Cardiovascular Research (2019) 115, 578–589 doi:10.1093/cvr/cvy215 The 4q25 variant rs13143308T links risk of atrial fibrillation to defective calcium homoeostasis Adela Herraiz-Martı́nez1,2, Anna Llach2,3, Carmen Tarifa1,2, Jorge Gandı́a1, Verónica Jiménez-Sabado4, Estefanı́a Lozano-Velasco5, Selma A. Serra1,2, Alexander Vallmitjana6, Eduardo Vázquez Ruiz de Castroviejo7, Raúl Benı́tez6, Amelia Aranega5, Christian Mu~ noz-Guijosa8, Diego Franco5, Juan Cinca2,3,4, and Leif Hove-Madsen1,2,4* 1 Biomedical Research Institute Barcelona CSIC-IIBB Antiguo Hospital de la Santa Creu i Sant Pau, Pabellon 11, St Antoni Ma Claret 167, 08025 Barcelona, Spain; 2IIB Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 3Department of Cardiology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 4CIBERCV, Spain; 5Department of Experimental Biology, University of Jaén, Spain; 6Department of Automatic Control, Universitat Politècnica de Catalunya, Barcelona, Spain; 7Cardiology Unit, Regional Hospital Ciudad de Jáen, Spain; and 8 Department of Cardiac Surgery, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Received 7 December 2017; revised 15 May 2018; editorial decision 16 August 2018; accepted 13 September 2018; online publish-ahead-of-print 30 May 2018 Time for primary review: 34 days 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 Western blotting, confocal calcium imaging, and patch-clamp techniques were used to identify mechanisms linking and results the 4q25 risk variants rs2200733T and rs13143308T to defects in the calcium homoeostasis in human atrial myo- cytes. 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 inci- dence 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 inde- pendently 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 prop- erties 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 exces- sive calcium release and spontaneous electrical activity linked to increased SERCA2 expression and RyR2 phosphorylation. 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 Keywords Human atrial myocytes • Single nucleotide polymorphisms • Sarcoplasmic reticulum calcium release • Ryanodine receptor • Spontaneous electrical activity * Corresponding author. Tel: þ34 93 556 5620; fax: þ34 93 556 5559, E-mail: leif.hove@iibb.csic.es C The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Cardiology. V This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Calcium homeostasis in 4q25 risk variants 579 .. 1. Introduction .. samples used for electrophysiological and/or molecular biological stud- .. ies, genotyping was done for batches of samples after experiments had .. Atrial fibrillation (AF) is the most common cardiac arrhythmia and a ma- .. been performed and analysed. Right atrial myocardial samples were col- jor contributory factor to increase mortality and risk of cerebrovascular .. lected from 342 genotyped patients undergoing cardiac surgery with .. embolism.1 Even though AF has been associated with a number of elec- .. extracorporeal circulation. Each patient gave written consent to obtain trophysiological, molecular, and structural alterations that favour the .. blood and tissue samples. The latter would otherwise have been dis- .. maintenance and self-perpetuation of the arrhythmia,2,3 current treat- .. carded during the surgical intervention. The study was approved by the ments of AF are often deficient or ineffective. .. Ethics Committees of the Spanish National DNA Bank (BNADN, .. Among the electrophysiological alterations, several studies have .. Salamanca), Hospital de la Santa Creu i Sant Pau (Barcelona), and of the reported disturbances in the calcium homoeostasis4–7 and malfunction- .. University of Jaén; and the investigation conforms to the principles out- .. ing of the sarcoplasmic reticulum (SR).4,5,7–10 Indeed, myocytes from .. lined in the Declaration of Helsinki. patients with AF depict a high rate of spontaneous SR calcium release,4 .. .. which has been linked to deficient phosphodiesterase activity,11 reduced .. phosphatase activity,12 or excessive protein kinase A5,9 or calmodulin ki- .. 