FGF23 promotes atrial fibrillation susceptibility through ETV1‐dependent sodium channel regulation and calcium handling abnormalities

Figures

• 

  FIGURE 1. Effect of FGF23 administration on atrial fibrillation susceptibility in mice. (a) Representative surface and intra‐oesophageal ECGs illustrating AF induction after atrial pacing in PBS and FGF23 groups. (b, c) Quantitative analysis of AF induction rate and AF duration in each group. n = 10. **P < 0.01. Abbreviation: AF, atrial fibrillation. Abbreviations: AF, atrial fibrillation; ECG, electrocardiogram; PBS, phosphate‐buffered saline; WT, wild‐type.

• 

  FIGURE 2. Effects of FGF23 on action potential properties and triggered activity in adult mouse atrial myocytes. (a) Typical action potential trajectories of the two groups of mice. (b–d) Quantitative analysis of the amplitude of APA (b), RP (c) and V max (d) in each group. n = 10 cells from six mice. (e–g) Quantitative analysis of APD20 (e), APD50 (f) and APD90 (g) in each group. n = 10 cells from six mice. (h) Representative action potential traces from the WT+FGF23 and WT+PBS groups. The rectangular enlarged area shows a prolonged action potential in WT+FGF23 group, and the straight segment indicates an increased incidence of DAD/TA in the WT+FGF23 group. (i) Incidence of TA or DAD in each group. n = 16 cells from nine mice. *P < 0.05, **P < 0.01 and ***P < 0.001. Abbreviations: APA, amplitude of action potential; ns, not significant; RP, resting potential; V max, maximum rate of action potential rise. Abbreviations: APA, action potential amplitude; APD20, action potential duration at 20% repolarization; APD50, action potential duration at 50% repolarization; APD90, action potential duration at 90% repolarization; DAD, delayed afterdepolarization; PBS, phosphate‐buffered saline; RP,…

• 

  FIGURE 3. Effect of FGF23 on sodium current (I Na) in adult mouse atrial myocytes. (a) Typical I Na trajectories of acutely isolated mouse atrial myocytes in voltage‐clamp mode, with stimulation procedure on the right. (b, c) I–V curve of I Na and quantitative analysis of peak sodium current density at −30 mV in PBS and FGF23 groups. n = 10 cells from 6 mice. (d–i) Steady‐state gating and recovery kinetics of I Na: (d) activation; (e) inactivation; and (f) recovery curves of I Na. Corresponding parameter statistics are shown as follows: (g) V 1/2 act and K act; (h) V 1/2 inact and K inact; and (i) recovery τ, n = 10. (j, k) Residual sodium current measured 3 ms after peak I Na at a test potential of −30 mV in atrial myocytes from both groups (black arrow indicates the current measured 3 ms after the peak current), n = 10 cells from six mice. (l) Window currents of sodium channels (black line indicates WT+PBS group; red line indicates WT+FGF23 group). (m, n) Representative western blotting and quantitative analysis of Nav1.5 proteins in atria of PBS or FGF23 mice. n = 5. ns, not significant, *P < 0.05 and ****P < 0.0001. Abbreviations: I Na, sodium current; NaV1.5, voltage‐gated sodium…

• 

  FIGURE 4. Effects of FGF23 stimulation on action potential properties and EAD incidence in primary atrial myocytes. (a) Typical action potential trajectories of the two groups of primary atrial myocytes. (b–d) Quantitative analysis of APA (b), RP (c) and V max (d) in each group. n = 10 cells from six mice. (e–g) Quantitative analysis of APD20 (e), APD50 (f) and APD90 (g) in each group. n = 10 cells from six mice. (h) Typical action potential trajectories of primary atrial myocytes in each group (the zoom‐in view shows EAD). (i) Statistics of the incidence of EAD in each group. n = 10 cells from six mice. ns, not significant, *P < 0.05, **P < 0.01 and ****P < 0.0001. Abbreviations: APA, action potential amplitude; APD20, action potential duration at 20% repolarization; APD50, action potential duration at 50% repolarization; APD90, action potential duration at 90% repolarization; EAD, early afterdepolarization; PBS, phosphate‐buffered saline; RP, resting membrane potential; Vmax, maximum upstroke velocity; WT, wild‐type.

