The 4q25 variant rs13143308T links risk of atrial fibrillation to defective calcium homoeostasis

Abstract

Aims: Single nucleotide polymorphisms on chromosome 4q25 have been associated with risk of atrial fibrillation (AF) but the exiguous knowledge of the mechanistic links between these risk variants and underlying electrophysiological alterations hampers their clinical utility. Here, we tested the hypothesis that 4q25 risk variants cause alterations in the intracellular calcium homoeostasis that predispose to spontaneous electrical activity.

Methods and results: Western blotting, confocal calcium imaging, and patch-clamp techniques were used to identify mechanisms linking the 4q25 risk variants rs2200733T and rs13143308T to defects in the calcium homoeostasis in human atrial myocytes. Our findings revealed that the rs13143308T variant was more frequent in patients with AF and that myocytes from carriers of this variant had a significantly higher density of calcium sparks (14.1 ± 4.5 vs. 3.1 ± 1.3 events/min, P = 0.02), frequency of transient inward currents (ITI) (1.33 ± 0.24 vs. 0.26 ± 0.09 events/min, P < 0.001) and incidence of spontaneous membrane depolarizations (1.22 ± 0.26 vs. 0.56 ± 0.17 events/min, P = 0.001) than myocytes from patients with the normal rs13143308G variant. These alterations were linked to higher sarcoplasmic reticulum calcium loading (10.2 ± 1.4 vs. 7.3 ± 0.5 amol/pF, P = 0.01), SERCA2 expression (1.37 ± 0.13 fold, P = 0.03), and RyR2 phosphorylation at ser2808 (0.67 ± 0.08 vs. 0.47 ± 0.03, P = 0.01) but not at ser2814 (0.28 ± 0.14 vs. 0.31 ± 0.14, P = 0.61) in patients carrying the rs13143308T risk variant. Furthermore, the presence of a risk variant or AF independently increased the ITI frequency and the increase in the ITI frequency observed in carriers of the risk variants was exacerbated in those with AF. By contrast, the presence of a risk variant did not affect the amplitude or properties of the L-type calcium current in patients with or without AF.

Conclusions: Here, we identify the 4q25 variant rs13143308T as a genetic risk marker for AF, specifically associated with excessive calcium release and spontaneous electrical activity linked to increased SERCA2 expression and RyR2 phosphorylation.

Keywords: Human atrial myocytes; Ryanodine receptor; Sarcoplasmic reticulum calcium release; Single nucleotide polymorphisms; Spontaneous electrical activity.

Figure 1

Frequency and segregation of the 4q25 loci rs2200733 and rs13143308. (A) The frequency of the rs2200733T (rs..733T) and rs13143308T (rs..308T) risk variants on chromosome 4q25. Number of patients genotyped are given for each risk variant in patients without (No AF) and with AF. (B) Relative frequency of rs..733 and rs..308 genotypes in 342 patients donating right atrial tissue (black bars). T at rs..733 indicates the presence of at least one risk allele, and T at rs..308 indicates the presence of at least one risk allele at this locus. Grey bars indicate the expected frequency of the same genotypes based on the allele frequencies. P-value for difference between observed and expected frequencies are given on the right.

Figure 2

The risk variant rs13143308T increases spontaneous electrical activity. (A) Spontaneous I_TI currents in patients with a normal or a risk variant at rs13143308. (B) I_TI frequency measured in myocytes without (normal) or with a risk variant. (C) I_TI amplitude measured in the myocytes from B. (D) Membrane potential recordings at −80 mV in myocytes from a patient with normal and one with a risk variant. (E) Frequency of spontaneous membrane depolarizations at −80 mV in patients with normal and risk variants. (F) Amplitude of the depolarizations in the myocytes from (E). P-values for the unpaired t-tests (C and F) or Mann–Whitney tests (B and E) are given above data points and the number of patients in parentheses.

Figure 3

The rs13143308T risk variant increases the calcium spark density. (A) Longitudinal plane of a human atrial myocyte from a patient without a 4q25 risk variant, with indication of two spark sites on the left (colored rectangles). Calcium signals are shown for each spark site on the right. Insets show calcium signals for individual sparks. (B) Sparks in a human atrial myocyte from a patient with a 4q25 risk variant. (C) Spark frequency. (D) Spark sites per cell. (E) Spark firing frequency. P-values from the unpaired t-tests (E) or Mann–Whitney tests (C and D) are given above data points and the number of patients in parentheses.

