Fig 2: Cardiac-myocyte specific Fgf23 knockout mice do not display changes in cardiac function and geometry. (A) Body length and heart size are not different between three months old Fgf23fl/fl/cre+ and Fgf23fl/fl/cre− control mice. (B) Fgf23fl/fl/cre+ mice have an increased mortality rate within six months of age. *p < 0.05 analyzed using Log-rank test; n = 54–79 mice per genotype. (C) Representative parasternal long-axis M-mode echocardiography images of 1, 3 and 6 months old Fgf23fl/fl/cre− and Fgf23fl/fl/cre+ mice. (D–H) Quantification of end-systolic and end-diastolic volume, end-systolic and end-diastolic pressure, and stroke work using echocardiography and Millar catheter, respectively, in six months old Fgf23fl/fl/cre− and Fgf23fl/fl/cre+ mice. Data is given as scatter dot plots with means; *p < 0.05 analyzed using unpaired t-tests according to Shapiro-Wilk normality test; n = 7–8 mice per group.

Fig 3: Serum of AAV-Fgf23 mice induces cardiac hypertrophy of NRVM. (A) Representative immunofluorescence images of isolated neonatal rat ventricular myocytes (NRVM) stimulated with 2% serum from AAV-Fgf23 (sAAV-Fgf23) or Ctrl (sCtrl) mice for 48 h. Myocytes are labeled with anti-α-actinin (red) and nuclei are counterstained with DAPI (blue) (original magnification ×20; scale bar 50 μm). (B) Quantification of the cross-sectional area of NRVM reveals hypertrophic cell growth due to treatment with sAAV-Fgf23 compared to sCtrl (mean of 100 cells per condition and isolation). (C–E) Quantitative real-time PCR analysis shows increased expression of ANP, BNP, and Rcan1 in sAAV-Fgf23-treated NRVM compared to sCtrl. Data are given as scatter dot plots with means; *p < 0.05, **p < 0.01, ***p < 0.001 analyzed using unpaired t-test or Mann–Whitney test according to Shapiro–Wilk normality test; n = 6 independent isolations of NRVM.

Fig 4: Dose- and time-dependent evaluation of AAV-Fgf23. (A) Relative cardiac Fgf23 mRNA expression is dose-dependently increased in AAV-Fgf23 mice compared to Ctrl, irrespective of the duration of exposure. (B) ELISA-based quantification of cardiac Fgf23 protein in heart tissue lysates shows increased intact Fgf23 (iFgf23) protein only in AAV-Fgf23 mice administered with 1012 vector genomic particles (vg) compared to Ctrl. (C) Representative immunoblotting of Fgf23 in cardiac tissue demonstrates increased iFgf23 protein in the 1012 vg AAV-Fgf23 group after 4 weeks compared to Ctrl. (D) Semi-quantitative PCR targeting AAV vector shows excessive virus transduction after 4 weeks in the heart and the liver of 1012 vg AAV-Fgf23 mice (A) compared to Ctrl (C). (E) Hepatic Fgf23 mRNA expression is significantly increased after 4 weeks in 1012 vg AAV-Fgf23 mice compared to Ctrl. (F) By injecting 5 × 1011 vg AAV-Fgf23, semi-quantitative PCR targeting AAV vector shows distinct virus transduction in the heart compared to Ctrl, whereas transduction in liver tissue was very low and absent in kidney, lung, spleen, brain, and bone tissue. (G) Immunofluorescent staining of cardiac mid-chamber sections demonstrates uniform Fgf23 (green) overexpression in almost all cardiac myocytes of the left ventricle in AAV-Fgf23 mice compared to Ctrl (original magnification ×20; scale bar, 500 and 250 μm, respectively). Data are given as scatter dot plots with means; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 analyzed using one-way ANOVA or Kruskal–Wallis test followed by Sidak’s or Dunn’s multiple comparisons post hoc tests, respectively, according to Shapiro–Wilk normality test; n = 3–7 mice per group.

Fig 5: Soluble klotho suppresses cardiac FGF23-induced cardiac hypertrophy in vitro. (A) Stimulation with oncostatin M (OSM) increases endogenous Fgf23 mRNA expression in isolated NRVM irrespective of cotreatment with soluble klotho (sKL). (B) ELISA-based quantification of intact Fgf23 (iFgf23) in conditioned medium of NRVM reveals enhanced iFgf23 release due to OSM treatment, while sKL costimulation does not alter OSM-mediated high iFgf23 secretion. (C,D) Immunofluorescence images of isolated NRVM and quantification of cross-sectional cell area observe hypertrophic growth of OSM stimulated NRVM. Phenylephrine (PE) serves as the positive control for cardiac hypertrophy. Cotreatment with sKL protects from cardiac hypertrophy induced by OSM but not by PE. Myocytes are labeled with anti-α-actinin (green) and nuclei are counterstained with DAPI (blue) (original magnification ×20; scale bar 100 μm). (E) Quantitative real-time PCR analysis shows increased expression of BNP in OSM and PE-treated NRVM compared to Ctrl that is only blocked by sKL in the OSM group. Data are given as scatter dot plots with means; ∗p < 0.05, ∗∗p < 0.01, and *⁣*⁣**p < 0.0001 analyzed using one-way ANOVA or Kruskal–Wallis test followed by Sidak’s or Dunn’s multiple comparisons post hoc tests, respectively, according to Shapiro–Wilk normality test; n = 6–9 independent isolations of NRVM; conditioned medium was used from n = 3–7 independent NRVM isolations.