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

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.Peer ReviewedPostprint (author's final draft

Novel PITX2 Homeodomain-Contained Mutations from ATRIAL Fibrillation Patients Deteriorate Calcium Homeostasis

Atrial fibrillation (AF) is the most common cardiac arrhythmia in the human population, with an estimated incidence of 1¿2% in young adults but increasing to more than 10% in 80+ years patients. Pituitary Homeobox 2, Paired Like Homeodomain 2 (PITX2c) loss-of-function in mice revealed that this homeodomain (HD)-containing transcription factor plays a pivotal role in atrial electrophysiology and calcium homeostasis and point to PITX2 as a candidate gene for AF. To address this issue, we recruited 31 AF patients for genetic analyses of both the known risk alleles and PITX2c open reading frame (ORF) re-sequencing. We found two-point mutations in the homedomain of PITX2 and three other variants in the 5¿untranslated region. A 65 years old male patient without 4q25 risk variants but with recurrent AF displayed two distinct HD-mutations, NM_000325.5:c.309G>C (Gln103His) and NM_000325.5:c.370G>A (Glu124Lys), which both resulted in a change within a highly conserved amino acid position. To address the functional impact of the PITX2 HD mutations, we generated plasmid constructs with mutated version of each nucleotide variant (MD4 and MD5, respectively) as well as a dominant negative control construct in which the PITX2 HD was lacking (DN). Functional analyses demonstrated PITX2c MD4 and PITX2c MD5 decreased Nppa-luciferase transactivation by 50% and 40%, respectively, similar to the PITX2c DN (50%), while Shox2 promoter repression was also impaired. Co-transactivation with other cardiac-enriched co-factors, such as Gata4 and Nkx2.5, was similarly impaired, further supporting the pivotal role of these mutations for correct PITX2c function. Furthermore, when expressed in HL1 cardiomyocyte cultures, the PITX2 mutants impaired endogenous expression of calcium regulatory proteins and induced alterations in sarcoplasmic reticulum (SR) calcium accumulation. This favored alternating and irregular calcium transient amplitudes, causing deterioration of the beat-to-beat stability upon elevation of the stimulation frequency. Overall this data demonstrate that these novel PITX2c HD-mutations might be causative of atrial fibrillation in the carrier.This work was supported by grants from The Spanish Ministry of Science Innovation and Universities [SAF2017-88019-C3-1-R] to L.H.-M. V.J.-S. was employed by CIBERCV [RD12/0042/0002] grant. Work was also supported by a PhD scholarship [FPU18/01250] to S.C., and partially funded by grants from Generalitat de Catalunya [SGR2017-1769] and Fundació Marato TV3 [20152030] to L.H.-M., a translational CNIC grant [2009/08] to D.F., R.C. and L.H.-M. and a grant-in-aid from the Junta de Andalucia Regional Council to D.F. and A.A. [CTS-446]

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

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 here tested the hypothesis that 4q25 risk variants cause alterations in the intracellular calcium homeostasis 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 homeostasis 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 (ITI) currents (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±0.5amol/pF, p¿=¿0.01), SERCA2 expression (1.37±0.13 fold, p¿=¿0.03) and RyR2 phosphorylation at s2808 (0.67±0.08 vs. 0.47±0.03, p¿=¿0.01) but not at s2814 (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: We here 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 phosphorylationThis 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 from Fundacio´ Marato´ TV3 [2015-20-30 to L.H.-M.]; and grants from the Spanish Ministry of Economy and Competition [SAF2014-58286-C2-1-R to L.H.-M.] and [DPI2013-44584-R to R.B.]; and from the Spanish Ministry of Health and Consume, Instituto de Salud Carlos III, Red de Investigacio´n Cardiovascular [RD12/0042/0002] and CIBERCV to J.C., and from Fondo Europeo de Desarrollo Regional (FEDER)

Atrial fibrillation in patients on haemodialysis in Andalusia. Prevalence, clinical profile and therapeutic management

