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Fibroblast growth factor 23 (FGF23) is an endocrine factor released primarily from osteocytes to regulate serum phosphate. FGF23 levels are increased during.

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Presentation on theme: "Fibroblast growth factor 23 (FGF23) is an endocrine factor released primarily from osteocytes to regulate serum phosphate. FGF23 levels are increased during."— Presentation transcript:

1 Fibroblast growth factor 23 (FGF23) is an endocrine factor released primarily from osteocytes to regulate serum phosphate. FGF23 levels are increased during CKD, and strong clinical associations exist between elevated FGF23 and the development of pathological cardiac hypertrophy and reduced ejection fraction (2). Increases in intracellular Ca 2+ in cardiomyocytes are known to increase cardiac contractility and play a role in inducing cardiac hypertrophy (1) Therefore, we hypothesized that acute exposure of adult, primary cardiomyocytes to FGF23 will increase intracellular Ca 2+ levels. Fibroblast Growth Factor 23 Directly Increases Intracellular Calcium in Adult Cardiomyocytes Brandon Carney, Michael Wacker, Chad Touchberry UMKC School of Medicine Acknowledgements This project was supported by: Sarah Morrison Research Grant to BC, NIH Grand Opportunities Grant (1RC2AR ) to MW, Missouri Research Board to MW, UMKC Center for Mineralized Tissue Research to MW, American Heart Association Grant (11SDG ) to MW, and UMKC School of Medicine. Results Conclusions and Significance FGF23-induced changes in intracellular Ca 2+ are receptor mediated, as indicated by reduced intracellular Ca 2+ levels with FGFR1 antagonist PD Ca 2+ free and verapamil experiments suggest that the increase in intracellular Ca 2+ induced by FGF23 is mediated by L-type calcium channels. During acute exposure to FGF23, the increase in Ca 2+ and subsequent cardiac contractility may be the bodys attempt to increase renal clearance of phosphate in conditions like CKD. However, chronic exposure of cardiomyocytes to high levels of FGF23 may induce pathological cardiac hypertrophy, which can lead to reduced ejection fraction and ultimately lead to heart failure. These findings have implications for conditions associated with elevated FGF23 levels. This is especially critical in patients with CKD who may have 1000 times the normal FGF23 levels, and thus FGF23 may be a critical link between CKD and heart disease and serve as a new form of therapeutic intervention for patients with CKD. Figure 4: FGF23 increases intracellular Ca 2+ in a receptor dependent manner that is dependent on extracellular Ca 2+. Panel A shows acute changes in fluorescence mediated by FGF23 and a significant decline in fluorescence during FGFR1 antagonism with PD (50nM). Panel B shows the average changes in fluorescence mediated by FGF23 treatment in the absence of extracellular Ca 2+ (0 mM, +0 Ca 2+ ) and following pretreatment with L-type Ca 2+ channel blocker verapamil. Measurements are indicated as a change in fluorescence after treatment divided by the initial fluorescence (F/F O ). Images were captured with a 40x objective. * denotes statistical significance from vehicle, and denotes statistical significance from FGF23 treated cells (n=15-20 cells from 2-3 animals; P<0.05). FGF23 Stimulates Extracellular Ca 2+ Entry FGF23 Induces Hypertrophy in Cardiomyocytes FGF23 Increases Intracellular Ca 2+ in Ventricular Cardiomyocytes Materials and Methods Adult primary cardiomyocytes were isolated using a modified Langendorff apparatus and a digestion buffer consisting of collagenase II (18,000 units), papain (20 units), and DNase (2,000 units). Isolated primary cardiomyocytes were loaded with Fluo-4 (1µM) and washed in Hanks balanced salt solution (HBSS). During Ca 2+ imaging, FGF23 was administered at 18,000 pg/ml via a drug delivery system and responses were recorded for 20 minutes. KCl (80mM) was added to test cell viability. Vehicle solutions containing HBSS served as negative control. All graphs were made and statistical procedures performed using GraphPad Prism 5.0. Data are presented as means ± standard error. Statistical significance was set at p<0.05. Figure 3: Isolated adult primary cardiomyocytes from CD1 mice were loaded with the green-fluorescent Ca 2+ indicator Fluo-4 AM. Following treatment with FGF23 (18,000 pg/ml), there was a large increase in intracellular Ca 2+ (warmer colors = more Ca 2+ ) in the presence of extracellular calcium. This effect was not seen in vehicle-treated cardiomyocytes. Figure 2: Fibroblast growth factor 23 (FGF23) increases cell size in a dose-dependent manner. Panel A. Representative forward- scatter histograms (FSC-H) of HL-1 cardiomyocytes treated with vehicle or FGF23 (900 pg/ml) for 48 h. Panel B. Summary of forward-scatter (FSC-H) data on cardiomyocytes treated with increasing doses of FGF23 (9–900 pg/ml) using flow cyctometry [T(X) = 63, P < 0.01]. FSC-H analysis of 10,000 live gated cells (n = 5 experiments). Results from independent experiments were normalized to vehicle controls and averaged. *Statistical difference from vehicle; statistical difference from FGF23 treatment (9 pg/ml). Figure 1: Panel A. FGF23 acutely increases contractile force in ex vivo ventricular muscle strips. Above are tracings of left ventricular muscle contractions at baseline and following treatment with either FGF23 or vehicle. Panel B. Mean, concentration- dependent changes in isometric tension data induced by FGF23 vs vehicle normalized to baseline contractions (n = 6–12 experiments, P < 0.05). *Statistical difference from vehicle. FGF23 Increases Cardiac Contractility Introduction B A maxresdefault.jpg References: 1) Touchberry, Chad. FGF23 is a novel regulator of intracellular calcium and cardiac contractility in addition to cardiac hypertrophy. Muscle Biology Group, School of Medicine, University of Missouri-Kansas City, Kansas City, Missouri. 26 Feb ) Stubbs, JR., Egwuonwu, S. Is fibroblast growth factor 23 a harbinger of mortality in CKD. Pediatr Nephrol, 2011


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