Fibroblast Growth Factor (FGF) 23 and FGF Receptor 4 promote cardiac metabolic remodeling in chronic kidney disease

Abstract

Chronic kidney disease (CKD) is a global health epidemic that significantly increases mortality due to cardiovascular disease. Left ventricular hypertrophy (LVH) is an important mechanism of cardiac injury in CKD. High serum levels of fibroblast growth factor (FGF) 23 in patients with CKD may contribute mechanistically to the pathogenesis of LVH by activating FGF receptor (FGFR) 4 signaling in cardiac myocytes. Mitochondrial dysfunction and cardiac metabolic remodeling are early features of cardiac injury that predate development of hypertrophy, but these mechanisms of disease have been insufficiently studied in models of CKD. Wild-type mice with CKD induced by adenine diet developed LVH that was preceded by morphological changes in mitochondrial structure and evidence of cardiac mitochondrial and metabolic dysfunction. In bioengineered cardio-bundles and neonatal rat ventricular myocytes grown in vitro, FGF23-mediated activation of FGFR4 caused a mitochondrial pathology, characterized by increased bioenergetic stress and increased glycolysis, that preceded the development of cellular hypertrophy. The cardiac metabolic changes and associated mitochondrial alterations in mice with CKD were prevented by global or cardiac-specific deletion of FGFR4. These findings indicate that metabolic remodeling and eventually mitochondrial dysfunction are early cardiac complications of CKD that precede structural remodeling of the heart. Mechanistically, FGF23-mediated activation of FGFR4 causes mitochondrial dysfunction, suggesting that early pharmacologic inhibition of FGFR4 might serve as novel therapeutic intervention to prevent development of LVH and heart failure in patients with CKD.

Keywords: CKD; FGF23; FGFR4; heart failure; metabolic remodeling; mitochondrial dysfunction.

Figure 1. Cardiac function, remodeling and changes to cardiac mitochondria in CKD.

Cardiac function of control and adenine fed mice was evaluated at the outset of the experiment at 8 weeks and at study termination at 16 weeks. Significant LVH was detectable at the final timepoint after 16 weeks CKD as indicated by left ventricular mass index (LVMI), posterior wall thickness (PW) and left ventricular end-systolic diameter (LVD;s) (A). After 12 weeks adenine diet, significant decrease in glomerular filtration rate (GFR) indicated kidney damage but no clear manifestation of structural LVH as indicated by the unchanged posterior wall thickness and overall wall thickness after 12 weeks on the adenine diet (B). Measurement of remodeling parameters Nppa, Timp1, Foxo1 and Pgc-1α indicate that hypertrophic and fibrotic remodeling had been initiated at 12 weeks CKD (C). Electron microscopy revealed clear changes in mitochondrial morphology with misalignment of mitochondria and changes in cristae appearance (D). Mitochondrial respiration after 12 weeks of adenine diet indicated that respiration through complex I and II was significantly increased before structural cardiac remodeling was detectable (E). Bar graphs represent mean with SEM and individual values included in the graph. * indicate p< 0.05. Scale bar in D is 500nm.

Figure 2. Changes to mitochondrial proteome and cardiac metabolome in CKD.

CKD leads to significant changes in the mitochondrial proteome of wildtype after 12 weeks of adenine diet, before structural remodeling becomes detectable (A,B). A total of 118 mitochondrial genes were significantly regulated in the mitoproteome of mice with CKD compared to controls (A). KEGG pathway analysis showed that, downregulated proteins represented fatty acid metabolism, and amino acid degradation (B, top). The up-regulated genes were enriched in ribosomal processes and translation (B, bottom). Analysis of metabolomics also showed significant changes in serum acylcarnitines, amino acids and keto acids (C, left). Among upregulated serum amino acids were phenylalanine and citrulline. These were also upregulated in cardiac tissue (C, right). Here, more amino acids were significantly downregulated, leucine and isoleucine showed a downward trend (p = 0.07). In contrast to serum, more cardiac acylcarnitines were significantly downregulated. For organic acids from cardiac tissue of CKD mice only pyruvate reached significance but lactate and citrate showed trends (p= 0.07).

Figure 3. 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.

Figure 4. FGFR4 mediates metabolic remodeling in cultured cardiomyocytes

Cultured 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.

Figure 5. Cardiac function, remodeling and metabolomics of FGFR4-Arg385 mice in the absence of CKD.

FGFR4-Arg385 mice did not have impaired kidney function as indicated by BUN values, but beginning LVH is detectable by increased wall thickness at 6 months of age. By 24 month of age, renal function remained unchanged and significant LVH/HFpEF was detected in FGFR4-Arg385 mice indicated by robust structural remodeling and increased fractional shortening (A). mRNA expression levels of remodeling, pro-fibrotic and pro-hypertrophic markers support initiation of cardiac remodeling at 6 months of age (B). Transmission electron microscopy showed similar changes in the mitochondria of six-month-old FGFR4-Arg385 mice as observed in mice with adenine induced CKD (C). Metabolomic analysis of FGFR4-Arg385 mice at 6 month of age showed significant increase in some serum acylcarnitines and reduction in cardiac MLACs (D). Similar to wildtype CKD animals, cardiac citrulline was upregulated while a number of other amino acids, including leucin and isoleucine, were downregulated (E). Reduction of serum keto acids was also in line with results obtained from the adenine CKD model. Organic acids also showed similar changes with a significant upregulation of pyruvate and a downregulation of lactate (F). Bar graphs represent mean with SEM and individual values included in the graph. * indicate p<0.05.

Figure 6. 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.

Figure 7. Cardiomyocyte expression of FGFR4 mediates metabolic remodeling in adenine-induced CKD.

After 16 weeks of adenine diet, control animals and α-MHCMerCreMer-FGFR4flox mice developed kidney damage to a similar degree with elevated FGF23. LV mass, wall thickness and ratio of heart weight/tibia length were significantly lower in α-MHCMerCreMer-FGFR4flox mice than control animals (A). Cardiac-specific deletion of FGFR4 also significantly reduced the cardiac expression of pro-hypertrophic and pro-fibrotic markers (B). Echocardiography showed no structural remodeling or abnormalizes in the hearts of α-MHCMerCreMer-FGFR4flox, whereas hearts of control animals showed significant wall thickening and remodeling (C). Analysis of the cardiac metabolome showed elevation of a greater number of MLAC when compared to control animals. Expression levels of organic acids indicate a normalization of glucose utilization in α-MHCMerCreMer-FGFR4flox mice and reduction in cardiac pyruvate and citrate concentrations (D). Amino acids were unchanged between groups. Bar graphs represent mean with SEM and individual values included in the graph. * indicate p<0.05.