Postmitotic Expression of SOD1(G93A) Gene Affects the Identity of Myogenic Cells and Inhibits Myoblasts Differentiation

Abstract

To determine the role of mutant SOD1 gene (SOD1(G93A)) on muscle cell differentiation, we derived C2C12 muscle cell lines carrying a stably transfected SOD1(G93A) gene under the control of a myosin light chain (MLC) promoter-enhancer cassette. Expression of MLC/SOD1(G93A) in C2C12 cells resulted in dramatic inhibition of myoblast differentiation. Transfected SOD1(G93A) gene expression in postmitotic skeletal myocytes downregulated the expression of relevant markers of committed and differentiated myoblasts such as MyoD, Myogenin, MRF4, and the muscle specific miRNA expression. The inhibitory effects of SOD1(G93A) gene on myogenic program perturbed Akt/p70 and MAPK signaling pathways which promote differentiation cascade. Of note, the inhibition of the myogenic program, by transfected SOD1(G93A) gene expression, impinged also the identity of myogenic cells. Expression of MLC/SOD1(G93A) in C2C12 myogenic cells promoted a fibro-adipogenic progenitors (FAPs) phenotype, upregulating HDAC4 protein and preventing the myogenic commitment complex BAF60C-SWI/SNF. We thus identified potential molecular mediators of the inhibitory effects of SOD1(G93A) on myogenic program and disclosed potential signaling, activated by SOD1(G93A), that affect the identity of the myogenic cell population.

Figure 1

Characterization of C2C12 MLC/SOD1^G93A. (a) Schematic representation of MLC/SOD1^G93A construct. (b) Western blot analysis for mutant human SOD1^G93A protein in C2C12 and C2C12 MLC/SOD1^G93A at different time points during differentiation. (c) Upper panel shows representative western blot analysis of gp91phox expression in C2C12 and C2C12 MLC/SOD1^G93A cells. Lower panel shows the relative densitometric analysis of C2C12 (white bars) and C2C12 MLC/SOD1^G93A (black bars). D2 and D5 referred to days of culture in differentiation condition. Data are represented as mean ± SEM.

Figure 2

Postmitotic expression of mutant SOD1 gene inhibits C2C12 cells differentiation. (a) Representative immunofluorescence images of C2C12 and C2C12 MLC/SOD1^G93A cells stained with anti-myosin heavy chain (MHC) antibody after 5 days in differentiation medium. (b) The histograms represent the differentiation index (left panel) and fusion index (right panel) in control (white bars) and transfected cells (black bars). (c) Upper panel shows representative western blot analysis of MHC expression in C2C12 and C2C12 MLC/SOD1^G93A cells. Lower panel shows densitometric analysis for MHC expression in C2C12 (white bars) and C2C12 MLC/SOD1^G93A (black bars). D0, D2, and D5 referred to days of culture in differentiation condition. Data are represented as mean ± SEM (^* P < 0.05).

Figure 3

Mutant SOD1 gene downmodulates the players involved in C2C12 differentiation. Real time PCR for MyoD (a), Myogenin (b), MRF4 (c), miR133a (d), miR206 (e), and miR1 (f) in both control (white bars) and transfected cells (black bars). D0, D2, and D5 referred to days of culture in differentiation condition. Data are represented as mean ± SEM (^* P < 0.05; ^** P < 0.005).

Figure 4

Muscle specific expression of human mutant SOD1 gene alters signaling pathways in muscle differentiation. (a) Representative western blot analysis for phospho-Akt (Thr 308) and total Akt expression (upper panel) and densitometric analysis (lower panel) of the ratio between phosphorylated Akt and the total form (lower panel) in C2C12 (white bars) and C2C12 MLC/SOD1^G93A (black bars). (b) Representative western blot analysis for phospho-P70 (Thr 389) and total P70 expression (upper panel) and densitometric analysis (lower panel) of the ratio between phospho-P70 and total P70 (lower panel) in C2C12 (white bars) and C2C12 MLC/SOD1^G93A (black bars). (c) Representative western blot analysis of phospho-p42/44 MAPK and p42/44 MAPK expression (upper panel) and densitometric analysis (lower panel) of the ratio between phospho-p42-44 MAPK and total p42/44 MAPK in C2C12 (white bars) and C2C12 MLC/SOD1^G93A (black bars). D0, D2, and D5 referred to days of culture in differentiation condition. Data are represented as mean ± SEM (^* P < 0.05).

Figure 5

C2C12 MLC/SOD1^G93A cells exhibit adipogenic features. Real time PCR analysis for Pax7 (a) and Pax3 (b). (c) Upper panel shows Oil Red O staining and phase contrast of control C2C12 and C2C12 MLC/SOD1^G93A transfected cells at day 5 in culture. (d) Upper panel shows representative western blot analysis of Plin2 and relative densitometric analysis (lower panel) of the ratio between Plin2 and α-tubulin in C2C12 (white bars) and C2C12 MLC/SOD1^G93A (black bars). D0, D2, and D5 referred to days of culture in differentiation condition. Data are represented as mean ± SEM (^* P < 0.05; ^*** P < 0.0005).

Figure 6

Expression of mutant SOD1 promotes a FAPS phenotype in C2C12 cells. (a) Flow cytometry profile for α7 integrin and Sca1 expression from control C2C12 and transfected C2C12 MLC/SOD1^G93A cells at days 2 and 5 in differentiation medium. (b, c) Histograms of the percentage of α7 integrin (b) and Sca1 (c) positive cells of C2C12 (white bars) and C2C12 MLC/SOD1^G93A (black bars) cells. D2 and D5 referred to days of culture in differentiation condition. Data are represented as mean ± SEM (^* P < 0.05; ^*** P < 0.0005).

Figure 7

The acquisition of fibro-adipogenic features involves a HDAC-regulated network. (a) Upper panel shows western blot analysis of HDAC4 expression in C2C12 and C2C12 MLC/SOD1^G93A cells and densitometric analysis (lower panel) of the ratio between HDAC4 and α-tubulin. (b) Real time PCR for BAF60C in C2C12 (white bars) and C2C12 MLC/SOD1^G93A (black bars). (c, d) Histograms of the percentage of Sca1 (c) and positive α7 integrin cells (d) of control C2C12 MLC/SOD1^G93A (black bars) cells and TSA treated C2C12 MLC/SOD1^G93A cells (grey bars). D2 referred to days of culture in differentiation condition. (e) Representative immunofluorescence analysis for MHC after 5 days in differentiation medium in untreated (left panel) and TSA treated (right panel) C2C12 MLC/SOD1^G93A cells. (f) Histograms of the differentiation index (left panel) and fusion index (right panel) in control (black bars) and treated cells (grey bars). D2 and D5 referred to days of culture in differentiation condition. Data are represented as mean ± SEM (^* P < 0.05; ^** P < 0.005).