2.2 Clinical characterization .. Clinical information relevant to this study and included in Table 1 was nase type II-dependent8,10,13 phosphorylation of the cardiac ryanodine .. receptor (RyR2). Importantly, spontaneous calcium waves can induce .. collected from the clinical records by a clinician. Medication refers to the .. patient medication prescribed before the surgical intervention. Patients arrhythmogenic membrane depolarizations in atrial myocytes from .. .. without any incidents of AF in the clinical record were considered free patients with AF,10 and occur both in resting and electrically stimulated .. of the arrhythmia. We cannot rule out that some patients with paroxys- human atrial myocytes.14,15 Moreover, transgenic mouse models with .. .. mal AF may not have been detected, but we assume that this error is high rates of spontaneous calcium release are more prone to present ar- .. similar for patients with and without risk variants. Patients were classified rhythmia10,16–18; supporting the notion that spontaneous SR calcium re- .. .. as having paroxysmal or chronic AF according to the information avail- lease plays a central role in these arrhythmogenic processes. .. able in the clinical record. Chronic AF included patients having persistent Thus, electrophysiological mechanistic studies have been narrowing .. . or long-term persistent AF. To reduce fractioning of the data, patients down the molecular mechanisms that underlie defective calcium homoeostasis in AF. Still, specific molecular triggers of AF are not well defined, except from mutations in ion channels associated to familial AF19–21 that only account for a minority of the patients with AF. Table 1 Clinical characterization of the patients included However, genome-wide association studies have identified several single in electrophysiological and molecular biological analyses nucleotide polymorphisms (SNPs) on chromosomes 4q25, 1q21, and Normal Risk P-value 16q22 that are associated with increased risk of AF,22,23 and recent (n 5 214) (n 5 128) meta-analyses have further expanded the number of risk variants.24 The ...................................................................................................... most striking arrhythmogenic variants remain those located at 4q2525 Age (years) 68.3 ± 0.7 68.0 ± 1 0.8 and some of them have been reported to modify the risk of AF recur- Height (cm) 164.4 ± 0.7 164.4 ± 0.8 1 rence after catheter ablation26 and the response to antiarrhythmic drug Weight (kg) 75.7 ± 0.9 75.3 ± 1.2 0.8 treatment.27 Furthermore, the bicoid-related homeodomain transcrip- Cardiovascular disease, % (n) tion factor Pitx2, which is located in the vicinity of the 4q25 variants has Arterial hypertension 65 (139) 72 (92) 0.2 been postulated as a molecular link between risk variants at 4q25 and Diabetes 41 (88) 27 (34) 0.01 AF.22 However, studies on Pitx2 expression in patients with 4q25 risk Aortic valve disease 49 (104) 57 (73) 0.15 variants28,29 and in patients with AF29–31 have not been able to link AF to Mitral valve disease 24 (51) 24 (31) 1 changes in Pitx2 expression or function caused by 4q25 risk variants. On Tricuspid valve disease 12 (26) 13 (16) 1 the other hand, no studies have so far investigated if the 4q25 risk var- Ischaemic heart disease 64 (136) 50 (64) 0.02 iants entail electrophysiological disturbances observed in atrial myocytes Echocardiography from patients with AF that have been associated to alterations in the Left atrial diameter (mm) 43.2 ± 0.6 43.0 ± 0.8 0.8 intracellular calcium homoeostasis. Therefore, here, we tested the Left atrial diameter index 2.40 ± 0.05 2.38 ± 0.05 0.8 hypothesis that risk variants on chromosome 4q25 cause alterations in LVEF (%) 55.7 ± 1 57.6 ± 1.2 0.2 the intracellular calcium homoeostasis that predispose to spontaneous Pharmacological treatment, % (n) electrical activity. ACE-inhibitors 37 (79) 35 (45) 0.8 ARB 18 (39) 21 (27) 0.6 b-blockers 49 (105) 52 (67) 0.6 2. Methods Calcium antagonists 22 (47) 25 (32) 0.6 Nitrates 25 (54) 23 (30) 0.8 2.1 Human biological samples and SNP Dicoumarin 25 (54) 30 (38) 0.4 genotyping Acetyl salicylic acid 45 (96) 46 (59) 0.9 A total of 605 blood samples were genotyped for the presence of the Statins 61 (130) 66 (85) 0.3 normal 4q25 variants rs2200733C and rs13143308G or for the corre- Values are represented as mean ± standard error or percentage and number (n) sponding AF risk variants rs2200733T and rs13143308T. Polymerase of patients with the condition. P-values for differences between the normal and risk groups are given on the right. chain reaction amplification of the SNPs rs2200733 and rs13143308 ACE-inhibitors, angiotensin converting enzyme inhibitors; ARB, angiotensin were carried out using flanking oligonucleotides (Supplementary material receptor blockers; LVEF, left ventricular ejection fraction. online, Methods section) followed by direct sequencing. For all atrial 580 A. Herraiz-Martı́nez et al. .. .. as described in the Supplementary material online, Methods section. .. Only elongated cells with clear cross striations and without abnormal .. .. granulation were used. .. .. .. .. .. 