Pik3ip1 mediates thyroid hormone-dependent regulation of the PI3K/Akt/mTOR axis in muscle atrophy

Figures

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  Figure 1. Transcriptomic characterization identifies Pik3ip1 as a Thyroid Hormone-dependent repressor of myogenic differentiation pathways. (A)Principal Component Analysis (PCA) of RNA-sequencing datasets derived from skeletal muscle samples of CTR, muscle-specific Dio2 knockout (mD2KO), and TRKO mice. (B) Heatmaps showing hierarchical clustering of Differentially Expressed Genes (DEGs) in mD2KO and TRKO muscles compared with CTR. DEGs were identified using p < 0.05, |–0.58 ≤ Log2 fold change ≥0.58| thresholds. Color scale represents normalized expression levels from minimum (purple) to maximum (yellow). (C–D) Volcano plots showing significantly deregulated transcripts in mD2KO versus CTR (C) and TRKO versus CTR (D) muscles. DEGs were identified using p < 0.05, |–1.0 ≤ Log2 fold change ≥1.0| thresholds. (E) Heatmap of selected genes associated with skeletal muscle differentiation and fusion pathways across CTR, mD2KO, and TRKO groups. (F)Gene Ontology (GO) enrichment analysis of deregulated transcripts in mD2KO/TRKO muscles. Downregulated pathways were predominantly associated with skeletal muscle differentiation, myoblast fusion, muscle cell fate commitment, and tissue regeneration,…

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  Figure 2. Thyroid Hormone signaling negatively regulates Pik3ip1 expression in skeletal muscle. (A) Schematic representation of predicted Thyroid Hormone Receptor Response Elements (TREs) identified within the murine Pik3ip1 promoter region. Putative TREs, their genomic localization, strand orientation, and prediction scores are shown. Highlighted sequences indicate the highest-confidence candidate binding motifs. (B) Sequence logos representing consensus TRE motifs identified within the Pik3ip1 regulatory regions. (C) Western Blot analysis of Pik3ip1 protein expression in skeletal muscle from CTR, mD2KO, and TRKO mice. Tubulin was used as loading control. Quantification of Pik3ip1/Tubulin ratio is shown on the right. (D) Representative immunofluorescence staining for Pik3ip1 (green) and WGA (magenta) in skeletal muscle sections from CTR, mD2KO, and TRKO mice. Nuclei were counterstained with DAPI (blue). Magnification 10x; scale bar 50 μm. (E) Relative Pik3ip1 mRNA expression in skeletal muscle from CTR, mD2KO, and TRKO mice. (F) Western Blot analysis of Pik3ip1 protein expression in skeletal muscle from euthyroid, hypothyroid, and hyperthyroid mice. Tubulin served as loading control.…

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  Figure 3. Pik3ip1 regulates TH-mediated activation of the Akt/mTOR signaling pathway in skeletal muscle cells. (A) Protein levels of Pik3ip1, phospho-Akt (Ser473), total Akt, phospho-mTOR (Ser2448), total mTOR, phospho-S6K1 (Thr389), and total S6K1 were evaluated by Western Blot in Control and Pik3ip1-overexpressing C2C12 cells treated with THs (T3 + T4, 30.0 nM each) for 24 or 48 h. Tubulin was used as loading control. (B) Representative immunofluorescence images of differentiated C2C12 myotubes transfected with either an empty vector (Control) or a Pik3ip1 expression plasmid and cultured in the absence or presence of THs (T3 + T4, 30.0 nM each) for 24 or 48 h. Cells were stained for Pik3ip1 (green), F-actin with Phalloidin (magenta), and nuclei with DAPI (blue). Dashed boxes indicate regions shown at higher magnification in the adjacent panels. Magnification 40x; scale bar 25 μm. (C) Protein levels of Pik3ip1, phospho-Akt (Ser473), total Akt, phospho-mTOR (Ser2448), total mTOR, phospho-S6K1 (Thr389), and total S6K1 were evaluated by Western Blot in differentiated C2C12 myotubes transfected with either a control shRNA plasmid (shCTR) or a Pik3ip1-targeting shRNA plasmid (shPik3ip1) and…

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  Figure 4. Pik3ip1 expression is induced during denervation-induced skeletal muscle atrophy in mD2KO and TRKO muscles. (A) Representative image of the sciatic nerve denervation procedure. Inset shows exposure and transection of the sciatic nerve. (B) Body weight measurements of CTR, mD2KO, and TRKO mice following denervation surgery over the indicated time course. (C)Tibialis Anterior (TA) muscle weight measured at the indicated time points after denervation. Percent reductions at day 14 are indicated. (D) TA muscle weight normalized to body weight following denervation. (E) Representative gross morphology and Hematoxylin and Eosin (H/E) staining of Innervated (Inn) and Denervated (Den) TA muscles from CTR, mD2KO, and TRKO mice collected at 4-, 10-, and 14-days post-denervation. Frequency distribution histograms of myofiber Cross-Sectional Area (CSA) are shown on the right (comparison inter genotypes over-time) and bottom panels (comparison intra genotypes over-time). Magnification 10x; scale bar 50 μm. (F) Relative Pik3ip1 mRNA expression in innervated and denervated TA muscles from CTR, mD2KO, and TRKO mice across the indicated time points. (G–I) The same dataset shown in panel F,…

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  Figure 5. Denervation-induced Pik3ip1 upregulation is associated with impaired AKT/mTOR signaling in mD2KO and TRKO skeletal muscle. (A) Representative immunofluorescence staining of Pik3ip1 (green) in Innervated (Inn) and Denervated (Den) Tibialis Anterior (TA) muscles from CTR, mD2KO, and TRKO mice collected at 4-, 10-, and 14-days post-denervation. Wheat Germ Agglutinin (WGA, magenta) was used to outline myofiber membranes and nuclei were counterstained with DAPI (blue). Quantification of Pik3ip1 integrated fluorescence intensity is shown on the right. Magnification 10x; scale bar 50 μm. (B–D) Western Blot analyses of Pik3ip1 and components of the AKT/mTOR signaling pathway in Innervated (Inn) and Denervated (Den) TA muscles from CTR, mD2KO, and TRKO mice collected at 4- (B), 10- (C), and 14- (D) days following denervation. Protein levels of phospho-Akt (S473), total Akt, phospho-mTOR (S2448), total mTOR, phospho-S6K (T389), total S6K1, and Pik3ip1 were evaluated. Tubulin was used as loading control.