Mitochondrial cardiolipin remodeling facilitates efficient myoblast differentiation

Figures

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  Fig. 1. Myoblast differentiation of Mito-HA-expressing C2C12 cells. A: Phase-contrast images of Mito-HA-expressing C2C12 cells cultured in the differentiation medium for 7 days. The scale bar represents 10 μm. B: The mRNA levels of MyoD, MyoG, Myh4, and Mck during C2C12 cell differentiation were quantified by qRT-PCR (n = 3). C and D: Immunoblot analysis of myoblast differentiation markers (MyoD, MyoG, and Myh) during C2C12 cell differentiation (C). The indicated antibodies were used, as appropriate. The protein expression levels were quantified (D) (n = 3). Mean ± SD; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant, one-way ANOVA with Dunnett’s multiple comparisons test.

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  Fig. 2. Comprehensive analysis of mitochondrial membrane phospholipids during myoblast differentiation of Mito-HA C2C12 cells. A: The proportion of each phospholipid in either the whole cell fraction or the IP fraction is shown as a heatmap with mean values (n = 3). B: The proportion of CL in the IP fraction for each fatty acid composition is shown as a heatmap with the mean value (n = 3). The number of linoleic acid moieties (18:2) is shown on the left side of the heatmap. C: The proportion of CL with 18:2 (n = 3). D: The proportion of CL with each number of 18:2 is shown as a heatmap with mean values (n = 3). E: The proportion of each phospholipid containing 18:2 is shown as a heat map with a mean value (n = 3). F: The amount of C18:2- (18:2 FA) and C18:2-containing lipids in the proliferation or differentiation media (n = 3). Mean ± SD; ∗∗P < 0.01; ns, not significant, one-way ANOVA with Dunnett’s multiple comparisons test (C) or unpaired Student’s t-test (F).

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  Fig. 3. Inhibition of myoblast differentiation does not affect the transition of fatty acid composition in CL. A and B: Immunoblot analysis of indicated proteins during myoblast differentiation of either siCtrl- or siMyoD-treated C2C12 cells (A). The protein expression levels were quantified (B) (n = 3). C: The proportion of CL with each fatty acid composition is shown as a heatmap with the mean value (n = 3). The number18:2 is shown on the left side of the heatmap. D: The proportion of CL with 18:2 (n = 3). E: The proportion of CL with each number of 18:2 is shown as a heatmap with mean values (n = 3). Mean ± SD; ∗P < 0.05; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant, one-way ANOVA with Dunnett’s multiple comparisons test (B) or Sidak’s multiple comparisons test (D).

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  Fig. 4. The inhibition of CL remodeling enzyme Taz suppressed the increase of CL with linoleic acid and the progress of myoblast differentiation. A and B: Immunoblot analysis of indicated proteins during myoblast differentiation of either siCtrl- or siTaz- (#1 or #2) treated C2C12 cells (A). The protein expression levels were quantified (B) (n = 3). C: The proportion of CL with each fatty acid composition is shown as a heatmap with the mean value (n = 3). The number 18:2 is shown on the left side of the heatmap. D: The proportion of CL with 18:2 (n = 3). E: The proportion of CL with each number of 18:2 is shown as a heatmap with mean values (n = 3). F and G: C2C12 cells were treated with 250 nM Torin 1 for 24 h and performed immunoblot analysis with indicated proteins (F). Taz expression levels were quantified (G) (n = 3). H: The proportion of CL with 18:2 (n = 3). I: The proportion of CL with each number of 18:2 is shown as a heatmap with mean values (n = 3). Mean ± SD; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗∗P < 0.0001; ns, not significant, one-way ANOVA with Sidak’s multiple comparisons test (B, D) or unpaired Student’s t-test (G, H).

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  Fig. 5. The inhibition of PRELID1, a lipid transfer protein for CL synthesis, suppressed the increase of CL with linoleic acid and the progress of myoblast differentiation. A and B: Immunoblot analysis of indicated proteins during myoblast differentiation of either siCtrl- or siPrelid1- (#1 or #2) treated C2C12 cells (A). The protein expression levels were quantified (B) (n = 3). C: The proportion of CL with each fatty acid composition is shown as a heatmap with the mean value (n = 3). The number 18:2 is shown on the left side of the heatmap. D: The proportion of CL with 18:2 (n = 3). E: The proportion of CL with each number of 18:2 is shown as a heatmap with mean values (n = 3). Mean ± SD; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; one-way ANOVA with Sidak’s multiple comparisons test.

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  Fig. 6. Inhibition of ATP synthase by oligomycin A (OA) affected myoblast differentiation and CL remodeling of C2C12 cells. A: The mRNA levels of Myh4 and Mck during the myoblast differentiation of OA-treated (10 nM, 3 days) C2C12 cells were quantified using qRT-PCR (n = 3). B: Immunoblot analysis of the indicated proteins during myoblast differentiation of OA-treated (10 nM, 3 days) C2C12 cells. Quantification of protein expression levels is shown in supplemental Fig. S6A. C: The proportion of CL with each fatty acid composition is shown as a heatmap with the mean value (n = 3). The number of linoleic acid molecules (18:2) is shown on the left side of the heatmap. D: The proportion of CL with 18:2 (n = 3). E: The proportion of CL with each number of 18:2 is shown as a heatmap with mean values (n = 3). Mean ± SD; ∗∗∗∗P < 0.0001; one-way ANOVA with Sidak’s multiple comparisons test.

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  Fig. 7. Linoleic acid supplementation partially rescued the defect of myoblast differentiation in siTaz-treated C2C12 cells. A and B: Immunoblot analysis of indicated proteins during myoblast differentiation of either siCtrl- or siTaz- (#1) treated C2C12 cells supplemented with oleic acid (18:1) or linoleic acid (18:2) (A). The protein expression levels were quantified (B) (n = 3). C: The proportion of CL with each fatty acid composition is shown as a heatmap with the mean value (n = 3). The number 18:2 is shown on the left side of the heatmap. D: The proportion of CL with 18:2 (n = 3). E: The proportion of CL with each number of 18:2 is shown as a heatmap with mean values (n = 3). Mean ± SD; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant, one-way ANOVA with Sidak’s multiple comparisons test.

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  Fig. 8. CL with linoleic acid was increased during the differentiation of isolated mouse primary myoblasts. A: Phase-contrast images of isolated mouse primary myoblasts cultured in a differentiation medium for 5 days. The scale bar represents 10 μm. B: The mRNA levels of Myh4 and Mck during isolated mouse primary myoblast differentiation were quantified by qRT-PCR (n = 3). C: Immunoblot analysis of myoblast differentiation markers and mitochondrial proteins during isolated mouse primary myoblast differentiation. The indicated antibodies were used, as appropriate. Quantification of protein expression levels is shown in supplemental Fig. S8A. D: The proportion of CL with each fatty acid composition is shown as a heatmap with the mean value (n = 3). The number 18:2 is shown on the left side of the heatmap. E: The proportion of CL with 18:2 (n = 3). F: The proportion of CL with each number of 18:2 is shown as a heatmap with mean values (n = 3). Mean ± SD; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant, one-way ANOVA with Dunnett’s multiple comparisons test.