2.4 Western blot and immunofluorescent .. .. labelling .. .. Proteins were separated by SDS-PAGE and electrotransferred onto .. polyvinylidene diflouride membranes. Membranes were incubated with .. .. primary antibodies against SERCA2a, calsequestrin-2(CSQ-2), NCX-1, .. phospholamban (PLB), PLB-s16, or PLB-t17 (Supplementary material .. .. online, Methods section for details). Detection was performed using .. horseradish peroxidase-labelled IgG and the Supersignal detection .. .. system. .. .. Isolated myocytes were fixed with paraformaldehyde for 5 min at .. room temperature. Subsequently, cells were inmunofluorescently la- .. .. belled with the primary antibodies mouse anti-RyR2 and rabbit anti- .. ser2808-P or anti-ser2814P. After labelling, cells were washed again and .. .. stored at 4 C until visualization (Supplementary material online, .. Methods section for further details). .. .. .. .. .. .. 2.5 Patch-clamp technique .. .. Calcium current (ICa) was elicited by a 200 ms depolarization to 0 mV, .. and spontaneous transient inward currents (ITI) were measured at hold- .. .. ing potentials of -80 or -50 mV using whole cell voltage-clamp in the .. perforated patch configuration with a HEKA EPC-10 amplifier .. .. (Supplementary material online, Methods section for further details). .. Experiments began when the series resistance did not decrease further, .. .. and cells were discarded if it was larger than 6 times the pipette .. resistance. Figure 1 Frequency and segregation of the 4q25 loci rs2200733 and .. rs13143308. (A) The frequency of the rs2200733T (rs..733T) and .. The SR calcium load was determined from the charge carried by the rs13143308T (rs..308T) risk variants on chromosome 4q25. Number .. Naþ-Ca2þ exchange (NCX) current elicited by rapid exposure to .. of patients genotyped are given for each risk variant in patients without .. 10 mM caffeine (for 5 s) that releases the calcium stored in the SR into (No AF) and with AF. (B) Relative frequency of rs..733 and rs..308 geno- .. the cytosol. The charge was converted to amoles (10-18 mol) of calcium .. types in 342 patients donating right atrial tissue (black bars). T at rs..733 .. released from the SR, assuming a stoichiometry of 3Naþ:1Ca2þ for the indicates the presence of at least one risk allele, and T at rs..308 .. NCX and normalized to the cell capacitance. The remaining calcium indicates the presence of at least one risk allele at this locus. Grey bars .. .. available for extrusion by the NCX at a given time point during the caf- indicate the expected frequency of the same genotypes based on the al- .. feine exposure was determined as the SR calcium load minus the NCX lele frequencies. P-value for difference between observed and expected .. .. time integral at that time point (Supplementary material online, frequencies are given on the right. .. .. Figure S1). Experimental protocols were linked and executed sequen- .. tially. Erroneous measurement(s) were eliminated, giving rise to varia- .. .. tions in the total number of experiments for different currents. Most of with paroxysmal and chronic AF were pooled into a single group of AF .. these cases occurred because exposure of myocytes to caffeine pro- .. patients. This grouping was done because unpublished data from our .. duced sudden increases in the leakage current or irreversible cell con- laboratory and published data7 show that spontaneous calcium release is .. tracture in some myocytes. .. already elevated in patients with paroxysmal AF. .. Membrane potentials were measured in the current-clamp configura- .. tion using Kþ-containing intra- and extracellular media. The holding cur- .. .. rent was varied in order to achieve a resting membrane potential near 2.3 Human atrial tissue and myocyte .. -80 mV and hence compare the effect of normal and risk variants on the .. isolation .. amplitude and frequency of spontaneous membrane depolarizations Samples were excised from the right atrial appendage of patients under- .. without interference of concurrent alterations in the membrane poten- .. going cardiac surgery with extracorporeal circulation, stored immedi- .. tial. We did not attempt to measure the native resting membrane poten- ately in cold oxygenated Tyrode solution and snap-frozen or used for .. tial because the cholinergic tone is washed out in isolated human atrial .. myocyte