Fig 2: MLX regulates the expression of several myokines. (A) Plot of RNA-seq data comparing log2 fold changes in gene expression observed in cells overexpressing MLX-DN (Y-axis) versus log2 fold changes seen in MLX shRNA cells (X-axis). Points are color-coded according to the log2 fold changes in gene expression seen in cells overexpressing MLX-WT. Many of the genes transcriptionally regulated by MLX encode for myokines; i.e., muscle-secreted proteins (31 out of 67 MLX-regulated annotated genes). See also Supplemental Table S1. (B) Enriched gene set analysis shows that MLX regulates predominantly the expression of extracellular and secreted proteins. (C) Fusion defects observed in myoblasts overexpressing MLX-DN are rescued by conditioned culture medium from control myoblasts. (D) Normalized expression of qPCR data indicates that MLX-WT and glucose strongly induce the expression of several myokines, whereas MLX shRNA and MLX-DN reduce their expression. Data from three biological replicates are shown. (E) Igf2 expression increases during myogenesis, suggesting that it is important for myoblast fusion and differentiation. Moreover, Igf2 expression is increased by MLX-WT and decreased by loss of MLX activity (MLX-DN and MLX shRNA). The histograms show the mean of three biological replicates ±SEM. (*) P < 0.05. See also Supplemental Figure S4.

Fig 3: Mlx-null mice have impaired muscle regeneration. (A) Representative histology images from uninjured and injured tibialis anterior muscles undergoing regeneration 7 d after cardiotoxin injection (D7 CTX) from Mlx-null (Mlx−/−) and wild-type (Mlx+/+) isogenic control mice at 12 wk of age. The top panels show hematoxylin/eosin staining, whereas the bottom panels show immunostaining for Laminin (green; a marker defining the external myofiber boundaries), nuclei (blue), and F-actin (red; which indicates viable myofibers, whereas its absence defines necrotic myofibers). In wild-type mice, muscle regeneration is completed 7 d after cardiotoxin injection (D7 CTX), resulting in the formation of myofibers completely filling external boundaries. Conversely, Mlx-null mice have impaired muscle regeneration, as indicated by persistence of numerous necrotic myofibers (indicated by asterisks) and the presence of small myotubes (indicated by arrows) that incompletely fill external boundaries left by prior myofibers. (B) The proportional muscle area (calculated as the percentage of total muscle area occupied by F-actin-positive myofibers) is similar in Mlx-null and wild-type muscles in uninjured conditions but is decreased significantly in regenerating Mlx-null muscles compared with wild-type controls. This indicates that muscle regeneration is impaired due to insufficient myogenesis in Mlx-null mice. Frequency distributions of myofiber and myotube diameters show a small shift to the right in uninjured Mlx-null myofibers compared with wild-type. However, there is an increased proportion of small myotubes (<30 µm in diameter) 7 d after cardiotoxin injection in Mlx-null muscles compared with wild-type controls, indicating impaired regeneration and insufficient fusion of Mlx-null myoblasts. Coincident changes are seen in the mean diameter of myotubes. Frequency distributions represent >2000 cell measurements pooled from individual animals for each genotype and condition. (C) qPCR analysis of Mlx and Txnip levels confirms that MLX activity is decreased in muscles from Mlx-null mice. Moreover, Igf2 up-regulation induced by cardiotoxin injection is significantly blunted in Mlx-null muscles, which explains their defective regeneration. The histograms show the mean of four or more biological replicates ±SEM. (*) P < 0.05 compared with the wild-type isogenic controls.

Fig 4: MLX promotes myogenesis via IGF2 signaling. (A) Of several MLX-induced myokines, only IGF2 induces myogenesis and rescues myoblast fusion defects caused by MLX-DN overexpression. Immunostaining for α-actin (red) and DAPI (4′,6-diamidino-2-phenylindole) (blue) is shown. See also Supplemental Figure S4. (B) ELISA measurement of IGF2 levels in conditioned cell culture medium and cellular lysates indicates that IGF2 protein levels increase as differentiation proceeds and in response to MLX activity (MLX-WT), whereas loss of MLX function (MLX-DN and MLX shRNA) decreases IGF2 protein levels. The histograms show the mean of three different experiments ±SEM. (*) P < 0.05. (C) Treatment of serum-deprived muscle cells (−) with recombinant mouse IGF2 (+) increases phospho-Akt levels (Ser473). Akt phosphorylation also increases during myogenesis ([Mb] myoblast; [D2] day 2 of differentiation; [D4] day 4 of differentiation) coincident with increased Igf2 mRNA and protein levels. Loss of MLX function (MLX-DN and MLX shRNA) decreases Akt phosphorylation, whereas MLX-WT increases it, indicating that MLX regulates IGF2/Akt signaling. (D) The dose response curve of the myoblast fusion index in response to IGF2 levels indicates that while exogenous addition of recombinant mouse IGF2 to culture medium stimulates fusion in control cells, myoblasts overexpressing MLX-WT are insensitive and appear to have already reached a maximum level of myoblast fusion. In addition, IGF-2 rescues myoblast fusion defects due to MLX-DN overexpression. (E) Myoblasts overexpressing IGF2 have increased fusion at low levels of glucose that are normally not permissive for fusion, similar to MLX-WT overexpression (shown in D). The mean of three biological replicates ±SEM is shown, with P < 0.05 compared with the respective control at the same concentration of IGF2/glucose.