Fig 2: FGFR4 regulates metabolic transcription and hypertrophy in bio-engineered cardio bundles.Treatment of neonatal rat ventricular myocyte cardio-bundles with FGF23 for 20 minutes significantly increased contractile force, while 7 days of chronic treatment led to a significant reduction in contractile force that could be rescued by co-application of BLU9931, a selective FGFR4 inhibitor (A). Electrophysiological function was evaluated by pacing of cardio-bundles and application of Di-4-ANEPPS as voltage sensitive dye. Chronic exposure of cardio-bundles to FGF23 lead to significantly longer action potential durations (B). FGF23-treated bundles exhibited significantly lower conduction velocity that was normalized after co-application of BLU9931 (C). Beside functional changes, chronic FGF23 treatment also led to cardio-bundle hypertrophy indicated by the significant rise in cross-section (D,G) and increased expression of hypertrophic mRNA markers Rcan1 and Trpc6 (E). Increased expression of Rcan1 and Trpc6 was blocked by parallel treatment with BLU9931. Metabolic transcription factors that were increased in CKD mice, also increased in cardio-bundles after FGF23 treatment (F). Representative images of cardio-bundles indicate cellular hypertrophy after FGF23 treatment by increased myocyte cross-sections (G). Gene set enrichment analysis of control and FGF23 treated cardio-bundles showed an enrichment of metabolic pathways, particularly fatty acid metabolism, adipogenesis and cholesterol homeostasis (H). Additional enrichment was detected in pathways related to mitochondrial function, such as oxidative phosphorylation, respiratory chain, organelle fission and organelle inner membrane (I). Downregulated pathways after FGF23 treatment include angiogenesis, vascular development TNFα signaling and P53.Bar graphs represent mean with SEM and individual values included in the graph. * indicate p<0.05. Scale bars in G are 10 μm.

Fig 3: FGFR4 mediates metabolic remodeling in cultured cardiomyocytesCultured neonatal rat ventricular myocytes (NRVM) esponded to 48h of FGF23 treatment with significant hypertrophy indicated by increased cross-sectional area and expression of pro-hypertrophic markers (A and B). Pro-hypertrophic mRNA expression and cellular hypertrophy could be mitigated by parallel treatment with the FGFR4-specific inhibitor BLU9931. Cardiac mitochondria isolated from NRVM treated with FGF23 for 1h, before observable hypertrophy takes place, were analyzed in a Seahorse XF analyzer for extracellular acidification rate (ECAR), elevated total proton efflux rates (PER) and glycolysis specific proton efflux rates (glycoPER) (C). ECAR was significantly higher in mitochondria from FGF23-treated cells, which could be reduced to control levels by BLU9931. PER showed elevated basal and compensatory glycolysis in mitochondria with FGF23 treatment; glycolysis-specific proton efflux was also increased. These FGF23 mediated effects were blocked by BLU9931 application. Seahorse mitochondrial stress test assay showed increased basal and maximal mitochondrial respiration after FGF23 treatment of NRVM (D). ATP production-linked, spare respiratory capacity and non-mitochondrial oxygen consumption rate increased in parallel after FGF23 treatment. The significant decrease in coupling efficiency and the increased proton leak indicate uncoupling of substrate oxidation and ATP synthesis after 1h of FGF23 treatment. Application of BLU9931 or the calcineurin inhibitor, cyclosporin A, prevented the changes to mitochondrial function caused by FGF23.Bar graphs represent mean with SEM and individual values included in the graph. * indicate p<0.05. Graphs in C represent 3 independent experiments.

Fig 4: Global deletion of FGFR4 prevents LHV and changes to cardiac mitoproteome in CKD.Mice with global deletion of FGFR4 develop CKD to the same degree as control mice after 16 weeks of adenine diet as indicated by the rise in FGF23 (A). Wildtype animals developed LVH at 16 weeks with increased LV mass, wall thickness and ratio of heart weight/tibia length. These changes were absent in FGFR4−/− mice (B). Cardiac mitoproteome of wildtype and FGFR4−/− mice was evaluated after 12 weeks adenine feeding, before overt remodeling is observed. 22 proteins were significantly regulated in wildtype CKD mice, but were not changed in FGFR4−/− CKD mice, with 9 proteins downregulated and 13 upregulated (B). Analysis showed enrichment in pathways connected to mitochondrial respiration and function (C). Additionally, 163 proteins were identified that were only regulated in FGFR4−/− CKD mice with 83 downregulated and 57 upregulated proteins (D). Enrichment analysis showed a partial normalization of mitochondrial proteins in FGFR4−/− mice (E).Bar graphs represent mean with SEM and individual values included in the graph. * indicate p<0.05.