FGF21-FGFR1 signaling protects against cardiac hypertrophy by regulating PINK1-mediated mitophagy pathway

Fig. 1. Deletion of FGF21 promotes the onset of cardiac hypertrophy and eventually causes heart failure. A. Schematic outline of animal experimental procedures: ten weeks WT and Fgf21-/- mice were subjected to sham or TAC operation for four weeks. B. Representative M−mode echocardiography from each group in mice. C. Measurement of LVEF in the indicated groups. D. Measurement of LVFS in the indicated groups. E. Gross hearts, scale bar = 2 mm and representative image of HE staining, scale bar = 2 mm (n = 7), representative image of WGA immunofluorescent staining (n = 5), scale bar = 20 µm, representative images of Masson's Trichrome staining (n = 7, 9), scale bar = 100 µm. F. Ratio of HW to BW. G. Quantitative analysis of cardiomyocyte cross-sectional area for each group. H. Quantitative analysis of interstitial fibrosis for each group. I, J. Relative ANP and BNP mRNA levels. K. ANP and BNP protein levels in the indicated groups (n = 4). All data represent mean ± SEM, and statistical significance was measured using the two-way ANOVA with the Bonferroni post-hoc test.

Fig. 2. Deletion of FGF21 inhibits mitophagy and impairs mitochondrial function in mice. A. Representative images from TEM (n = 5–7), scale bar = 1 µm. B.Quantitative analysis of mitochondrial area for each group. C. Relative TMRM fluorescence in the indicated groups. D. ATP level in the indicated groups. E, F. Complex I, II, III, IV, and V protein levels in the indicated groups. Quantitative analysis was measured using ImageJ software. G, H. PGC-1α, Mfn2, Drp1, P62, LC3, PINK1, PARKIN, Tom20 protein levels in indicated groups. Quantitative analysis was measured using ImageJ software. All data represent mean ± SEM, and statistical significance was measured using the two-way ANOVA with the Bonferroni post-hoc test.

Fig. 3. Knockdown of FGF21 promotes cardiomyocyte hypertrophy and impairment of mitophagy and decreases PINK1 level in PE-induced NRCMs. Neonatal rat cardiomyocytes were infected with si-NC or si-FGF21 and then treated with control or 100 μM PE for 24 h. A. Representative images of F-actin immunofluorescent. B. Quantitative analysis of cardiomyocyte cross area for each group, scale bar = 20 µm. C. Relative ANP mRNA level. D. Relative BNP mRNA level. E. ATP level in the indicated group. F. Fluorescence intensity of MitoSOX. G. si-NC or si-FGF21 were infected with adenovirus encoding LC3 for 24 h, then subjected to PE stimulation for 24 h. These cells were stained using MitoTracker Red dye, scale bar = 20 µm. H. LC3 and PINK1 protein levels. I. PINK1 protein levels in lysates of cytosolic and mitochondrial fractions. All data represent mean ± SEM, and statistical significance was measured using the two-way ANOVA with the Bonferroni post-test.

Fig. 4. Absence of PINK1 blocks FGF21-promoted mitophagy in PE-treated NRCMs. Neonatal rat cardiomyocytes were infected with si-NC or si-PINK1, and then treated with control or 100 μM PE for 24 h, followed by vehicle or FGF21 protein. A. Relative PINK1 mRNA level. B. LC3 and PINK1, FGF21 protein levels. C. PINK1 protein level in lysates of cytosolic and mitochondrial fractions. D. si-NC and si-PINK1 NRCMs were infected with adenovirus encoding LC3 for 24 h, then subjected to 100 µM PE stimulation for 24 h, followed by vehicle or FGF21 protein. These cells were stained using MitoTracker Red dye, scale bar = 20 µm. E. Mitochondria and lysosomes were stained with MitoTracker green dye and LysoTracker Deep Red dye, scale bar = 20 µm. F. Relative ANP mRNA level. G. Relative BNP mRNA level. All data represent mean ± SEM, and statistical significance was measured using the two-way ANOVA with the Bonferroni post-test.

