FGF23 induces left ventricular hypertrophy

Abstract

Chronic kidney disease (CKD) is a public health epidemic that increases risk of death due to cardiovascular disease. Left ventricular hypertrophy (LVH) is an important mechanism of cardiovascular disease in individuals with CKD. Elevated levels of FGF23 have been linked to greater risks of LVH and mortality in patients with CKD, but whether these risks represent causal effects of FGF23 is unknown. Here, we report that elevated FGF23 levels are independently associated with LVH in a large, racially diverse CKD cohort. FGF23 caused pathological hypertrophy of isolated rat cardiomyocytes via FGF receptor-dependent activation of the calcineurin-NFAT signaling pathway, but this effect was independent of klotho, the coreceptor for FGF23 in the kidney and parathyroid glands. Intramyocardial or intravenous injection of FGF23 in wild-type mice resulted in LVH, and klotho-deficient mice demonstrated elevated FGF23 levels and LVH. In an established animal model of CKD, treatment with an FGF-receptor blocker attenuated LVH, although no change in blood pressure was observed. These results unveil a klotho-independent, causal role for FGF23 in the pathogenesis of LVH and suggest that chronically elevated FGF23 levels contribute directly to high rates of LVH and mortality in individuals with CKD.

Figure 1. Elevated circulating FGF23 levels are associated with LVH in patients with CKD.

(A) The distribution of FGF23 levels in baseline samples of 3,070 participants who enrolled in the CRIC study and underwent echocardiography 1 year later. The median FGF23 was 142 RU/ml. Fifty-eight participants with FGF23 of more than 1,000 RU/ml (range 1,054–14,319 RU/ml), who were included in the analysis, are not shown here. (B) Ascending quartiles of FGF23 were associated with significantly decreased ejection fraction (P for linear trend < 0.001), but the differences between groups were modest, and the mean (± SEM) ejection fraction for each quartile was normal (>50%). (C) Ascending quartiles of FGF23 were associated with significantly increased mean (± SEM) left ventricular mass index (P for linear trend < 0.001). (D) With increasing quartiles of FGF23, the prevalence of concentric (gray) and eccentric (green) LVH increased at the expense of normal left ventricular geometry (white) and left ventricular remodeling (blue) (P < 0.001). Numbers in the bars represent the percentages of prevalence for each condition.

Figure 2. FGF23 increases the surface area of isolated NRVMs and activates hypertrophic gene programs.

(A) Surface area of isolated NRVMs increases after FGF23 and FGF2 treatment, as revealed by immunocytochemical analysis using antibodies to α-actinin (red). DAPI (blue) identifies nuclei (original magnification, ×100; scale bar: 25 μm). (B) Compared with that of untreated control cells (white), 48 hours of treatment with FGF23 (blue) or FGF2 (red) significantly increases cell surface area (mean ± SEM). Fifty cells per group per isolation were analyzed by morphometry (n = 3 isolations of NRVMs; *P < 0.01, compared with untreated). (C) Compared with those of untreated control cells (white), FGF23 (blue) or FGF2 (red) significantly increase α-actinin protein levels normalized to GAPDH in isolated NRVMs, as determined by immunoblotting (mean ± SEM; n = 3 isolations of NRVMs; *P < 0.01). (D) Compared with that of untreated control cells (white), FGF23 (blue) or FGF2 (red) decrease expression of α-MHC and MCAD mRNA and increase β-MHC, ANP, and BNP) mRNA (mean ± SEM; n = 3 isolations of NRVMs quantified by RT-PCR normalized to Gapdh; *P < 0.01, compared with untreated).

Figure 3. FGF23 and FGF2 use different signaling pathways to induce hypertrophy of NRVMs.

