Cardiolipin metabolism regulates expression of muscle transcription factor MyoD1 and muscle development

Figures

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  Figure 1. Muscle differentiation pathways are decreased in TAZ-KO myoblasts. A, scatter plot indicating changes in gene expression between undifferentiated TAZ-KO myoblasts and WT controls. Results are the average of three biological replicates. Genes with a fold change >1.5 and FDR < 0.05 were considered significant. Of 21,516 analyzed genes, 326 were downregulated (red dots) and 487 were upregulated (green dots). MyoD1 is one of the top downregulated genes in TAZ-KO cells (blue dot indicated by black arrow). B, gene ontology analysis of the top 100 downregulated genes based on their fold change in TAZ-KO myoblasts (versus WT controls) determined by RNA-seq. ClueGO (20) was used to identify significantly altered biological processes (p < 0.01). Cardiocyte differentiation and muscle cell development are among the most significantly suppressed pathways in TAZ-KO cells. C, representative decreased pathways in TAZ-KO myoblasts predicted by PANTHER pathway analysis based on RNA-seq data (22, 23). Pathways with a fold change >2 and p <0.05 were considered significant. The p-value for each pathway is shown on the respective bar. D, immunoblot images of MHC protein in WT and TAZ-KO cells after 5,…

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  Figure 2. MyoD1 is downregulated in TAZ-KO myoblasts and TAZ-KO mouse hearts.A, fragments per kilobase million (FPKM) values determined by RNA-seq (left) and qPCR analysis of MyoD1 mRNA levels (right) in WT and TAZ-KO myoblasts. β-actin (ACTB) was used as an internal control for qPCR. B, qPCR analysis of MyoD1 mRNA levels before and after differentiation induction. Adapter-relatedprotein complex3subunitdelta 1 (Ap3d1) was used as an internal control. C, qPCR analysis of MyoD1 mRNA levels in WT and TAZ-KO mouse hearts. ACTB was used as an internal control. D, immunoblot images of MyoD1 protein in WT and TAZ-KO mouse hearts (upper panel), and their respective quantification (lower panel). All data shown are mean ± S.D. ∗p < 0.05, ∗∗∗p < 0.001. MyoD1, myoblast determination protein 1; qPCR, quantitative PCR; RNA-seq, RNA sequencing; TAZ-KO, TAFAZZIN-knockout.

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  Figure 3. Exogenous MyoD1 rescues the myogenesis defects of TAZ-KO cells. A, MyoD1 protein is not detectable in TAZ-KO cells but can be restored by exogenous expression. Upper panel, WT, and TAZ-KO myoblasts were transfected with an empty vector (EV) (61) or CMV-MyoD plasmid (60), and after 7 days, cells were harvested for immunoblotting analysis against MyoD1. Lower panel, quantification of MyoD1 protein levels as previously described. B, MHC protein levels are rescued by exogenous expression of MyoD1 in TAZ-KO cells. Upper panel, cells were induced to differentiate by culturing in differentiation medium for 5 or 7 days, then harvested for immunoblotting against the myotube marker MHC. Lower panel, quantification of MHC levels 5 days after induction of differentiation. All data shown are mean ± S.D. ∗p < 0.05, ∗∗p < 0.01. MHC, myosin heavy chain; MyoD1, myoblast determination protein 1; TAZ-KO, TAFAZZIN-knockout.

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  Figure 4. Increased MyoD1 negative regulator MKX in TAZ-KO cells. A, Mkx fragments per kilobase million (FPKM) values of WT and TAZ-KO myoblasts determined by RNA-seq (left), and qPCR analysis of Mkx mRNA levels in WT and TAZ-KO cells before and after differentiation induction (right). Ap3d1 was used as an internal control. B, left: Mkx mRNA levels are significantly reduced in TAZ-KO cells transfected with MKX-targeting siRNAs compared to scrambled-treated cells. Right: MyoD1 mRNA levels are increased in MKX siRNA-treated TAZ-KO cells compared to scrambled-treated cells. Data were obtained by qPCR analysis, and Ap3d1 was used as an internal control. All data shown are mean ± S.D. ∗p < 0.05, ∗∗p <0.01, ∗∗∗p < 0.001. MKX, Mohawk; MyoD1, myoblast determination protein 1; qPCR, quantitative PCR; RNA-seq, RNA sequencing; TAZ-KO, TAFAZZIN-knockout.

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  Figure 5. Wnt signaling is decreased in TAZ-KO cells. A, Wls mRNA levels determined by RNA-seq (left) and qPCR analysis (right) in WT and TAZ-KO cells before and after differentiation induction. B, qPCR analysis showed that Porcn (left) and Axin2 (right) mRNA levels are decreased in TAZ-KO cells after 1 and 7 days of differentiation induction. Ap3d1 was used as an internal control. C, left: MyoD1 protein levels are rescued by Wnt agonist treatment. After 5 days of differentiation induction, cells were treated with 5 μM or 10 μM Wnt agonist I in DMSO (CAS 853220–52–7, Calbiochem) for 48 h before subjecting to immunoblotting analysis against MyoD1. Right: quantification of MyoD1 protein levels as previously described. All data shown are mean ± S.D. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <0.001. MyoD1, myoblast determination protein 1; qPCR, quantitative PCR; RNA-seq, RNA sequencing; TAZ-KO, TAFAZZIN-knockout; Wls, Wntless

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  Figure 6. Proposed mechanism of MyoD1 repression in TAFAZZIN-deficient myoblasts. Wnt signaling is an essential signal transduction pathway required for functional muscle development. Wnt signaling and mitochondrial function regulate one another through positive feedback. We propose that decreased mitochondrial function in the absence of TAFAZZIN leads to decreased Wnt signaling and a concomitant increase in MKX-mediated repression of MyoD1. MyoD1 is the master transcription factor that regulates muscle development and homeostasis. Therefore, repression of MyoD1 via the above mechanism may serve as a link between TAFAZZIN deficiency and skeletal myopathy in BTHS patients. Image was created withBioRender.com. BTHS, Barth syndrome; MKX, Mohawk; MyoD1, myoblast determination protein 1.

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