Semaglutide Modulates Proinflammatory Epicardial Adipogenesis With Paracrine Effects on hiPSC-Atrial Cardiomyocytes

Figures

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  Figure 1. Comparison Between EAT and SAT Biopsies or Stromal Cells Dots of individual values with bar plots with mean ± SD show mRNA expression levels of PGC1α and PPARγ, paired Student's t-test, n = 35 (A), GLUT4 and DEFA3, paired Student's t-test and Wilcoxon signed-rank test respectively, n = 30 (B), glucose consumption and L-lactate production levels (mg/dL) (a.u.), paired Student's t-test and Wilcoxon signed-rank test respectively, n = 46 in epicardial adipose tissue (EAT) and subcutaneous adipose tissue (SAT). (C) Dots of individual values with bar plots with mean ±SD mRNA expression levels of PGC1α and PPARγ, paired Student's t-test, n = 12 (D), GLUT4 and CD68, paired Student's t-test, n = 11 (E), and extracellular acidification rate (ECAR) and mRNA expression levels of GLP1R, paired Student's t-test, n = 6, n = 11, respectively (F), in EAT and SAT stromal cells. Statistical significance ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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  Figure 2. Adipogenesis Validation and Proteomics Comparison Representative fluorescence microscopy images from Adipored (both EAT and SAT for the same patient, scale bar = 100 μm) (A), dots of individual values with bar plots with mean ± SD of AdipoRed staining (adipogenesis area/cell number) for EAT and SAT stromal cells with (IDMT) or without (control) adipogenesis induction. Statistical significance ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 12, paired Student's t-test (B). Volcano plot showing proteins decreased (red) and increased (green) after adipogenesis induction according to SWATH-MS analysis (P < 0.05, n = 6) (C), interaction of the proteins after adipogenesis induction (D). Abbreviations as in Figure 1.

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  Figure 3. Adipocytes, Differentiation, and Inflammation Markers mRNA Levels Regarding ACTB as Arbitrary Units Based on 2-(ACTB/Gene), in EAT Cells With IDMT Induction With/out Semaglutide Treatment Dots of individual values with bar plots with mean ± SD show mRNA expression levels of FABP4, ADIPOQ, and PLIN1 (n = 12, n = 6, n = 9 respectively) (A); PGC1α (n = 11), PPARγ (n = 12), and CITED1 (n = 9) (B); CD68 (n = 12), IL6 (n = 9), and sPLA2 (n = 11) (C); and MMP9, aSMA (n = 12), and INHBA (n = 11) (D). Statistical significance ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, Friedman test with Dunn’s multiple comparison test for PGC1α and INHBA, and repeated measures 1-way analysis of variance with Tukey’s multiple comparison test for other genes. S = semaglutide; other abbreviations as in Figures 1 and 2.

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  Figure 4. Glucose Transporters, CPT1, and GLP1R mRNA Levels Regarding ACTB as Arbitrary Units Based on 2-(ACTB/Gene), in EAT Cells After IDMT Induction With/out Semaglutide Treatment, and GLP1R Immunostaining Dots of individual values with bar plots with mean ± SD show mRNA levels of GLUT1 and GLUT4 (n = 12 and n = 9 respectively, repeated measures 1-way analysis of variance with Tukey’s multiple comparison test), and CPT1(A), of GLP1 receptor (GLP1R) (n = 12, Friedman test with Dunn’s multiple comparison test), and representative fluorescence microscopy images of GLP1R immunostaining (Scale bar = 100μm), GLP1R stained area per cell (B). S = semaglutide; other abbreviations as in Figures 1 and 2.

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  Figure 5. Proteomic Analysis of Stromal Cells After Adipogenesis Induction With and Without Semaglutide Treatment Volcano plot showing proteins decreased (red) and increased (green) with semaglutide (S) treatment after adipogenesis induction (IDMT) in EAT stromal cells (P < 0.05, n = 6) (A); network of differential proteins (String software) (B); commonly and differentially detected proteins among control, IDMT, and IDMT+S (Scaffold analysis) (C); and normalized total spectra of voltage-dependent anion channel 3 (VDAC3) protein shown for each sample (D).

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  Figure 6. Paracrine Effect of Conditioned Medium of Adipogenized (IDMT) Epicardial Stromal Cells With and Without Semaglutide on hiPSC-aCMs, mRNA Levels of GLP1R, Cardiomyocyte Markers, Sodium, and Potassium Channels Immunocytofluorescence staining of human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) with MYL7 and atrial natriuretic peptide (ANP) antibodies and nucleus staining using 4',6-diamidino-2-phenylindole (DAPI) (Scale bar = 100 μm) (A); line plot of MYL7, NPPA, TNNT2, KCNA5, KCNJ3, and KCNJ5 mRNA expression levels in different passages of hiPSC-aCM (B); hiPSC-aCM monitored with Olympus Provi CM20 through the treatment, graph shows the confluency percentage of the cardiomyocytes for different treatment groups (n = 3), the vehicle (Veh) for adipogenesis control medium, IDMT for adipogenesis, and IDMT+S for adipogenesis with semaglutide (1 nmol/L) treatment (C); dots of individual values with bar plots with mean ± SD show mRNA expression levels regarding ACTB as arbitrary units (a.u.) based on 2-(ACTB/gene) algorithm, with fold changes over basal cardiomyocyte for GLP1R, MYL7, and NPPA for cardiomyocytes cultured with vehicle (Veh), IDMT, and IDMT+S…

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  Supplemental Figure.