(A) Klotho expression is detectable in mouse brain (Br) and kidney (K) by nested RT-PCR but not in heart (H), isolated cardiomyocytes (CM), or in the absence of template (blank [Bl]). (B) FGFR1–FGFR4 are detectable in liver (L), heart, and isolated cardiomyocytes by RT-PCR but not in the absence of template. (C) Surface area of isolated NRVMs after 48 hours of FGF treatment alone and in the presence of inhibitors. The lower edge of the bars represents the mean (± SEM) area, and their height represents the difference in area compared with that of cells that were treated with FGF alone (red). Inhibiting FGFR (gray) prevents any increase in area regardless of FGF concentration. ERK inhibition (orange shades) completely prevents FGF2-induced hypertrophy but only partially prevents FGF23-induced hypertrophy. PI3K/Akt inhibition (green shades) partially prevents FGF2-induced hypertrophy but has no effect on FGF23-treated cells. PLCγ/calcineurin inhibition (blue shades) prevents FGF23-induced hypertrophy but not FGF2-induced hypertrophy (150 cells per condition; *P < 0.01, compared with corresponding FGF concentration without inhibitor). (D) FGF2 stimulates a greater increase in ERK phosphorylation (p-ERK) and Egr-1 expression than FGF23 relative to total ERK (t-ERK). (E) FGF23 but not FGF2 increases NFAT activity in C2C12 myoblasts. Cyclosporine A (CsA) blocks the effect of FGF23 (values represent fold change ± SEM compared with vehicle; *P < 0.01, compared with vehicle). (F) Only FGF2 induces an increase in Akt phosphorylation (p-Akt) relative to total Akt (t-Akt).

Figure 4. Intramyocardial injection of FGF23 induces LVH in mice.

(A) Intramyocardial injection of FGF23 induces a significantly increased ratio of heart weight to tibial length by day 14 (*P < 0.01, compared with vehicle). (B) Intramyocardial injection of FGF23 induces significantly increased thickness of the left ventricular free wall that is detectable at day 7 and progresses by day 14. Hypertrophy is significantly more pronounced at the injection site in the free wall compared with the interventricular septum at 14 days (*P < 0.01, compared with vehicle at corresponding date and site; **P < 0.01, comparing free wall to septum). (C) Representative gross pathology section from the cardiac mid-chamber (MC; hematoxylin and eosin stain; original magnification, ×5; scale bar: 200 μm) and WGA-stained sections from the mid-chamber free wall (original magnification, ×63; scale bar: 50 μm) demonstrate FGF23-indcued LVH at 14 days, confirmed by M-mode echocardiography. (D) Intramyocardial injection of FGF23 induces significantly increased cross-sectional surface area of individual cardiomyocytes (*P < 0.01, compared with vehicle; **P < 0.01, comparing day 14 versus 7). (E) Echocardiography at baseline and at 1 and 2 weeks after injection of FGF23 or vehicle reveals no change in ejection fraction but significantly decreased left ventricular internal diameter in diastole and increased relative wall thickness by day 14 in the mice injected with FGF23, consistent with concentric LVH (*P < 0.01, compared with vehicle). All values are mean ± SEM; n = 3 mice per group for morphological analyses; n = 100 cells per group for WGA analysis.

Figure 5. Intravenous injection of FGF23 results in LVH in mice.

(A) Intravenous injection of FGF23 results in a significant increase in serum FGF23 levels (mean ± SEM; n = 11 mice per group; *P < 0.01, compared with vehicle). (B) Intravenous injection of FGF23 results in a significant increase in cardiac weight/tibial length (mean ± SEM; n= 11 mice per group; *P < 0.01, compared with vehicle). (C) Representative gross pathology (hematoxylin and eosin stain; original magnification, ×5; scale bar: 200 μm) and WGA-stained sections (original magnification, ×63; scale bar: 50 μm) from the mid-chamber of the left ventricle demonstrate LVH in mice that received intravenous injections of FGF23. (D) Mice that received intravenous injections of FGF23 manifest a significant increase in left ventricular wall thickness (mean ± SEM; n = 5 mice per group; *P < 0.01, compared with vehicle). (E) Mice that received intravenous injections of FGF23 manifest a significant increase in cross-sectional surface area of individual cardiomyocytes (mean ± SEM; n = 100 cells per group; *P < 0.01, compared with vehicle). (F) Intravenous injection of FGF23 results in a significant decrease in expression of α-MHC and MCAD mRNA and increased β-MHC, ANP, and BNP mRNA (mean ± SEM; n = 3 mice per group quantified by RT-PCR normalized to Gapdh; *P < 0.05, compared with vehicle).

