Increased Circulating FGF23 Does Not Lead to Cardiac Hypertrophy in the Male Hyp Mouse Model of XLH

Abstract

Serum levels of fibroblast growth factor 23 (FGF23) markedly increase with renal impairment, with FGF23 levels correlating with the presence of left ventricular hypertrophy (LVH) and mortality in patients with chronic kidney disease (CKD). FGF23 activates calcineurin/nuclear factor of activated T cell (NFAT) signaling and induces hypertrophy in murine cardiomyocytes. X-linked hypophosphatemia (XLH) is characterized by high circulating levels of FGF23 but, in contrast to CKD, is associated with hypophosphatemia. The cardiac effects of high circulating levels of FGF23 in XLH are not well defined. Thus, studies were undertaken to define the cardiac phenotype in the mouse model of XLH (Hyp mice). Echocardiographic and histological analyses demonstrated that Hyp left ventricles (LVs) are smaller than those of wild-type mice. Messenger RNA expression of cardiac hypertrophy markers was not altered in the LV or right ventricle of Hyp mice. However, the Hyp LVs had increased expression of the NFAT target genes NFATc1 and RCAN1. To determine whether phosphate alone can induce markers of hypertrophy, differentiated C2C12 myocytes were treated with phosphate. Phosphate treatment increased expression of cardiac hypertrophy markers, supporting a primary role for phosphate in inducing LVH. Although previous studies showed that increased circulating FGF23 and phosphate levels are associated with LVH, our results demonstrated that in XLH, high circulating levels of FGF23 in the setting of hypophosphatemia do not induce cardiac hypertrophy.

Analyses of hearts in the Hyp mouse model of XLH demonstrated that high serum levels of FGF23 in the setting of hypophosphatemia do not lead to left ventricular hypertrophy.

X-linked hypophosphatemia (XLH) is characterized by a mutation in PHEX, leading to rickets and osteomalacia (1, 2). Affected individuals have increased circulating levels of fibroblast growth factor 23 (FGF23) (2–4), a key regulator of phosphate homeostasis that is made predominantly by osteocytes and osteoblasts in bone (4, 5). It binds to fibroblast growth factor receptors (FGFRs) 1 and 4 with α-Klotho as a cofactor to decrease brush border sodium phosphate transporters 2a and 2c in the renal proximal tubule, suppress vitamin D 1-α-hydroxylase, and stimulate vitamin D 24-hydroxylase. This results in an increase in urinary phosphate excretion, a decrease in 1,25-dihydroxyvitamin D, and hypophosphatemia (6–10).

Cardiac left ventricular hypertrophy (LVH) is characterized by a thickening and/or lengthening of the left ventricle (LV) walls and can lead to congestive heart failure, arrhythmias, myocardial ischemia, and death from acute hemodynamic collapse (11–14). Health conditions such as hypertension and chronic kidney disease (CKD) increase the risk of LVH and its associated cardiovascular comorbidities (15–19). In particular, CKD, which affects approximately 13% of people in the United States, leads to multiple mineral ion and hormone abnormalities and increased risk for all-cause mortality (15, 16, 20–22). Circulating levels of FGF23 markedly increase with worsening renal function in CKD, with studies demonstrating an association between FGF23 levels, mortality, and LVH in patients with CKD (23, 24). FGF23 has also induced LVH in mice as well as activated calcineurin/nuclear factor of activated T cell (NFAT) signaling and increased cell size in cultured cardiomyocytes, suggesting that cardiomyocyte-specific FGF23 actions contribute to the pathogenesis of LVH in CKD (24, 25).

It has not been established whether the high levels of FGF23 in XLH are associated with LVH. Therefore, the current studies were undertaken to determine whether mice with XLH (Hyp) exhibit cardiac hypertrophy and activation of signaling pathways and genes that regulate cardiac remodeling. Distinct from other mouse models with elevated FGF23 levels (24–26), mice with X-linked hypophosphatemia (Hyp mice) do not have high phosphate levels or CKD (1, 27). Therefore, these investigations also addressed the cardiac effects of FGF23 in the absence of hyperphosphatemia.

Materials and Methods

Animal studies

Murine studies were approved by the institutional animal care and use committee and were conducted according to accepted standards of humane animal care. All mice were in the C57BL/6J background. They were maintained in a virus- and parasite-free barrier facility, exposed to a 12-hour light/12-hour dark cycle, weaned on day 18 onto house chow (1% calcium, 0.6% phosphate; 3003219-249; PMI Nutrition International, LLC, St. Louis, MO), and housed up to five mice per cage. Wild-type (WT) and Hyp male mice were examined at 30 weeks of age.