Figure 4

Calcium spark properties do not depend on the genotype at rs13143308. (A) Ca²⁺ transient from a single spark site. Consecutive 8 × 8 μm images of the calcium spark are shown below the Ca²⁺ transient during its rise and decay (white window area). (B) Enlarged image of the calcium spark in A, recorded at its maximal amplitude. The profile of the calcium spark is shown on the right for each of the four coloured axes outlined in the image on the left. The width of the calcium spark at half maximum was calculated as the average of the four axes. (C) Calcium spark amplitude. (D) Calcium spark duration at half maximum. (E) Width of the calcium spark at half maximum. Values are from the same myocytes as in Figure 2. P-values for the unpaired t-tests (C) or Mann–Whitney tests (D and E) are given above data points and the number of patients is given in parentheses.

Figure 5

RyR2 phosphorylation, SR calcium loading, and SERCA2a expression is increased in patients with a rs13143308T risk variant. (A) Visualization of total RyR2 clusters (green), ser2808 phosphorylated clusters (red), and overlay of the two channels in a human atrial myocyte. The degree of ser2808 phosphorylation was estimated as the ratio of the ser2808/RyR2 intensity for each RyR2 cluster (right panel). (B) Visualization of total RyR2 clusters (green), ser2814 phosphorylated clusters (red), and overlay of the two channels in a human atrial myocyte. The degree of ser2814 phosphorylation was estimated as the ratio of the ser2814/RyR2 intensity for each RyR2 cluster (right panel). White scale bars: 20 μm. (C) Caffeine-induced NCX currents (top) their time integral (middle) and the calcium available for extrusion by the NCX (bottom) for a normal and a risk variant. (D) Caffeine releasable SR calcium content. (E) Relationship between the caffeine-induced NCX current and the calcium available for extrusion by the NCX for a normal and a risk variant. (F) Mean slope of the relationship between NCX activity and calcium available for extrusion. (G) Immunoblots of SERCA2a, CSQ-2, NCX-1, and GADPH in right atrial samples from patients with normal or risk variants at 4q25. GADPH served as reference. (H) Mean SERCA2a, CSQ-2, and NCX-1 expression levels. Values were normalized to the mean expression level in the normal variant. P-values for the unpaired t-tests (D, F, and H) or Mann–Whitney tests (A and B) are given above graphs and the number of patients is given in parentheses.

Figure 6

Risk variants at 4q25 do not modify the L-type calcium current in isolated human atrial myocytes. (A) I_Ca recordings from patients that had normal variants (black traces) or at least one risk variant (grey traces) in the absence of presence of AF. The mean I_Ca is shown below each trace. (B) Superimposed normalized I_Ca traces (top panel) and the fast time constant for I_Ca inactivation (bottom). (C) I_Ca recordings at different test potentials (top) in myocytes from patients carrying a risk variant, one of them with AF and one without AF (no AF). Current–voltage curves for each patient group (bottom). *indicates a significant difference between AF-patients and those with No AF. (D) I_Ca tracings recorded with different pre-potentials (top) in myocytes from patients without AF with a normal or a risk variant at 4q25. The voltage for half-maximal I_Ca inactivation is shown below. The number of patients is indicated for each data set in parentheses and P-values for comparisons are given above (in B) or below graphs (in A and D).

Figure 7

Alterations in the calcium homoeostasis that may confer a higher risk of atrial fibrillation to carriers of the 4q25 risk variant rs13143308T. Schematic outline of the mechanisms identified in the present study (blue rectangle) that may contribute to increase the risk of atrial fibrillation in carriers of the rs13143308T risk variant. Briefly, carriers of this variant present higher SERCA2a expression and RyR2 phosphorylation at ser2808 than carriers of the normal rs13143308G variant, which may account for the higher spark frequency in myocytes from patients with the risk variant. The higher incidence of spontaneous calcium release, in turn, will stimulate electrogenic Na⁺-Ca²⁺ exchange, giving rise to the higher incidence of I_TI currents in carriers of the risk variant, and hence increase the frequency of DADs large enough to trigger spontaneous atrial electrical activity and induce atrial fibrillation.