Fibroblast growth factor 23 does not directly influence skeletal muscle cell proliferation and differentiation or ex vivo muscle contractility

Abstract

Skeletal muscle dysfunction accompanies the clinical disorders of chronic kidney disease (CKD) and hereditary hypophosphatemic rickets. In both disorders, fibroblast growth factor 23 (FGF23), a bone-derived hormone regulating phosphate and vitamin D metabolism, becomes chronically elevated. FGF23 has been shown to play a direct role in cardiac muscle dysfunction; however, it is unknown whether FGF23 signaling can also directly induce skeletal muscle dysfunction. We found expression of potential FGF23 receptors ( Fgfr1-4) and α-Klotho in muscles of two animal models (CD-1 and Cy/+ rat, a naturally occurring rat model of chronic kidney disease-mineral bone disorder) as well as C₂C₁₂ myoblasts and myotubes. C₂C₁₂ proliferation, myogenic gene expression, oxidative stress marker 8-OHdG, intracellular Ca²⁺ ([Ca²⁺]_i), and ex vivo contractility of extensor digitorum longus (EDL) or soleus muscles were assessed after treatment with various amounts of FGF23. FGF23 (2-100 ng/ml) did not alter C₂C₁₂ proliferation, expression of myogenic genes, or oxidative stress after 24- to 72-h treatment. Acute or prolonged FGF23 treatment up to 6 days did not alter C₂C₁₂ [Ca²⁺]_i handling, nor did acute treatment with FGF23 (9-100 ng/ml) affect EDL and soleus muscle contractility. In conclusion, although skeletal muscles express the receptors involved in FGF23-mediated signaling, in vitro FGF23 treatments failed to directly alter skeletal muscle development or function under the conditions tested. We hypothesize that other endogenous substances may be required to act in concert with FGF23 or apart from FGF23 to promote muscle dysfunction in hereditary hypophosphatemic rickets and CKD.

Keywords: chronic kidney disease; fibroblast growth factor 23; hypophosphatemic rickets; intracellular Ca2+; myogenesis.

Fig. 1.

Fibroblasti growth factor (Fgf) receptors (Fgfr), α-Klotho, and Fgf23 are expressed in skeletal muscle. A–C: are representative real-time RT-PCR amplification plots showing values of fluorescence at each cycle number of Fgfr1–4, α-Klotho, and Fgf23 (each reaction in triplicate) in isolated extensor digitorum longus (EDL; A) and soleus (B) muscles from 4- to 5-mo-old CD-1 male mice and in differentiated C₂C₁₂ myotubes (C). D: summary data showing average $2^{{}^{–\Delta C_T}}$ values (×10⁶) of Fgfr1–4, α-Klotho, and Fgf23 in EDL and soleus muscles from 4- to 5-mo-old CD-1 male mice (n = 3) and in C₂C₁₂ myoblasts and myotubes (n = 1) expressed relative to β-actin on log₁₀ scale.

Fig. 2.

Fgfr4 expression is elevated in the Cy/+ rat model for chronic kidney disease (CKD). Differences in gene expression of Fgfr1, r4, and α-Klotho in EDL muscle from Cy/+ rats or normal littermates (NL) were calculated using the $2^{{}^{–\Delta \Delta C_T}}$ method (n = 6). *P < 0.05 vs. NL, 1-way ANOVA with Bonferroni post hoc analysis.

Fig. 3.

FGF23 treatment has no effect on C₂C₁₂ proliferation. A and B: cellular proliferation of undifferentiated C₂C₁₂ myoblasts (A) and differentiated C₂C₁₂ myotubes (B) treated with FGF23 (100 ng/ml), soluble Klotho (1 µg/ml), FGF23 + Klotho, or FGF2 (100 ng/ml, positive control) for 24 and 48 h, as assessed by absorbance at 490 nm using CellTiter 96 AQueous One kit (n = 3; 3 separate experiments). C: MTT assay of undifferentiated C₂C₁₂ myoblast proliferation after treatment with FGF23 (2–50 ng/ml) or FGF2 (50 ng/ml) for 24, 48, and 72 h (n = 4; repeated twice). D: hemocytometer-based cell count of undifferentiated C₂C₁₂ myoblasts after treatment with FGF23 (2–50 ng/ml) or FGF2 (50 ng/ml) for 24, 48, and 72 h (n = 3; repeated twice). *P < 0.05, **P < 0.01, and ***P < 0.001, 1-way ANOVA with Bonferroni post hoc analysis.

Fig. 4.

FGF23 treatment does not alter expression of myogenic markers or oxidative stress. A–D: expression of myogenic gene markers Pax7 (A), Myod (B), Myogenin (C), and Myostatin (D) in differentiated C₂C₁₂ myotubes after treatment with FGF23 (100 ng/ml) or vehicle for 24 and 48 h. Gene expression was calculated using the $2^{{}^{–\Delta \Delta C_T}}$ method (n = 6). E: positive control data showing expression of Pax7, Myod, Myogenin, and Myostatin in differentiated C₂C₁₂ myotubes after treatment with FGF2 (100 ng/ml) or vehicle for 48 h (n = 3). *P < 0.05 and ***P < 0.001, 1-way ANOVA with Bonferroni post hoc analysis. F: oxidative stress was assessed by measuring the DNA oxidative damage marker 8-hydroxy-2′-deoxyguanosine (8-OHdG) in differentiated C₂C₁₂ myotubes after 24-h treatment with FGF23 (100 ng/ml) or vehicle (n = 6). P > 0.05, Student’s t-test.

Fig. 5.