isolation within 5–10 min after excision. For myocyte isolation, .. myocytes where acetyl choline activated potassium channels play a key the sample was cut into small fragments and digested in a calcium-free .. role in setting the resting membrane potential32 and because their ex- .. Tyrode solution with collagenase, proteinase, and fatty acid-free albumin . pression and activation is altered in AF.32,33 Calcium homeostasis in 4q25 risk variants 581 Figure 2 The risk variant rs13143308T increases spontaneous electrical activity. (A) Spontaneous ITI currents in patients with a normal or a risk variant at rs13143308. (B) ITI frequency measured in myocytes without (normal) or with a risk variant. (C) ITI 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. .. Unless otherwise stated, values were averaged for each patient. Mean 2.6 Confocal calcium imaging .. Confocal calcium images (512  140 pixels) were recorded at 90 Hz. To .. values are indicated with black horizontal bars and 95% confidence inter- .. vals with grey horizontal bars in dot plots or given as mean ± SEM, when detect calcium sparks, a wavelet-based detection method was applied to .. the normalized time-dependent fluorescence signal zi(t) at every pixel in .. indicated. Statistical significance was evaluated using the Fisher’s exact .. test for categorical data. The Student’s t-test was used for paired or un- order to detect candidates for Ca2þ release events (Supplementary ma- .. terial online, Methods section for details on signal processing). After su- .. paired comparisons and a Mann–Whitney test was used for data that did .. not have a normal distribution. ANOVA analysis or a linear regression pervised validation and merging, sparks that coincided spatially (within a .. radius of 2 mm) were pooled into a common spark site and calcium .. model was used for comparison of multiple effects as indicated. For the .. traces were generated for each spark site. Each spark was characterized .. statistical analysis of the effects of the different genotypes containing dif- by its amplitude, its duration characterized as the full duration at half .. ferent SNPs, we used a linear regression model taking into account the .. maximum and its width characterized as the full width at half maximum. .. effects of the genotype, the presence of AF, and whether the patients .. were undergoing mitral valve replacement. Based on the linear regres- The spark frequency was calculated as the number of sparks/cell/s or .. normalized to the cell area within the confocal plane and expressed as .. sion model, the effect of AF and the most common genotypes was esti- .. mated and given as mean ± SEM. Statistically significant effects are sparks/mm2/s. Additionally, the number of spark sites were expressed as .. sites/cell or normalized to the cell area and given as sites/mm2. The aver- .. indicated with P-values or *P < 0.05, **P < 0.01, and ***P < 0.001. .. age spark frequency per site was given as sparks/site/s. .. .. .. 3. Results 2.7 Statistical analysis .. .. 3.1 4q25 risk variants in the study Electrophysiological and molecular biological analyses of human atrial .. samples were performed without knowledge about clinical data or geno- .. population .. types and clinicians gathering the clinical data did not know the experi- .. Genotyping was done in 605 patients for the 4q25 risk variants mental results. Data sets were analysed with SPSS statistical software. .. rs2200733T and rs13143308T that are highly associated with AF 582 A. Herraiz-Martı́nez et al. 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. risk.22,34 The rs13143308T risk variant was more frequent in patients .. .. 3.2 Impact of the 4q25 risk variant with AF (P = 0.004) while the rs2200733T risk variant depicted a non- .. rs13143308Ton spontaneous electrical significant trend (Figure 1A). Furthermore, the rs2200733T risk variant .. .. activity co-segregated with rs13143308T (P < 0.001; Figure 1B). .. Atrial tissue samples and/or myocytes from 342 of these patients .. Since the rs2200733 and rs13143308 variants are located next to each .. other30 and the rs2200733T risk variant occurred together with the were used for further clinical, electrophysiological, and/or molecular .. biological analysis. The clinical and echocardiographic data as well as .. rs13143308T risk variant in myocytes isolated from all but one of the .. patients, the specific effect of the rs2200733T variant could not be mea- pharmacological treatments of these patients are summarized in .. Table 1. .. sured directly. Therefore, this analysis focused on how the presence of a Calcium homeostasis in 4q25 risk variants 583 Figure 4 Calcium spark properties do not depend on the genotype at rs13143308. (A) Ca2þ transient from a single spark site. Consecutive 8  8 lm images of the calcium spark are shown below the Ca2þ 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. .. rs13143308T risk variant affected the calcium homoeostasis and sponta- .. 