Dynamics of the skeletal muscle secretome during myoblast differentiation

Abstract

During recent years, increased efforts have focused on elucidating the secretory function of skeletal muscle. Through secreted molecules, skeletal muscle affects local muscle biology in an auto/paracrine manner as well as having systemic effects on other tissues. Here we used a quantitative proteomics platform to investigate the factors secreted during the differentiation of murine C2C12 skeletal muscle cells. Using triple encoding stable isotope labeling by amino acids in cell culture, we compared the secretomes at three different time points of muscle differentiation and followed the dynamics of protein secretion. We identified and quantitatively analyzed 635 secreted proteins, including 35 growth factors, 40 cytokines, and 36 metallopeptidases. The extensive presence of these proteins that can act as potent signaling mediators to other cells and tissues strongly highlights the important role of the skeletal muscle as a prominent secretory organ. In addition to previously reported molecules, we identified many secreted proteins that have not previously been shown to be released from skeletal muscle cells nor shown to be differentially released during the process of myogenesis. We found 188 of these secreted proteins to be significantly regulated during the process of myogenesis. Comparative analyses of selected secreted proteins revealed little correlation between their mRNA and protein levels, indicating pronounced regulation by posttranscriptional mechanisms. Furthermore, analyses of the intracellular levels of members of the semaphorin family and their corresponding secretion dynamics demonstrated that the release of secreted proteins is tightly regulated by the secretory pathway, the stability of the protein, and/or the processing of secreted proteins. Finally, we provide 299 unique hydroxyproline sites mapping to 48 distinct secreted proteins and have discovered a novel hydroxyproline motif.

Fig. 1.

Myogenic commitment of C2C12 myoblasts using dFBS. A, experimental overview. C2C12 cells were grown to confluence (day 0), and myoblast differentiation was induced by reducing the amount of dFBS to 2%. CM were collected on days 0, 2, and 5 of differentiation of C2C12 cells. “Day 0 cells” were cultured using regular Arg0 and Lys0, “day 2 cells” were cultured using Arg6 and Lys4, and “day 5 cells” were cultured using Arg10 and Lys8. CM were concentrated by ultrafiltration, and proteins were separated by one-dimensional gel electrophoresis. Excised gel bands from the entire lane were subjected to in gel-digestion and subsequently analyzed on an LTQ-Orbitrap-XL mass spectrometer. B and C, efficiency of differentiation was verified by examining the expression of muscle markers either by confocal laser scanning microscopy (B) or Western blotting (C). B, immunostaining of C2C12 cells at days 0, 2, and 5 of differentiation with an antibody against troponin T and visualized by a green fluorescence-tagged secondary antibody. Nuclei were counterstained with Sytox. The scale bar represents 95 μm. C, Western blot analysis of whole cell extracts prepared from C2C12 myoblasts at days 0, 2, and 5 of myogenesis using the indicated antibodies.

Fig. 2.

Functional analyses of identified secreted proteins from C2C12 skeletal myoblasts. A and B, identified secreted proteins from C2C12 myoblasts were compared with a reference list of secreted proteins obtained from the entire list of IPI entries. For selected biological processes (A) and molecular functions (B), the frequency of the terms in the data and the IPI database is illustrated. Only significantly overrepresented categories (p < 0.05) are shown.

Fig. 3.

Dynamic and functional characterization of differentially regulated secreted proteins. Cluster analysis of regulated proteins according to their secretion profile is shown. The heat map summarizes the occurrences of the indicated biological process and molecular function GO terms within each cluster. Only GO terms with five or more occurrences in at least one cluster are shown.

Fig. 4.

Secretion and expression of TGF-β1, -β2, and -β3 and IGF-1 and -2 are differentially regulated during the course of skeletal muscle differentiation. A, dynamic secretion profiles, including ratios obtained by mass spectrometry analyses, for the proteins TGF-β1, -β2, -β3, LTBP-3, IGF-1, IGF-2, IGFBP-2, and IGFBP-4 during the course of C2C12 differentiation. B, mRNA expression of TGF-β and IGF isoforms at days 0, 2, and 5 of C2C12 differentiation analyzed by Q-PCR. The level of mRNA expression of selected genes was normalized to the mRNA level of TBP. Gene expression data are given relative to day 0, and data are shown as mean ± S.D. (n = 3). Error bars represent the Standard Deviation S.D.

Fig. 5.

Differential secretion of different subclasses of semaphorin proteins during the process of myogenesis. A, quantitative profiles of semaphorin secretion during the course of C2C12 differentiation, including their ratios, applying quantitative proteomics. B and C, Western blot analysis of selected semaphorin proteins in conditioned media (B) or whole cell extracts (C) at days 0, 2, and 5 of C2C12 differentiation. D, mRNA expression for Sema3A, Sema3E, Sema6A, and Sema7A by real time Q-PCR. Gene expression data were normalized to the expression of TBP and are presented as mean ± S.D. (n = 3) relative to day 0. E, dynamic profile of MMP-2 secretion as observed by MS. F, gelatin zymography assessing MMP-2 activity in conditioned media from C2C12 myoblasts at days 0, 2, and 5 of differentiation (pro-MMP-2, 72 kDa; and active MMP-2, 62 kDa). * represents the glycosylated form of Sema7A.

Fig. 6.

Analyses of hydroxyproline sites identified from proteins secreted by C2C12 myoblasts and myotubes. A, distribution of hydroxyproline sites on the different collagen subtypes. B, distribution of the regulation (SILAC ratios) of hydroxyproline sites at days 2 (mean, −1.49) and 5 (mean, −0.32) of differentiation. C, hydroxyproline motif analysis. Sites matching the prototypical collagen hydroxyproline motif (top panel), sites with a tryptophan in the −3 position, all on fibronectin (middle panel), and the remaining sites not matching a significant shared motif (bottom panel) are shown.