Journal: bioRxiv

Article Title: microRNA-1 Regulates Metabolic Flexibility in Skeletal Muscle via Pyruvate Metabolism

doi: 10.1101/2024.08.09.607377

Figure Lengend Snippet: (A) Ratio of mitochondrial protein to total protein abundance across samples, referred to as Mitochondrial Enrichment Factor (MEF). (B) Quantification of the OXPHOS protein complexes generated by the summed abundance of all subunits within a given complex. Data are presented as a percentage of the max for each complex. (C) Volcano plot depicting changes in the skeletal muscle mitochondrial proteome. Red color indicates significance ( p < 0.05), and differentially expressed proteins involved in pyruvate metabolism are labeled. Data in (A-C) from n=4 WT and n=4 miR-1 KO female mice (post-tamoxifen treatment and an 8-week washout). (D) Representative Western blot of phosphorylation of Ser 293 on the PDHE1a subunit [p-PDHE1a(Ser293)], total PDHE1a, and corresponding total protein levels in WT and miR-1 KO gastrocnemius muscle lysates. (E) Quantification of p-PDHE1a/total PDHE1a protein levels after densitometric analysis of the levels of each sample normalized to corresponding total protein levels, expressed as a ratio. (F) Quantification of total PDHE1a protein levels after densitometric analysis of the levels of each sample normalized to corresponding total protein levels, expressed as FC. (G) Principal component analysis (PCA) scores plot generated using MetaboAnalyst based on gastrocnemius metabolites in n=4 WT and n=4 miR-1 KO female mice (post-tamoxifen treatment and an 8-week washout). Predictive component (PC) 1 and PC2 can differentiate the WT and miR-1 KO muscle. (H) Summary of altered metabolic pathways analysis with MetaboAnalyst reflecting the impact on the pathway and the level of significance. The colors of dots (varying from yellow to red) indicates the significance of the metabolites in the data, and the size of the dot is positively corelated with the impact of the metabolic pathway. Top 6 pathways labeled. (I) Study design for in vitro studies, created using Biorender.com. (J) miR-1 expression of 4-Hydroxytamoxifen (4-OH TAM)-treated myotubes from WT or miR-1 KO mice (n=3 untreated female mice per group). (K) Extracellular acidification rate (ECAR) trace over time after injection of indicated glycolytic modulators in WT (n=3 female) and miR-1 KO (n=4 female)-derived myotubes. Oligo: oligomycin, 2-DG: 2-deoxy-D-glucose. (L) Quantification of basal ECAR (before Oligo addition), and (M) maximal ECAR (after Oligo addition). (N) Pkm mRNA expression of WT and miR-1 KO myotubes. Data in (E-F, J, L-N) analyzed using independent t -tests. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The primary antibodies and respective concentrations used were as follows: anti-PKM1 (1:1000, 7067, Cell Signaling, Danvers, MA) anti-PKM2 (1:1000, 4053, Cell Signaling), anti-phospho-PDHE1a (Ser293) (1:1000, NB110-93479, Novus Biologicals, Centennial, CO, USA) and anti PDHE1a (1:1000, NBP2-33922, Novus Biologicals).

Techniques: Generated, Labeling, Western Blot, In Vitro, Expressing, Injection, Derivative Assay