Fig 1: FGF21 activates ERK1/2 in cultured cardiac myocytes in the presence of high glucose and in heart tissue of mice. (a–c) Length, width and width to length ratio for ARVMs treated with BSA (control), FGF21 (25 ng/ml), increased glucose and/or the MEK inhibitor PD98059 (20 µM) for 48 h. Bars and colored symbols indicate average mean and means of independent experiments using different myocyte preparations, respectively. (d–f) Western blot analysis of ARVMs treated with BSA (control) or mouse recombinant FGF21 (25 ng/ml) with or without 10 mM glucose for 6 h. ERK1 is p44 and ERK2 is p42. (g) Analysis of cardiac tissue from FGF21 Tg mice and wild-type littermates at 8–12 weeks of age by Western blotting. (h, i) qRT-PCR for Egr-1 and c-Fos mRNA using total RNA from heart tissue of FGF21 Tg mice and wild-type littermates at 8–12 weeks of age. (j) Binding of 1 µg of soluble β-klotho (βKL) or PBS, 500 ng of Fc-tagged FGFR 1c, 2c, 3c, or 4 to 96-well plates coated with 200 ng of FGF21. (k) qRT-PCR for FGFR4 mRNA using total RNA isolated from ARVMs treated with BSA (control), FGF21 (25 ng/ml), and/or increased glucose (15.6 mM total). Comparison between groups was performed in form of a one-way (a–c, k) or two-way (e–f) ANOVA followed by post-hoc Tukey test or a two tailed t-test (h, i). All values are expressed as mean ± SEM. (a–c) N = 3, ^p ≤ 0.05 vs. BSA CTR, *p ≤ 0.05 vs. Glucose + FGF21 CTR; (e, f) N = 5, ^p ≤ 0.05 vs. BSA CTR, #p ≤ 0.05 vs. FGF21 CTR; (h, i) N = 9−19, *p ≤ 0.05 vs. WT; (k) N = 4, *p ≤ 0.05 vs. CTR. All Western blots are cropped, and original blots are presented in Supplementary Fig. 3.
Fig 2: FGFR4 blockade protects diabetic mice from pathologic cardiac remodeling. db/db mice and wild-type (WT) littermates were treated for 24 weeks with either FGFR4 blocking antibody (25 mg/kg) or vehicle (PBS) on a bi-weekly basis, starting at 4 weeks of age. (a) Body weight, (b) blood glucose levels, and (c) serum FGF21 levels. (d) left ventricular (LV) posterior wall thickness in diastole, and (e) LV mass from 4 weeks until 28 weeks of age, as determined by serial echocardiography. (f) Gravimetric heart weight/tibia length ratio, (g) wheat germ agglutinin-stained LV tissue section (scale bar = 25 µm), and (h) cardiac myocyte cross-section area. Comparison between groups was performed in form of a one-way ANOVA (a–c, f, h) or a 2-way ANOVA (d, e) followed by a post-hoc Tukey test. All values are expressed as mean ± SEM. (a, c, f) N = 5–12; (b) N = 6–13; (h) N = 5–9, *p ≤ 0.05 vs. WT, #p ≤ 0.05 vs. db/db. (d, e) N = 6–13, *p ≤ 0.05 vs. WT of same age, #p ≤ 0.05 vs. db/db of same age. For the complete set of echocardiography parameters see Supplementary Table 3.
Fig 3: FGF21 induces hypertrophic growth of cultured cardiac myocytes in the presence of high glucose via FGFR4. (a) Transmitted light images of primary adult rat ventricular myocytes (ARVMs) treated with BSA (control), and mouse recombinant FGF21 (25 ng/ml) with or without 10 mM increased glucose (final 5.6 or 15.6 mM) for 48 h (scale bar = 25 µm), and (b–d) myocyte length, width, and width to length ratio. FGFR4-specific blocking antibody (anti-FGFR4; 10 mg/ml) was included as indicated. (e) Images of ARVMs treated with BSA (control), and FGF21 (25 ng/ml) with or without phenylephrine (PE; 20 µM) or isoproterenol (Iso; 10 µM) for 48 h (scale bar = 25 µm), and (f–h) myocyte length, width, and width to length ratio. Comparison between groups was performed in form of a one-way (b–d, f–h) ANOVA followed by post-hoc Tukey test. All values are expressed as mean ± SEM. (b–d) N = 5, ^p ≤ 0.05 vs. BSA Control, #p ≤ 0.05 vs. FGF21 Control, *p ≤ 0.05 vs. Glucose Control. (f–h) N = 4, *p ≤ 0.05 vs. respective Control.
Fig 4: AlphaScreen binding assay data for 1 and 15 toward FGFR1c/KLB (A), FGFR3c/KLB (B), and FGFR4/KLB (C). Data are mean ± SEM (n = 3).
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