3.3 Impact of the 4q25 risk variant .. rs13143308Ton calcium homoeostasis neous electrical activity. First, we analysed the frequency of ITI resulting .. from extrusion by the NCX of spontaneously released calcium in .. Using frame-scanning confocal calcium imaging to visualize local calcium .. patients without AF in order to avoid confounding effects of the arrhyth- .. release from RyR2 clusters (calcium sparks) in a longitudinal plane of mia on calcium homoeostasis and electrical activity. In these patients, the .. myocytes from patients without risk variants at rs2200733 and .. ITI frequency was significantly higher in myocytes from carriers of .. rs13143308 (Figure 3A) and with at least one risk variant at rs13143308 the rs13143308T risk variant (Figure 2A and B) while the ITI-amplitudes .. (Figure 3B) revealed that the spark frequency was significantly higher in .. were similar for patients with and without risk alleles (Figure 2C). .. carriers of the risk variant (Figure 3C). This was due to a higher density of Current-clamp experiments, performed in myocytes from a subset of .. spark sites (Figure 3D) rather than a higher firing frequency per site .. patients to determine the net impact of calcium release-induced NCX .. (Figure 3E). Analysis of the kinetics (Figure 4A) and the width (Figure 4B) on the membrane potential, showed a significantly higher frequency of .. of calcium sparks showed that the 4q25 risk variants tended to reduce .. spontaneous membrane depolarizations (DADs) (Figure 2D-E) with .. the spark amplitude (Figure 4C) but did neither affect the duration unchanged amplitudes (Figure 2F) in patients carrying the risk variant. In .. (Figure 4D) nor the width of the calcium sparks (Figure 4E). .. three of the patients with risk variants, some of the DADs were large .. Immuofluorescent labelling of total and ser2808 or ser2814 phos- enough to trigger spontaneous action potentials. .. phorylated RyR2 clusters revealed that the presence of a risk variant did . 584 A. Herraiz-Martı́nez et al. 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 phos- phorylation 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 lm. (C) Caffeine-induced NCX currents (top) their time in- tegral (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 sam- ples 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. not affect the density of RyR2 clusters (0.59 ± 0.10 vs. 0.56 ± 0.07 clus- .. but not at ser2814. The weak ser2814 phosphorylation in the human .. ters/mm2). However, it significantly increased the ser2808/RyR2 .. atrial myocytes is in contrast to a clear ser2814 labelling in mouse ven- (Figure 5A) but not ser2814/RyR2 (Figure 5B) intensity ratios, suggesting .. tricular myocytes under the same conditions (Supplementary material .. that the rs13143308T variant favours RyR2 phosphorylation at ser2808 . online, Figure S2), suggesting that it is not a methodological problem. Calcium homeostasis in 4q25 risk variants 585 .. The caffeine releasable SR calcium load estimated from the time integral .. homoeostasis using a linear regression model. Analysis of myocytes from of the caffeine-induced NCX current (Figure 5C) was also significantly .. 77 patients showed that both the rs13143308T genotype (P = 0.004) and .. higher in atrial myocytes from patients with a risk variant (Figure 5D), .. AF (P = 0.003) independently increased the ITI frequency. Comparison of which could potentially increase the number of RyR2 clusters that reach .. specific patient groups showed that patients with No AF carrying a risk .. the threshold for spontaneous calcium release and thereby the density ... allele had a five-fold higher ITI frequency than those with No AF and of calcium spark sites. By contrast, plotting the NCX current against the .. normal alleles at rs2200733 and rs13143308 (Table 2, P < 0.001). On remaining calcium available for extrusion by the NCX in order to assess .. the other hand AF increased the ITI frequency three-fold in patients .. the NCX activity at different calcium levels (Figure 5E) showed no differ- .. with the normal allele (P = 0.002). Moreover, the effects of AF and ence between the resulting slopes for patients with normal and risk .. 