• 

  FIGURE 5. Effects of FGF23 stimulation on I Na and electrophysiological properties in primary atrial myocytes. (a) Typical I Na trajectories of primary atrial myocytes in the voltage‐clamp mode. (b, c) I–V curve (b) and quantitative analysis of peak sodium current density at −20 mV (c) in control and FGF23 groups. n = 10 cells from six mice. (d, e) I Na steady‐state activation curves (d) and parameter statistics (e) in control and FGF23 group. n = 10. (f, g) Steady‐state inactivation curves (f) and parameter statistics (g) in acutely isolated mouse atrial myocytes. n = 10 cells from six mice. (h) Residual sodium current measured 3 ms after peak I Na at a test potential of −20 mV in primary atrial myocytes from both groups (black arrow indicates the current measured 3 ms after the peak current). (i) Quantitative analysis of residual sodium current measured 3ms after peak I Na, expressed as a percentage of peak I Na, n = 10 cells from 6 mice. (j, k) Representative western blotting (j) and quantitative analysis of Nav1.5 proteins (k). n = 6. (l) Window currents of sodium channels (black line indicates control group; yellow line indicates FGF23 group). ns, not significant, *P < 0.05 and **P <…

• 

  FIGURE 6. Regulation of intracellular calcium homeostasis by FGF23 in adult mouse atrial myocytes. (a) Typical I Ca,L trajectories of acutely isolated mouse atrial myocytes in voltage‐clamp mode, with stimulation procedure on the right. (b–c) I–V curve (b) and quantitative analysis of peak calcium current density (I Ca,L) at 0 mV in WT+PBS and WT+FGF23 group (c). n = 10 cells from six mice. (d–i) Steady‐state gating and recovery kinetics of I Ca: (d) activation; (e) inactivation; and (f) recovery curves of I Ca. Corresponding parameter statistics are shown as follows: (g) V 1/2 act and K act; (h) V1/2 inact and K inact,; and (i) recovery τ, n = 10–12 cells from six mice. (j) Representative images of calcium spark captured using confocal microscopy in the WT+FGF23 and WT+PBS groups. The black rectangles represent regions with fewer calcium sparks, appearing as flat plains in the 3D images, and the red rectangles represent regions with more calcium sparks, appearing as clustered peaks in the 3D images. (k) Quantitative analysis of calcium spark frequency in each group. n = 15 cells from eight mice. (l) The cardiomyocytes of WT+FGF23 and WT+PBS groups were stimulated at 1 Hz and recorded for…

• 

  FIGURE 7. Regulation of intracellular calcium by FGF23 in primary atrial myocytes from neonatal mice. (a) Typical I Ca,L trajectories of primary atrial myocytes in voltage‐clamp mode, with stimulation procedure on the right. (b, c) I–V curve (b) and quantitative analysis of peak calcium current density (I Ca,L) at 0 mV in PBS and FGF23 groups (c). n = 10 cells from six mice. (d–i) Steady‐state gating and recovery kinetics of I Ca: (d) activation; (e) inactivation; and (f) recovery curves of I Ca. Corresponding parameter statistics are shown as follows: (g) V 1/2 act and K act; (h) V 1/2 inact and Kinact; and (i) recovery τ, n = 10 cells from six mice. (j–o) Representative western blotting and quantitative analysis of SERCA2a, CAMKII, RyR2, Cav1.2 and NCX1 proteins in each group. n = 8. ns, not significant, *P < 0.05, **P < 0.01 and ***P < 0.001. Abbreviations: CaMKII, calcium/calmodulin‐dependent protein kinase II; CaV1.2, voltage‐gated calcium channel 1.2; I Ca, calcium current; I Ca,L, L‐type calcium current; NCX, sodium‐calcium exchanger; PBS, phosphate‐buffered saline; RyR2, ryanodine receptor 2; SERCA2a, sarco/endoplasmic reticulum Ca²⁺‐ATPase 2a.

