D) Quantification of the percentage of dMHC positive myofibers in WT andNfix-null mice after 7 and 14 days following CTX injection (n = 6 mice for time point 7 and n = 5 mice for time point 14). The data are presented as mean ± SD (∗p < 0.05;∗∗p < 0.01; and two-tailed unpaired t test). See also Figure S2E.
Absence of Nfix in SCs Determines the Delay in Muscle Regeneration (A) H&E staining and immunofluorescence for dMHC (green) on transverse sections of regeneratingTibialis anterior muscles fromTg:Pax7-CreERT2:Nfix+/+ andTg:Pax7-CreERT2:Nfixfl/−mice with (TMX) or without (NO TMX) tamoxifen treatment. The muscles were collected and stained after 5, 7, and 14 days (d) from CTX injection. The laminin is marked in red and Hoechst was used to stain nuclei (n = 3 mice for each group). The scale bar represents 100 μm. (B) Western blot analysis of Myogenin expression on regenerating muscles fromTg:Pax7-CreERT2:Nfix+/+ (Nfix+/+) andTg:Pax7-CreERT2:Nfixfl/− mice (Nfixfl/−) with (TMX) or without (NO TMX) tamoxifen treatment after 3, 5, 7, and 14 days (d) from CTX injection. Myogenin and GAPDH in the right panel come from two separate gels but using the same samples and protein amount. GAPDH was used to normalize. See also Figure S3.
Nfix Regulates Myostatin Expression in Differentiating Myoblasts through a Direct Binding to its Promoter (A) Real-time qPCR showing Myostatin upregulation in differentiating SC-derived myoblasts. The SCs were isolated by FACS and plated in differentiation medium for a time course analysis from 1 to 3 days (DM1, DM2, and DM3) (n = 4 DM1 WT, n = 2 DM1Nfix-null, n = 5 DM2 WT, n = 3 DM2Nfix-null, n = 4 DM3 WT, and n = 4 DM3Nfix-null). The data are presented as mean ± SD (not significant, ns;∗p < 0.05;∗∗p < 0.01; and two-tailed unpaired t test). (B) Western blot analysis of Nfix expression in C2C12 myoblasts transduced with a control vector (C2C12 scramble) or with a vector carrying a shRNA targeting Nfix (C2C12 shNfix). GAPDH was used to normalize. (C) Real-time qPCR analysis of Myostatin expression in scramble and shNfix C2C12 in a time course from 3 to 7 days in differentiation medium. The values are plotted as relative expression and normalized to GAPDH (n = 3 independent samples for each time point). The data are presented as mean ± SD (not significant, ns;∗p < 0.05;∗∗p < 0.01; and two-tailed unpaired t test). (D) ChIP on differentiated C2C12 transduced with a vector expressing a HA-tagged Nfix2 isoform to test binding to putative Nfix binding sites on Myostatin promoter located at −87/−212 bp and −274/−548 bp from transcription start site. Binding on Nfatc4 promoter and on an intergenic region were used as positive and negative controls, respectively. The data are means of two independent experiments and expressed as fold enrichment (mean ± SD) relative to the IgG signal (n = 3 independent ChIP). (E) Real-time qPCR for Myostatin in WT andNfix-null SC-derived myoblasts transduced with a control vector (scramble) or with a vector overexpressing the Nfix2 isoform (Nfix2-HA). The values are plotted as relative expression and normalized to GAPDH (n = 3 samples for each group). The data are presented as mean ± SD (∗p < 0.05;∗∗p < 0.01; and two-tailed unpaired t test).
In Vivo Silencing of Myostatin in RegeneratingNfix Null Muscles Rescues their Regeneration Defects (A) Immunofluorescence analysis of dMHC expression (green) on regenerating WT andNfix-nullTibialis anterior muscles after muscle electroporation with a control plasmid (scramble) or with a plasmid carrying an shRNA targeting Myostatin (shmstn). The muscles were electroporated after 4 days following CTX injection and were collected and stained after 7, 10, and 14 days (d) following CTX injection. The laminin is marked in red and Hoechst was used to stain nuclei (n = 3 mice for each group). The scale bar represents 100 μm. (B) Quantification of the percentage of dMHC positive myofibers inNfix-null mice electroporated with scramble or shmstn plasmids after 7 and 14 days following CTX injection (n = 5 mice for each group). The data are presented as mean ± SD (∗p < 0.05 and two-tailed unpaired t test). (C) Real-time qPCR showing Myostatin expression in myoblasts re-isolated fromTibialis anterior electroporated with control (scramble) or shMyostatin (shMstn) plasmids. The myoblasts were isolated the day after in vivo transfection, and cultures were stopped 2 days later to perform qRT-PCR. The data are presented as mean ± SD. See also Figure S4.
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