Neither acute nor long-term FGF23 treatment has an effect on C₂C₁₂ myotube [Ca²⁺]_i. A: representative fluo-4 fluorescent images in C₂C₁₂ cells after vehicle, FGF23 (20 ng/ml), KCl (80 mM), or caffeine (20 mM) perfusion. Note the increase in fluorescence after KCl and caffeine treatment but not FGF23. B: representative fluo-4 fluorescence signal and perfusion protocol showing acute C₂C₁₂ myotube [Ca²⁺]_i responses to FGF23 (20 ng/ml) (left) and KCl (80 mM) and caffeine (20 mM) perfusion (right). Arrowheads indicate points of perfusion of specific treatments. The fluorescence signal is expressed relative to baseline (F/F_O). C: data summary of average peak change in fluo-4 fluorescence after perfusion with FGF23 (20 ng/ml) or vehicle expressed relative to baseline (F/F_O) (n = 3 dishes/group with 7–8 myotubes/dish averaged). P > 0.05, Student’s t-test. D: average change in fluo-4 fluorescence in response to perfusion with KCl (80 mM) and caffeine (20 mM) in myotubes incubated with FGF23 (20 ng/ml) for 24 h or 6 days (n = 3–6 dishes/group with 5–11 myotubes/dish averaged). Horizontal dashed line indicates the fluorescence level at baseline before stimulation of Ca²⁺ release. P > 0.05, 1-way ANOVA. E: Rhod-3 fluorescence in differentiated C₂C₁₂ myocytes after treatment with FGF23 (100 ng/ml) for 4 h (n = 6). P > 0.05, Student’s t-test.

Fig. 6.

Acute FGF23 administration does not alter CD-1 mouse EDL muscle contractile properties. A: representative ex vivo contractility force data obtained from 1 muscle (x-axis: time; y-axis: force) showing an entire contraction protocol and time of FGF23 addition. B: maximal tetanic force output (at 200-Hz stimulation) from CD-1 mouse EDL muscles after treatment with vehicle or FGF23 expressed relative to values before vehicle or FGF23 application. C: force-frequency relationship of vehicle- or FGF23-treated EDL muscles stimulated to contract with frequencies in the range of 1–220 Hz. Forces at each frequency are expressed relative to the maximal force obtained. D: time course of maximal tetanic force decline during a fatiguing protocol in vehicle- or FGF23-treated EDL muscles. Force at each time point is expressed relative to force just before fatigue. E: maximal tetanic force recovery during various time points postfatigue and with the addition of 5 mM caffeine in vehicle- or FGF23-treated EDL muscles. F: half-maximal tetanic force output (at 100-Hz stimulation) from CD-1 mouse EDL muscles after treatment with vehicle or FGF23 expressed relative to values before vehicle or FGF23 application. G: time course of half-maximal tetanic force decline during a fatiguing protocol in vehicle- or FGF23-treated EDL muscles. Force at each time point is expressed relative to force just before fatigue. H: half-maximal tetanic force recovery during various time points postfatigue and with the addition of 5 mM caffeine in vehicle- or FGF23-treated EDL muscles. I: maximal and half-maximal tetanic force after exposure of EDL muscles to 10 mM caffeine or 5 µM ryanodine, as positive controls, expressed as %force before vehicle or FGF23 treatment (however, error bars that are present in each group may be too small to be seen). Dashed lines in B and F represent points at which FGF23 was added to the muscle contractility bath; n = 3–10 muscles/group. **P < 0.01 and ***P < 0.001, 2-way ANOVA with Bonferroni post hoc analysis.

Fig. 7.

Acute FGF23 administration does not alter CD-1 mouse soleus muscle contractile properties. A: maximal tetanic force output (at 140–160 Hz stimulation) from CD-1 mouse soleus muscles after treatment with vehicle or FGF23 expressed relative to values before vehicle or FGF23 application. B: force-frequency relationship of vehicle- or FGF23-treated soleus muscles stimulated to contract with frequencies in the range of 1–220 Hz. Forces at each frequency are expressed relative to the maximal force obtained. C: time course of maximal tetanic force decline during a fatiguing protocol in vehicle- or FGF23-treated soleus muscles. Force at each time point is expressed relative to force just before fatigue. D: maximal tetanic force recovery during various time points postfatigue and with the addition of 5 mM caffeine in vehicle- or FGF23-treated soleus muscles. E: half-maximal tetanic force output (at 40 Hz stimulation) after treatment with vehicle or FGF23 expressed relative to values before vehicle or FGF23 application. F: time course of half-maximal tetanic force decline during a fatiguing protocol in vehicle- or FGF23-treated soleus muscles. Force at each time point is expressed relative to force just before fatigue. G: half-maximal tetanic force recovery during various time points postfatigue and with the addition of 5 mM caffeine in vehicle- or FGF23-treated soleus muscles. H: maximal and half-maximal tetanic force after exposure of soleus muscles to 10 mM caffeine or 5 µM ryanodine, as positive controls, expressed as a %force before vehicle or FGF23 treatment (however, error bars that are present in each group may be too small to be seen). Dashed lines in A and E represent points at which FGF23 was added to the muscle contractility bath; n = 5–10 muscles/group. ***P < 0.001, 2way ANOVA with Bonferroni post hoc analysis.

Fig. 8.

Acute FGF23 administration increases isolated heart contractility. A: representative force tracings of isolated mouse heart muscle paced at 1 Hz at baseline (before vehicle or FGF23 treatment) and after 30 min of exposure to vehicle or 9 ng/ml FGF23. B: summary data of whole heart contractile force output after 30 min of vehicle or 9 ng/ml FGF23 exposure. Data are expressed as fold change of contractile force relative to baseline; n = 4–6 hearts/group. *P < 0.05, 2-tailed t-test.