Figure 6. Klotho-deficient and klotho heterozygous mice develop LVH.

(A) kl/kl mice demonstrate significant increases in serum levels of FGF23, phosphate, and 1,25-dihydroxyvitamin D compared with those of wild-type mice. Only FGF23 was significantly increased in kl/+ mice. (B) Representative gross pathology of sagittal and mid-chamber sections of the heart (hematoxylin and eosin stain; original magnification, ×5; scale bar: 200 μm) and WGA-stained sections from the left ventricular mid-chamber free wall (original magnification, ×63; scale bar: 50 μm) demonstrate LVH in kl/kl and kl/+ mice. (C) kl/kl and kl/+ mice manifest significant increases in left ventricular wall thickness. (D) kl/kl and kl/+ mice manifest significant increases in the ratio of heart weight to total body weight. (E) kl/kl and kl/+ mice manifest significant increases in left ventricular relative wall thickness. (F) kl/kl and kl/+ mice manifest significant increases in cross-sectional surface area of individual cardiomyocytes. (G) kl/kl and kl/+ mice demonstrate decreased levels of α-MHC and MCAD mRNA and increased β-MHC, ANP, and BNP mRNA. All values are mean ± SEM (n = 6 mice per group for all laboratory and morphological analyses; n = 3 mice per group for RT-PCR analyses; n = 100 cells per group for WGA analysis; *P < 0.01, compared with WT; **P < 0.01, compared with kl/+).

Figure 7. Pharmacological inhibition of FGFR attenuates LVH in an animal model of CKD.

(A) PD173074 attenuates the increases in left ventricular mass (by echocardiography) and cardiac weight/body weight that develop in 5/6 nephrectomized rats treated with vehicle (*P < 0.05, compared with sham; **P < 0.05, compared with 5/6 nephrectomy treated with vehicle). (B) Representative gross pathology sections (hematoxylin and eosin stain; original magnification, ×2.5; scale bar: 400 μm), M-mode echocardiography images, and WGA-stained sections (original magnification, ×63; scale bar: 50 μm) from the left ventricular mid-chamber at day 14 after 5/6 nephrectomy demonstrate that PD173074 attenuates LVH compared with vehicle. (C) PD173074 attenuates the effects of 5/6 nephrectomy to increase left ventricular anterior wall thickness and relative wall thickness (by gross pathology), to increase cross-sectional surface area of individual cardiomyocytes (by WGA staining), and to decrease ejection fraction and LV end diastolic volume (by echocardiogram; *P < 0.05, compared with sham; **P < 0.05, compared with 5/6 nephrectomy treated with vehicle). All values are mean ± SEM (n = 6 rats per group).

Figure 8. Schematic representation of FGF23 signaling in classic target cells and cardiomyocytes.

In the kidney and parathyroid glands, FGF23 signaling requires FGFR and the coreceptor klotho. FGF23-klotho binding to FGFR stimulates autophosphorylation of the receptor tyrosine kinase and induces signaling through 3 major pathways: Ras-MAPK, PI3K-Akt, and PLCγ-PKC. FGF23 regulates phosphorus balance by altering expression of genes involved in parathyroid, vitamin D, and phosphorus metabolism. In cardiomyocytes, FGF2 signaling requires FGFR and heparan sulfate proteoglycans (HSP) as coreceptor and signals primarily through the Ras-MAPK pathway. Binding of FGF23 to FGFR on cardiomyocytes stimulates autophosphorylation of the receptor tyrosine kinase independent of klotho, which is not expressed in cardiomyocytes, and signals primarily through the PLCγ-calcineurin pathway. Whether HSP acts as coreceptor remains to be determined.