Histology

Hearts dissected from mice were horizontally transected. The superior half of the heart was fixed in 10% formalin and processed for paraffin sectioning. For picro‒Sirius Red staining, sections were rehydrated, stained with hematoxylin, and then immersed in picro‒Sirius Red solution [1% Sirius Red (Sigma, St. Louis, MO) in 1.3% picric acid (Sigma)] for 1 hour. Sections were washed in acidified water and dehydrated in 100% alcohol. LVs and right ventricles (RVs) were isolated from the apex of each heart and frozen at −80°C for RNA analyses.

Noninvasive measurement of systolic blood pressure

Systolic blood pressure (SBP) was measured with a noninvasive blood pressure system (CODA; Kent Scientific, Torrington, CT) in awake WT and Hyp mice as previously described (28). Briefly, the mouse was initially placed in a restrainer (Kent Scientific) for 1 minute and then maintained in the restrainer for longer times to allow it to acclimate to the device, as judged by the absence of agitation. After a few days of these acclimation sessions, the mouse remained comfortable for prolonged periods, and SBP was measured and recorded.

Echocardiography

Transthoracic echocardiography was performed with a commercially available echocardiography system (Vivid 7; GE Medical, Milwaukee, WI) using a 13-MHz linear ultrasonography probe as previously described (29, 30). Anterior wall thickness, posterior wall thickness, LV end-diastolic diameter, LV end-systolic diameter, and fractional shortening were obtained from M-mode tracings at the level of the papillary muscles (30). LV ejection fraction was calculated from the two-dimensional parasternal long-axis view using the prolate ellipsoid method (31).

Cardiac RNA analyses

LVs or RVs were homogenized in Trizol (Thermo Fisher Scientific, Waltham, MA). Total RNA was precipitated using 100% ethanol and purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was reverse transcribed with PrimeScript^TM (Takara-Clontech, Mountain View, CA), and quantitative real-time polymerase chain reaction (PCR) was performed using the QuantiTect SYBR Green Real-Time PCR Kit (Qiagen) on the DNA Engine Opticon (MJ Research, Waltham, MA). Gene expression was normalized to that of WT for each sample, using the methods of Livak and Schmittgen (32).

Immunohistochemistry and antibody

Phospho–extracellular signal–regulated kinase (ERK) 1/2 (9101; Cell Signaling, Danvers, MA) immunohistochemistry was performed on paraffin sections as previously described (33, 34). The following antibody was used: p44/42 MAP Kinase (Research Resource Identifier: AB_331646; Cell Signaling).

Cell culture

Undifferentiated C2C12 myoblasts (35) were allowed to proliferate in growth medium (20% fetal bovine serum, 5% penicillin-streptomycin in Dulbecco’s modified Eagle medium; Gibco, Waltham, MA). Upon reaching 50% to 60% confluence, they were then maintained in differentiation medium (2% donor equine serum, 5% penicillin-streptomycin, and 1 μM of insulin in Dulbecco’s modified Eagle medium) for 72 hours. Differentiated C2C12 cells were serum starved overnight and treated with 7 mM of sodium sulfate (control) or sodium phosphate for 12 hours. RNA was isolated from treated cells, reverse transcribed, and subjected to real-time quantitative PCR analyses as described.

Statistical Analysis

All data shown are reported as mean ± standard deviation. One-way analysis of variance followed by Fisher least significant difference test was used to analyze significance between all groups. Significance was defined as P < 0.05.

Results

Hyp hearts did not exhibit hypertrophy

Previous studies demonstrated that FGF23 increased blood pressure by enhancing membrane abundance of the sodium chloride (Na⁺/Cl⁻) cotransporter and thus renal sodium reabsorption in the renal distal tubule (36). Although Hyp mice have elevated circulating levels of FGF23, tail cuff measurements did not reveal any alterations in SBP (Fig. 1A). Because FGF23 induces cardiomyocyte hypertrophy (24), transthoracic echocardiography and histological analyses were performed to evaluate whether Hyp hearts exhibit hypertrophic changes. Hyp mice had a normal heart rate as shown by M-mode echocardiograms (Fig. 1A). Transthoracic echocardiography analyses also demonstrated that the LVs of Hyp mice were smaller than those of WT mice, with a significant decrease in anterior and posterior wall thickness, LV mass, and end-diastolic diameter. However, no change in end-systolic diameter was observed (Fig. 1B). Because hypophosphatemia leads to growth retardation and rickets in Hyp mice (37), resulting in significantly decreased body weights (Fig. 1C), LV mass was normalized to body weight. These analyses confirmed the absence of LVH in Hyp mice (Fig. 1C). Ejection fraction and fractional shortening were decreased in Hyp mice (Fig. 1D).