• 

  FIGURE 8. Effects of ETV1 on action potential in primary atrial myocytes. (a) Typical action potential trajectories in each group. (b–m) Quantitative analysis of APA, RP, V max, APD20, APD50 and APD90 in each group. n = 9–10 cells from six mice. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Abbreviations: APA, action potential amplitude; APD20, action potential duration at 20% repolarization; APD50, action potential duration at 50% repolarization; APD90, action potential duration at 90% repolarization; NC, negative control; OE, overexpression; PBS, phosphate‐buffered saline; RP, resting membrane potential; si, small interfering RNA; Vmax, maximum upstroke velocity.

• 

  FIGURE 9. Effects of ETV1 on I Na in primary atrial myocytes. (a) Typical I Na trajectories of primary atrial myocytes in the voltage‐clamp mode in each group. (b, c) I–V curve (b) and quantitative analysis of peak sodium current density at −20 mV in each group (c). n = 8–10 cells from six mice. (d–o) Steady‐state gating and recovery kinetics of I Na: (d, g) activation; (e, h) inactivation; and (f, i) recovery curves of I Na in each group (d–f, knockdown; g–i, overexpression). Corresponding parameter statistics are shown as follows: (j, m) V 1/2 act and K act; (k, n) V 1/2 inact and K inact; and (l, o) recovery τ. (j–l, knockdown; m–o, overexpression), n = 8–10 cells from six mice. (p–s) Representative western blotting and quantitative analysis of Nav1.5 proteins in each group. n = 3. ns, not significant, *P < 0.05, **P < 0.01 and ***P < 0.001. Abbreviations: I Na, sodium current; NaV1.5, voltage‐gated sodium channel 1.5; NC, negative control; OE, overexpression; PBS, phosphate‐buffered saline; Si, small interfering RNA.

• 

  FIGURE 10. Effects of ETV1 knockout and FGF23 stimulation on AF susceptibility and action potential properties. (a) Representative surface ECG traces illustrating AF induction after atrial pacing in each group. (b, c) Statistics of transoesophageal AF induction rate and duration of AF in mice, n = 8–10. (d) Typical action potential trajectories in each group. (e–j) Quantitative analysis of APA, RP, V max, APD20, APD50 and APD90 in each group. n = 10 cells from six mice. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Abbreviations: AF, atrial fibrillation; APA, action potential amplitude; APD20, action potential duration at 20% repolarization; APD50, action potential duration at 50% repolarization; APD90, action potential duration at 90% repolarization; ECG, electrocardiogram; KO, knockout; PBS, phosphate‐buffered saline; RP, resting membrane potential; Vmax, maximum upstroke velocity.

• 

  FIGURE 11. Effects of ETV1 knockout and FGF23 stimulation on sodium channel properties and Nav1.5 expression. (a) Typical I Na trajectories in the voltage‐clamp mode in acutely isolated mouse atrial myocytes of WT+PBS, ETV1‐KO+PBS and ETV1‐KO+FGF23 mice. (b, c) I–V curve (b) and quantitative analysis of peak sodium current density at −20 mV in each group (c). n = 10 cells from six mice. (d, e) I Na steady‐state activation curves (b) and parameter statistics (c) in acutely isolated mouse atrial myocytes in each group. n = 10 cells from six mice. (f, g) Steady‐state inactivation curves (f) and parameter statistics (g) in acutely isolated mouse atrial myocytes in each group. n = 10 cells from six mice. (h, i) Recovery kinetic curves (h) and parameter statistics (i) in each group. n = 10 cells from six mice. (j, k) Representative western blotting (j) and quantitative analysis of Nav1.5 proteins (k) in each group. (l) Window currents of sodium channels (black line indicates WT+PBS group; blue line indicates ETV1‐KO+PBS group; and red line indicates ETV1‐KO+FGF23 group). ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Abbreviations: KO, knockout; PBS,…