Loss of emerin alters myogenic signaling and miRNA expression in mouse myogenic progenitors

Abstract

Emerin is an integral membrane protein of the inner nuclear membrane. Mutations in emerin cause X-linked Emery-Dreifuss muscular dystrophy (EDMD), a disease characterized by skeletal muscle wasting and dilated cardiomyopathy. Current evidence suggests the muscle wasting phenotype of EDMD is caused by defective myogenic progenitor cell differentiation and impaired muscle regeneration. We obtained genome-wide expression data for both mRNA and micro-RNA (miRNA) in wildtype and emerin-null mouse myogenic progenitor cells. We report here that emerin-null myogenic progenitors exhibit differential expression of multiple signaling pathway components required for normal muscle development and regeneration. Components of the Wnt, IGF-1, TGF-β, and Notch signaling pathways are misexpressed in emerin-null myogenic progenitors at both the mRNA and protein levels. We also report significant perturbations in the expression and activation of p38/Mapk14 in emerin-null myogenic progenitors, showing that perturbed expression of Wnt, IGF-1, TGF-β, and Notch signaling components disrupts normal downstream myogenic signaling in these cells. Collectively, these data support the hypothesis that emerin is essential for proper myogenic signaling in myogenic progenitors, which is necessary for myogenic differentiation and muscle regeneration.

Figure 1. mRNA expression profiling identifies changes in Notch, Wnt, TGF-β and IGF pathways.

mRNA microarray expression data from emerin-null myogenic progenitors was normalized to mRNA expression in wildtype progenitors. Dotted line represents no change in expression. A) Notch pathway. B) Wnt pathway. C) TGF-β pathway. D) IGF pathway. All changes in gene expression were statistically significant (p≤0.05).

Figure 2. Validation of selected components of the Notch, Wnt, TGF-β and IGF pathways.

Validation of mRNA expression changes of selected Notch, Wnt, TGF-β and IGF pathways by qPCR. Dotted line represents no change in expression. A) Notch and p38 components. B) Wnt components. C) IGF and TGF-β components. All changes in gene expression were statistically significant (p≤0.05). D) Whole cell lysates of wildtype and emerin-null H2K myogenic progenitors were separated by SDS-PAGE and western-blotted for GSK3β, Kat2b and γ-tubulin. E,F) Densitometry was performed on blots in panel D and GSK3β and Kat2b levels were normalized to γ-tubulin. Emerin-null protein expression was normalized to protein expression in wildtype progenitors. G) GFP-emerin or GFP was expressed in emerin-null cells and Gsk3b, Mapk14, Igfbp3 and Gapdh were measured by qPCR. Data is depicted as fold-change in GFP-emerin expressing cells compared to GFP-transfected cells.

Figure 3. Downstream target genes of the Wnt and TGF-β are also altered in emerin-null myogenic progenitors.

mRNA expression of selected downstream targets of the Wnt and TGF-β pathways was measured using qPCR. Dotted line represents no change in expression. A) Wnt targets. B) TGF-β targets. All changes in gene expression were statistically significant (p≤0.05).

Figure 4. Expression of miRNAs was altered in emerin-null myogenic progenitors.

miRNA expression was determined for emerin-null and wildtype myogenic progenitors by microarray. miRNA expression in emerin-null myogenic progenitors was normalized to wildtype miRNA expression in wildtype progenitors. Dotted line represents no change in expression. All changes in expression were statistically significant (p≤0.05).

Figure 5. miRNA-downregulation causes increased expression of target proteins.

A) Whole cell lysate was separated by SDS-PAGE and western-blotted for Myf5, Smad2 and γ-tubulin. B,C) Densitometry was done on blots in panel A and Myf5 and Smad2 were normalized to γ-tubulin. Protein expression in wildtype myogenic progenitors was set to one.

Figure 6. IGF signaling is activated in emerin-null myogenic progenitors.

Protein expression of p38 and phosphorylated p38 in emerin-null myogenic progenitors was monitored by western blotting (A) and quantitated by densitometry normalized to γ-tubulin (B) or total p38 (C) levels in wildtype progenitors. Antibodies specific for p38 (B) or phosphorylated p38 (C) were used to monitor activation of the IGF-1 pathway.

Figure 7. Model of predicted effects of emerin loss on myogenic signaling during differentiation.

A) Notch, Wnt, TGF-β and IGF signaling act at specific times during myogenic differentiation to ensure proper differentiation. B) Our data predicts that activation of Notch and IGF-1 signaling pathways and attenuation of Wnt and TGF-β signaling pathways will lead to impaired differentiation by maintaining the progenitors in a proliferative state and exhausting the satellite cell niche.