Figure 1.

Hyp hearts were smaller than WT hearts. (A) Systolic blood pressure (BP) measured by noninvasive tail cuff and heart rate measured by echocardiography in 30-week-old mice. Data are representative of those obtained from 10 mice per genotype. (B) Measurements of cardiac parameters in WT and Hyp mice obtained by transthoracic echocardiography (anterior wall thickness, posterior wall thickness, LV mass, end-diastolic diameter, and end-systolic diameter). (C) Body weight (BW) and normalization of LV mass (LVM; quantitated from echocardiography analyses) to BW. (D) Ejection fraction and fractional shortening as measured by echocardiography. *P value <0.05. Data are represented as mean ± standard deviation.

On hematoxylin and eosin staining, neither the RV nor the LV of Hyp hearts appeared different from those of WT hearts (Fig. 2A). Consistent with the lack of hypertrophy, no increase in fibrosis was observed in the Hyp hearts on picro‒Sirius Red stain (Fig. 2B).

Figure 2.

Hyp mice did not have cardiac hypertrophy. (A) Hematoxylin and eosin (H&E) and (B) picro‒Sirius Red stain of cardiac sections of 30-week-old mice. Data are representative of those obtained from five mice per genotype.

Calcineurin signaling was increased in the LV of Hyp mice

FGF23 activates FGFR4 in cardiomyocytes to induce calcineurin/NFAT signaling, leading to induction of genes that regulate cardiac remodeling and myocyte hypertrophy (24, 25). Therefore, the messenger RNA expression of the NFAT target genes NFATc1 and RCAN1 (38, 39) was quantitated in the LVs and RVs of WT and Hyp hearts. Although RV gene expression was unchanged in Hyp mice, LV gene expression of NFATc1 and RCAN1 was significantly increased (Fig. 3A). Consistent with the lack of hypertrophy on echocardiographic and histological analyses, LV and RV expression levels of markers of cardiac hypertrophy, including atrial natriuretic peptide, brain natriuretic peptide, and β-myosin heavy chain, were similar in Hyp and WT mice (Fig. 3B) (40, 41).

Figure 3.

Hyp LVs had increased expression of calcineurin/NFAT targets. (A) Messenger RNA (mRNA) expression of NFATc1 and RCAN1 in WT and Hyp LVs and RVs. (B) mRNA expression of markers of cardiac hypertrophy (ANP, BNP, β-MHC) in WT and Hyp LVs and RVs. Gene expression is normalized to that of WT. Data are representative of those obtained from five mice per genotype. *P value <0.05. Data are represented as mean ± standard deviation. (C) Immunohistochemistry for p-ERK1/2 in WT and Hyp LVs. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; p-ERK1/2, phospho–extracellular signal–regulated kinase 1/2; β-MHC, β-myosin heavy chain.

FGFR signaling has been shown to activate the mitogen-activated protein kinase (MAPK) signaling cascade in cardiomyocytes, leading to phosphorylation of ERK1/2 and activation of genes that promote cardiac hypertrophy (24). Despite the increased circulating levels of FGF23 in Hyp mice, Hyp cardiomyocytes did not exhibit increased phospho-ERK1/2 immunoreactivity (Fig. 3C).

Phosphate increased expression of cardiac hypertrophy markers in differentiated myocytes

Previous murine studies demonstrating an association of high FGF23 levels with LV hypertrophy were performed in models that also had high serum phosphate levels (24–26). To address the hypothesis that phosphate alone can induce the molecular signature of cardiac hypertrophy, differentiated C2C12 myocytes were treated with 7 mM of sodium sulfate (control) or sodium phosphate. Phosphate treatment led to a significant induction in messenger RNA expression of markers of cardiac hypertrophy, including atrial natriuretic peptide, brain natriuretic peptide, and β-myosin heavy chain. However, expression of NFAT-signaling target genes was not enhanced. Rather, phosphate decreased NFATc1 expression in C2C12 myocytes and did not alter RCAN1 expression (Fig. 4). These results suggest that cooperative interactions between phosphate and FGF23 promote cardiac remodeling in the setting of CKD.

Figure 4.

