Download Is fibroblast growth factor 23 a harbinger of mortality in CKD

Fibroblast Growth Factor 23 Directly Increases Intracellular Calcium in Adult Cardiomyocytes
Brandon Carney, Michael Wacker, Chad Touchberry
UMKC School of Medicine
Introduction
Results
FGF23 Increases Cardiac Contractility
FGF23 Stimulates Extracellular Ca2+ Entry
• Fibroblast growth factor 23 (FGF23) is an endocrine factor released primarily from
osteocytes to regulate serum phosphate.
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• 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 Ca2+ 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,
cardiomyocytes to FGF23 will increase intracellular Ca2+ levels.
primary
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, concentrationdependent 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.
Figure 4: FGF23 increases intracellular Ca2+ in a receptor dependent manner that is dependent on extracellular
Ca2+. Panel A shows acute changes in fluorescence mediated by FGF23 and a significant decline in fluorescence
during FGFR1 antagonism with PD-166866 (50nM). Panel B shows the average changes in fluorescence mediated
by FGF23 treatment in the absence of extracellular Ca2+ (0 mM, +0 Ca2+) and following pretreatment with L-type
Ca2+ channel blocker verapamil. Measurements are indicated as a change in fluorescence after treatment divided by
the initial fluorescence (F/FO). 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 Induces Hypertrophy in Cardiomyocytes
Conclusions and Significance
•
Materials and Methods
• Ca2+ free and verapamil experiments suggest that the increase in intracellular Ca2+
induced by FGF23 is mediated by L-type calcium channels.
• 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).
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• Isolated primary cardiomyocytes were loaded with Fluo-4 (1µM) and washed in
Hanks’ balanced salt solution (HBSS).
• During acute exposure to FGF23, the increase in Ca2+ and subsequent cardiac
contractility may be the body’s attempt to increase renal clearance of phosphate in
conditions like CKD.
Figure 2: Fibroblast growth factor 23 (FGF23) increases cell size in a dose-dependent manner. Panel A. Representative forwardscatter 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).
FGF23 Increases Intracellular Ca2+ in Ventricular Cardiomyocytes
• During Ca2+ 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.
• Statistical significance was set at p<0.05.
• 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.
Acknowledgements
This project was supported by: Sarah Morrison Research Grant to BC, NIH Grand
Opportunities Grant (1RC2AR058962-01) to MW, Missouri Research Board to MW,
UMKC Center for Mineralized Tissue Research to MW, American Heart Association
Grant (11SDG5330016) to MW, and UMKC School of Medicine.
• 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.
FGF23-induced changes in intracellular Ca2+ are receptor mediated, as indicated
by reduced intracellular Ca2+ levels with FGFR1 antagonist PD 166866.
Figure 3: Isolated adult primary cardiomyocytes from CD1 mice were loaded with the green-fluorescent Ca2+ indicator Fluo-4
AM. Following treatment with FGF23 (18,000 pg/ml), there was a large increase in intracellular Ca2+ (warmer colors = more Ca2+)
in the presence of extracellular calcium. This effect was not seen in vehicle-treated cardiomyocytes.
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 2014.
2) Stubbs, JR., Egwuonwu, S. Is fibroblast growth factor 23 a harbinger of mortality in CKD. Pediatr Nephrol, 2011