4q25 risk variants were additive with the ITI frequency being three-fold .. variants (Figure 5F) suggesting that the NCX rate is not affected by the .. higher in patients with AF and a risk variant than in patients with AF but rs13143308T risk variant. .. with normal variants (P = 0.008) and two-fold higher than in patients .. Western blot analysis of SR calcium regulatory proteins revealed that .. with a risk variant but without AF (P = 0.04). Analysis of the DAD expression of the cardiac sarco-endoplasmatic reticulum calcium .. frequency (Table 2) showed effects of risk variants and AF similar to .. ATPase (SERCA2a) was significantly higher in patients with a .. those observed on the ITI frequency, i.e. the presence of a risk variant rs13143308T risk variant, whereas expression of the SR calcium buffer- .. doubled the DAD frequency (P = 0.04) in patients without AF; and .. ing protein CSQ-2 and the NCX-1 were similar in patients with and .. when the risk variant occurred in patients with AF the DAD frequency without risk variants (Figure 5G–H). Additional analysis revealed that nei- .. was 2.7-fold higher than in risk carriers without AF (P = 0.01). Analysis .. ther PLB expression (72 ± 11% of control, P = 0.24) nor PLB phosphory- .. of the caffeine-induced NCX current in a subset of 53 patients lation at ser-16 (80 ± 27% of control, P = 0.54) or at thr-17 (69 ± 9% of .. revealed that the SR calcium load was significantly modified by the .. control, P = 0.09) were different from the levels in controls without risk .. genotype (P = 0.001) but not by AF (P = 0.128). Comparison of patient .. variants. .. groups revealed a significantly higher SR calcium load in patients .. without AF that carry the rs13143308T risk variant. There were no .. .. differences in SR calcium loading or in NCX-activity between patients 3.4 Combined and independent .. with AF carrying normal or risk variants (Table 2). effects of the rs13143308T risk variant .. .. Analysis of the L-type ICa, which is strongly reduced in patients with and AF .. chronic AF, revealed that the risk variants had no effect on ICa amplitude .. To determine if the rs13143308T risk variant exacerbates calcium- .. (Figure 6A), the decay of the ICa (Figure 6B), the current-voltage relation- handling disturbances in AF, we determined whether the presence of .. ship (Figure 6C), or the voltage for half-maximal ICa inactivation .. the risk variant and/or AF had independent effects on the calcium . (Figure 6D) in patients with or without AF. Table 2 Effect of rs13143308 variants on ITI and DAD frequency, SR calcium load, and NCX activity in patients without (No AF) and with AF ITI DAD .................................................................................... ..................................................................................... 4q25: Normal Risk P-value Normal Risk P-value .............................................................................................................................................................................................................................. No AF, 0.26 ± 0.09, 1.33 ± 0.24, <0.001 0.56 ± 0.17, 1.22 ± 0.26, 0.04 95% CI 0.10–0.41 0.91–1.75 0.22–0.89 0.71–1.72 (n) (28) (23) (34) (17) AF, 0.84 ± 0.24, 2.49 ± 0.61, 0.008 0.95 ± 0.30, 3.27 ± 1.24, 0.02 95% CI 0.65–1.62 0.76–3.40 0.36–1.54 0.84–5.70 (n) (16) (10) (12) (4) P-value 0.002 0.04 0.25 0.01 Load NCX .................................................................................... .................................................................................... 4q25: Normal Risk P-value Normal Risk P-value .............................................................................................................................................................................................................................. No AF, 7.28 ± 0.54, 10.2 ± 1.4, 0.03 0.81 ± 0.07, 0.70 ± 0.10, 0.37 95% CI 6.23–8.34 7.50–12.82 0.67–0.95 0.50–0.91 (n) (22) (16) (22) (16) AF, 6.93 ± 0.77, 5.2 ± 0.6, 0.09 0.86 ± 0.09, 1.00 ± 0.25, 0.49 95% CI 5.42–8.44 3.95–6.44 0.69–1.03 0.51–1.50 (n) (9) (6) (8) (6) 0.72 0.19 0.73 0.22 The ITI and DAD frequencies are given in events/min, the SR calcium load in amol/pF, and the NCX activity in s-1. Values are represented as mean ± SEM and 95% CI. P-values for comparison of the normal and risk variants are given in columns to the right of each comparison. Risk refers to the presence of at least one rs13143308T risk allele. Normal refers to patients with normal rs2200733C and rs13143308G alleles. P-values for comparison of No AF and AF are given below. CI, confidence interval. 