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. 2021 Nov 22;6(11):834–850. doi: 10.1016/j.jacbts.2021.09.004

Figure 1.

Figure 1

WNK Inhibition Combats RV Glucotoxicity in MCT Rats

(A) Representative Western blots and quantification of protein abundance in right ventricular (RV) extracts from control, monocrotaline-vehicle (MCT-V), and WNK463 rats demonstrate WNK463 trends toward normalizing expression of WNK1 (control rats: 1.0 ± 0.4; MCT-V: 1.5 ± 0.4; WNK463: 1.1 ± 0.5 expression relative to control rats; n = 5 RVs per group for all proteins evaluated), glucose transporter 1 (GLUT1) (control rats: 1.0 ± 0.3; MCT-V: 1.4 ± 0.5; WNK463: 0.9 ± 0.4), GLUT4 (control rats: 1.0 ± 0.2; MCT-V: 1.5 ± 0.3; WNK463: 0.9 ± 0.4), 160 kDa substrate of the Akt serine/threonine kinase (AS160) (control rats: 1.0 ± 0.2; MCT-V: 0.6 ± 0.2; WNK463: 0.7 ± 0.2), phosphorylated AS160 (control rats: 1.0 ± 0.6; MCT-V: 1.9 ± 0.4; WNK463: 1.4 ± 0.5), and phosphorylated AS160 normalized to AS160 (control rats: 1.0 ± 0.7; MCT-V: 2.8 ± 0.5; WNK463: 1.9 ± 1.0). Western blot results were normalized to the myosin heavy chain band in the Coomassie brilliant blue (CBB) stained post-transfer gel. Values are expression relative to control rats. Representative immunofluorescence images from RV free wall sections show WNK463 significantly reduced the amount of (B) cytoplasmic WNK1 expression (red arrows) (wheat germ agglutinin staining in white, WNK1 staining in green) and (C) GLUT1 receptors (red arrows) and (D) GLUT4 receptors (red arrows) at the cell membrane. Scale bar 10 μm in B and 20 μm in C and D. n = 3 RVs per group and 7-10 areas assessed per RV in B to D. (E) Hexosamine biosynthetic pathway, glycolysis, and pentose phosphate pathway intermediates are outlined. RV metabolomics studies demonstrate WNK463 partially restores the levels of (F) hexosamine biosynthesis pathway, (G) glycolysis, and (H) pentose phosphate pathway intermediates as depicted by hierarchical cluster analysis. One-way analysis of variance (ANOVA) with Dunnett post hoc analysis was completed in (A). ∗∗∗∗P < 0.0001, and NS by Brown-Forsythe and Welch ANOVA with Dunnett multiple comparison test in B and C and 1-way ANOVA with Tukey post hoc test in D.