Phosphate increased expression of cardiac hypertrophy markers in myocytes. C2C12 myocytes were differentiated for 72 hours and treated with 7 mM of sodium sulfate (S) or sodium phosphate (Pi) for 12 hours. Expression of cardiac hypertrophy markers (ANP, BNP, β-MHC) and targets of calcineurin/NFAT signaling (NFATc1, RCAN1) were quantitated. Data normalized for actin in each sample represent RNA obtained from three independent experiments. *P value <0.05. Data are represented as mean ± standard deviation. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, β-myosin heavy chain.

Discussion

The current studies demonstrate that despite increased circulating levels of FGF23, Hyp mice did not exhibit cardiac hypertrophy. These results are consistent with clinical studies reporting unaltered LV size on electrocardiographic and echocardiographic analyses in children and adults with XLH, regardless of previous treatment with phosphate and calcitriol (42, 43). When LV mass assessed by echocardiography was normalized to patient height, there was a suggestion that children with XLH may be at risk for developing LVH (44). However, these data may reflect the shorter height of patients with XLH or the presence of hyperparathyroidism in 11 of 13 study subjects (44). Alternatively, on the basis of our finding that phosphate induced a hypertrophic signature in C2C12 myocytes, phosphate treatment of the children with XLH we studied could be responsible for the increase in normalized LV mass observed (44).

Recent studies suggest that enhanced FGF23 action in Hyp mice increases distal renal tubule reabsorption of sodium, leading to hypertension and LVH, as quantified by total heart weight normalized to body weight (36, 45). The disparity between these findings and our data may reflect the disproportionately lower weight of Hyp mice relative to their cardiac weight and differences in the sodium and phosphate content of the mouse chow used. Consistent with our findings, studies in DMP1-knockout (KO) mice, characterized by hypophosphatemia in the setting of increased FGF23 level (46–48), revealed a decrease in cardiac weight without evidence of hypertrophy (49), supporting our conclusion that increased FGF23 in the setting of hypophosphatemia does not lead to cardiac hypertrophy.

Activation of FGFRs was associated with development of LVH in mice (24, 50–52). In vitro treatment of cardiomyocytes with FGF23 increased cell size and expression of genes that mediate cardiac hypertrophy, and hearts from mice injected with FGF23 for 2 weeks exhibited increased weight and LV wall thickness (24). FGF23 activated calcineurin/NFAT signaling to mediate these effects on the LV (24, 25). Consistent with FGF23 activation of calcineurin/NFAT signaling in cardiomyocytes, the LVs of Hyp mice had increased expression of NFATc1; however, there were no histological or molecular features of hypertrophy. The lack of LVH in Hyp mice in the presence of high serum levels of FGF23 suggests actions of FGF23 alone are not sufficient to induce LVH. Consistent with this hypothesis, blocking FGF23 action with an anti-FGF23 antibody did not prevent LV hypertrophy in rats with CKD induced by 5/6 nephrectomy (53).

In humans and rats with CKD (24), in mice fed a high-phosphate diet (25, 26), and in mice lacking Klotho, the FGF23 cofactor (24), hyperphosphatemia leads to a compensatory increase in circulating levels of FGF23, which in turn is associated with development of LVH. In contrast, the high circulating levels of FGF23 in mice and humans with XLH, which lead to hypophosphatemia (1, 54), and in DMP1-KO mice (49) do not increase LV size or fibrosis. The different cardiac phenotypes in these human and rodent models suggest that increased FGF23 in the presence of high serum phosphate level underlies the pathogenesis of the LVH observed in CKD. Studies in mice with CKD due to Alport syndrome (Col4a3 KO mice) further support our findings that increased FGF23 does not lead to hypertrophy in the absence of hyperphosphatemia. Although Col4a3 KO mice exhibit increased serum levels of FGF23 by 6 weeks of age, their LV size is not increased at 12 weeks old when they develop hyperphosphatemia (55). In conclusion, our in vivo studies in Hyp mice and in vitro analyses in C2C12 myocytes suggest that cooperative interactions between phosphate and FGF23 promoted cardiac remodeling in the setting of CKD.

Glossary

Abbreviations:

CKD

chronic kidney disease

ERK

extracellular signal–regulated kinase

FGF23

fibroblast growth factor 23

FGFR

fibroblast growth factor receptor

Hyp mice

mice with X-linked hypophosphatemia

KO

knockout

LV

left ventricle

LVH

left ventricular hypertrophy

MAPK

mitogen-activated protein kinase

NFAT

nuclear factor of activated T cell

PCR

polymerase chain reaction

RV

right ventricle

SBP

systolic blood pressure

WT

wild-type

XLH

X-linked hypophosphatemia