586 A. Herraiz-Martı́nez et al. Figure 6 Risk variants at 4q25 do not modify the L-type calcium current in isolated human atrial myocytes. (A) ICa recordings from patients that had nor- mal variants (black traces) or at least one risk variant (grey traces) in the absence of presence of AF. The mean ICa is shown below each trace. (B) Superimposed normalized ICa traces (top panel) and the fast time constant for ICa inactivation (bottom). (C) ICa 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) ICa 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 ICa 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). .. 4. Discussion .. carrying the rs13143308T risk variant display increased SERCA2a .. expression, SR calcium load, and RyR2 phosphorylation, which .. 4.1 Main findings .. likely cause the abnormally high incidence of both calcium release- This study identifies for the first time a common SNP on chromo- .. induced ITI and spontaneous membrane depolarizations observed in .. these patients (see Figure 7). Since these electrophysiological altera- some 4q25 (rs13143308T) that is associated with functional .. changes in the calcium homoeostasis and electrical activity of hu- .. tions are recognized hallmarks of AF,4,10 the fact that we observe .. them even in patients without AF suggests that the rs13143308T man atrial myocytes, affording an electrophysiological mechanism .. that may explain the higher incidence of AF in carriers of this vari- .. variant is a genetic risk marker for AF associated with excessive .. spontaneous calcium release-induced electrical activity. ant. Specifically, we show that human atrial myocytes from patients .. Calcium homeostasis in 4q25 risk variants 587 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 Naþ-Ca2þ exchange, giving rise to the higher incidence of ITI 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. .. been found in patients with paroxysmal AF7 but not in patients with per- 4.2 Mechanisms promoting spontaneous .. SR calcium release in the 4q25 risk variant .. manent AF.4,5,7,8,10 .. rs13143308T .. .. 4.3 The 4q25 risk variant rs13143308T as Confocal calcium imaging in human atrial myocytes from our patients .. revealed that the higher incidence of spontaneous calcium waves in car- .. a genetic risk marker of AF associated .. riers of the 4q25 risk variant rs13143308T was due to a larger number of .. with spontaneous electrical activity calcium spark sites. Since calcium sparks are produced by opening of the .. Electrophysiological analysis showed that the presence of a single risk al- .. RyR2, a high open probability of the RyR2 has been alluded to account .. lele at rs13143308 was associated with a five-fold increase in the ITI fre- for an increased incidence of spontaneous calcium sparks and waves.35,36 .. .. quency (Figure 2A and B) and in the frequency of spontaneous DADs Mechanistically, an increased RyR2 opening may result from several fac- .. (Figure 2D–E). These findings may provide a mechanistic understanding tors, such as luminal calcium activation,37–39 hyperphosphorylation of .. .. of observations on the efficacy of ablation therapy in carriers of the RyR2,5,8–10,40 and/or increased SR calcium loading.7,41 In accordance .. rs10033464 or rs2200733 risk variants43 that are located next to the .. with this, we found that carriers of the rs13143308T risk variant free of .. rs13143308 locus studied here and expected to co-segregate with AF had higher RyR2 phosphorylation at ser2808, higher expression of .. it.34,44 Thus, Shoemaker et al.43 found that ablation therapy was less effi- .. SERCA2a without changes in PLB expression or phosphorylation, and .. cient in carriers of the rs2200733 risk variant, which might be expected if higher SR calcium loading. These findings, combined with the lack of con- .. the ectopic activity is caused by calcium release-induced DADs that are .. current increase in the SR calcium buffering protein CSQ-2, suggest that .. likely to occur throughout the atrial myocardium. Interestingly, Parvez a greater number of RyR2 could reach the threshold for spontaneous .. et al.27 also found that antiarrhythmic drug therapy was less efficient in .. calcium release42 and consequently, lead to an increased calcium spark .. patients carrying a 4q25 risk variant at the rs10033464 