Fig. 5. The protective effects of FGF21 against TAC-induced cardiac hypertrophy are abolished in the absence of PINK1 in mice. Ten-week-old WT and Pink1-/- mice underwent sham or TAC surgery. One week after the TAC, some TAC groups were subcutaneously injected with saline, and other TAC groups were subcutaneously injected with Fc-FGF21 fusion protein (Efruxifermin). The injection is once a week for four weeks. A. Representative M−mode echocardiography of the left ventricle of WT or Pink1-/- mice in the indicated groups. B. Measurement of LVEF in the indicated groups. C. Measurement of LVFS in the indicated groups. D. Ratio of HW to BW. E. Quantitative analysis of cardiomyocyte cross-sectional area for each group (n = 3, 4). F. Quantitative analysis of interstitial fibrosis for each group. G. Representative image of HE staining, scale bar = 2 mm, and WGA immunofluorescent staining, scale bar = 20 µm, and Masson's Trichrome staining, scale bar 100 µm (n = 4). H, I. ANP and BNP mRNA levels in the indicated group. J. ANP and BNP protein levels in the indicated group. All data represent mean ± SEM, and statistical significance was measured using the two-way ANOVA with the Bonferroni post-hoc…

Fig. 6. The activation of PINK1-mediated mitophagy prevents Fgf21-/- mice from TAC-induced heart failure. Ten-week-old Fgf21-/- mice underwent sham or TAC surgery. Three weeks after the TAC, the sham and TAC groups were intraperitoneally injected with solvent, and the other TAC group was intraperitoneally injected with rapamycin for two weeks. A. PINK1 and PARKIN, LC3 protein levels in lysates of mitochondrial fractions. B. Representative images from TEM (n = 3,7), scale bar = 1 µm. C. Quantitative analysis of the number of autophagosomes containing mitochondria for each group. D. Representative M−mode echocardiography of the left ventricle of Fgf21-/- mice after sham or TAC surgery or TAC surgery treated with rapamycin. E. Measurement of LVEF in the indicated groups. F. Measurement of LVFS in the indicated groups. G. Gross hearts, scale bar = 2 mm and representative image of HE staining, scale bar = 2 mm (n = 5, 6), representative image of WGA immunofluorescent staining (n = 5, 9), scale bar = 20 µm and representative images of Masson's Trichrome staining (n = 4, 5), scale bar = 100 µm. H. Ratio of HW to BW. I. Quantitative analysis of cardiomyocyte cross-sectional area for each group.…

Fig. 7. FGFR1 is necessary for FGF21-mediated mitophagy. A. FGFR1, FGFR2, FGFR3, and FGFR4 mRNA levels in the heart of WT mice. B. Neonatal rat cardiomyocytes were infected with si-NC or si-FGFR1. Relative FGFR1 mRNA level. C. Neonatal rat cardiomyocytes were infected with si-NC or si-FGFR1, and then treated with 100 μM PE for 24 h, followed by vehicle or FGF21 protein, LC3, PINK1, and Tom20 protein levels. D. si-NC and si-FGFR1 NRCMs were infected with adenovirus encoding LC3 for 24 h, then subjected to 100 μM PE stimulation for 24 h, followed by vehicle or FGF21 protein. These cells were stained using MitoTracker Red dye, scale bar = 20 µm. E. Mitochondria and lysosomes were stained with MitoTracker green dye and LysoTracker Deep Red dye, scale bar = 20 µm. F. Relative FGF21 mRNA level after TAC at different times. G. Relative FGFR1 mRNA level after TAC at different times. All data represent mean ± SEM, and statistical significance was measured using the One-way ANOVA followed by Dunnett's post-hoc test and the two-way ANOVA with the Bonferroni post-test.