locus, and that frequency. Interestingly the increase in SR calcium loading and SERCA2a .. carriers of the ancestral (normal) variant with AF responded better to .. expression was only observed in risk carriers free of AF. This suggests .. Class III antiarrhythmic drugs while carriers of the risk variant with AF that the rs13143308T mediated increase in SR calcium loading may pre- .. responded better to Class I antiarrhythmic drugs (sodium channel block- .. cede the occurrence of AF and might contribute to the initiation of the .. ers). Given that carriers of the rs10033464 risk variant are expected to arrhythmia. In support of this assumption, a higher SR calcium load has .. carry the rs13143308 risk variant also34,44 and hence have a high 588 A. Herraiz-Martı́nez et al. .. incidence of spontaneous calcium release-induced DADs, Class I antiar- .. rs13143308T variant settles the bases for testing whether current or rhythmic drugs would be expected to attenuate the ability of these .. novel pharmacological therapies targeting SR calcium release are more .. DADs to trigger spontaneous action potentials by increasing the thresh- .. efficient in preventing AF in patients with this variant than in patients old for sodium channel activation. .. with normal rs13143308G alleles. .. Considering that the ITI amplitude is expected to reflect electrogenic .. In conclusion, our results identify the rs13143308T risk variant on Naþ-Ca2þ exchange and hence determine the amplitude of the mem- .. chromosome 4q25 as a potential biomarker for AF linked to arrhythmo- .. brane depolarization, our findings of proportionality between the ITI and .. genic calcium release and a new key to understand the complex DAD amplitudes in patients with and without the risk variant (Figure 2C .. molecular mechanisms that link AF to perturbations in the calcium .. and F) supports the notion that the DADs could be produced by calcium .. homoeostasis. release-induced ITIs. On the other hand, we cannot rule out that the .. .. rs13143308 risk variant also affects other ion channels that could modify .. .. the frequency or amplitude of calcium release-induced DADs. In this .. Supplementary material context, patients with AF have previously been reported to present a re- .. duction in potassium currents in the right atrium that normally help .. Supplementary material is available at Cardiovascular Research online. .. repolarizing the myocyte and stabilize the resting membrane poten- .. tial.32,45 Thus, the present findings warrant future studies analysing the .. .. effect of 4q25 risk variants on the expression and activity of potassium .. Acknowledgements channels that stabilize the resting membrane potential. .. .. The collaboration of the Department of Cardiac Surgery at Hospital de A prominent reduction in the ICa density is another characteristic fea- .. la Santa Creu i Sant Pau is greatly appreciated. ture of atrial myocytes from patients with AF.5,6,31 However, analysis of .. .. Conflict of interest: none declared. the rs13143308T and rs2200733T risk variants showed no effects on the .. .. amplitude or the properties of ICa in patients with or without AF; sug- .. gesting no role for L-type channels as a functional link between these .. .. Funding 4q25 risk variants and AF. .. This work was supported by multi-centric grants from Centro Nacional de .. .. Investigaciones Cardiovasculares [CNIC-2009-08 to L.H.-M. and D.F.]; a grant 4.4 Study limitations .. from Fundació Marató TV3 [2015-20-30 to L.H.-M.]; and grants from the Due to ethical constrains, our study was limited to the use of human .. .. Spanish Ministry of Economy and Competition [SAF2014-58286-C2-1-R to right atrial specimens because they can be routinely obtained at the time .. L.H.-M.] and [DPI2013-44584-R to R.B.]; and from the Spanish Ministry of of right atrial cannulation during the majority of surgical cardiac interven- .. Health and Consume, Instituto de Salud Carlos III, Red de Investigación .. tions that use extracorporeal circulatory support. In contrast, tissue sam- .. Cardiovascular [RD12/0042/0002] and CIBERCV to J.C., and from Fondo ples from the left atrium are only available in patients undergoing .. Europeo de Desarrollo Regional (FEDER). .. interventions that require opening of the left atrium, such as mitral valve .. surgery. Even in these cases, the left atrium is often dilated and affected .. .. References structurally and this has been reported to disturb the calcium homoeo- .. 1. 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