Atrial fibrillation (AF) represents an important social and healthcare problem. There is wide variability in the prevalence of this arrhythmia in studies analysing patients on haemodialysis (HD). Objective: To investigate the prevalence, clinical profile and therapeutic management of patients with AF on HD in Andalusia. Methods: We asked the public healthcare system of Andalusia to provide us with the number of patients who were being treated with HD. We asked attending nephrologists from all hospital and outpatient centers in 5 of the 8 Andalusian provinces to perform an electrocardiogram and to fill out a questionnaire on patients selected by simple random sampling. Results: A total of 2348 patients were being treated with HD in the 5 provinces included in the study. The estimated sample size was 285 patients. We obtained an electrocardiogram and information from 252 patients (88.4%); mean age 65.3 ± 16 years; 40.9% women. Sixty-three patients (25%) had AF. Of these, 36 (14.3%) had AF in the recorded ECG and in the rest it had been documented previously. In the multivariate analysis, older age (OR: 1.071; 95% CI: 1.036–1.107; p = 0.000) and greater time on HD (OR: 1.009; 95% CI: 1.004–1.014; p = 0.000) were independently associated with the presence of AF. Of the patients with AF, 41.3% were on anticoagulant treatment at the time of the study; and 41.2% were on antiplatelet agents. Conclusions: AF in dialysis units is an important finding. Establishing the risk–benefit ratio of anticoagulant treatment constitutes a real challenge. Well-designed clinical trials are pivotal in order to define the rational use of antithrombotic drugs. Resumen: La fibrilación auricular (FA) es un importante problema social y sanitario. Existe una amplia variación en la prevalencia de esta arritmia en los estudios que analizan a los pacientes en hemodiálisis (HD). Objetivo: Investigar la prevalencia, perfil clínico y manejo terapéutico de los pacientes con FA en HD en Andalucía. Métodos: Solicitamos al sistema sanitario público de Andalucía el número de pacientes que estaban siendo tratados con HD. Pedimos a los nefrólogos responsables de todos los centros hospitalarios y extrahospitalarios de 5 de las 8 provincias de Andalucía que realizaran un electrocardiograma y cumplimentaran un cuestionario en pacientes seleccionados por un muestreo aleatorizado simple. Resultados: Estaban en HD 2.348 pacientes en las 5 provincias incluidas. El tamaño muestral estimado fue 285 pacientes. Obtuvimos electrocardiograma e información de 252 (88,4%). Edad media 65,3 ± 16 años; 40,9% mujeres. Tenían FA 63 pacientes (25%). De estos, 36 (14,3%) tenían FA en el registro realizado y en el resto había sido documentada previamente. En el análisis multivariante, mayor edad (OR: 1,071; IC 95%: 1,036-1,107; p = 0,000) y mayor tiempo en HD (OR: 1,009; IC 95%:1,004-1,014; p = 0,000) se asociaron de forma independiente con la FA. De los pacientes con FA, el 41,3% estaban en tratamiento anticoagulante en el momento del estudio y el 41,2% con antiagregantes. Conclusiones: La FA en las unidades de diálisis es un importante hallazgo. Establecer la relación riesgo-beneficio del tratamiento anticoagulante constituye un auténtico reto. Son necesarios ensayos clínicos bien diseñados para establecer el uso racional del tratamiento antitrombótico. Keywords: End-stage chronic kidney failure, Haemodialysis, Atrial fibrillation, Antithrombotic therapy, Palabras clave: Insuficiencia renal crónica terminal, Hemodiálisis, Fibrilación auricular, Tratamiento antitrombótic

Fibrilación auricular en los pacientes en hemodiálisis en Andalucía. Prevalencia, perfil clínico y manejo terapéutico

Resumen: La fibrilación auricular (FA) es un importante problema social y sanitario. Existe una amplia variación en la prevalencia de esta arritmia en los estudios que analizan a los pacientes en hemodiálisis (HD). Objetivo: Investigar la prevalencia, perfil clínico y manejo terapéutico de los pacientes con FA en HD en Andalucía. Métodos: Solicitamos al sistema sanitario público de Andalucía el número de pacientes que estaban siendo tratados con HD. Pedimos a los nefrólogos responsables de todos los centros hospitalarios y extrahospitalarios de 5 de las 8 provincias de Andalucía que realizaran un electrocardiograma y cumplimentaran un cuestionario en pacientes seleccionados por un muestreo aleatorizado simple. Resultados: Estaban en HD 2.348 pacientes en las 5 provincias incluidas. El tamaño muestral estimado fue 285 pacientes. Obtuvimos electrocardiograma e información de 252 (88,4%). Edad media 65,3 ± 16 años; 40,9% mujeres. Tenían FA 63 pacientes (25%). De estos, 36 (14,3%) tenían FA en el registro realizado y en el resto había sido documentada previamente. En el análisis multivariante, mayor edad (OR: 1,071; IC 95%: 1,036-1,107; p = 0,000) y mayor tiempo en HD (OR: 1,009; IC 95%:1,004-1,014; p = 0,000) se asociaron de forma independiente con la FA. De los pacientes con FA, el 41,3% estaban en tratamiento anticoagulante en el momento del estudio y el 41,2% con antiagregantes. Conclusiones: La FA en las unidades de diálisis es un importante hallazgo. Establecer la relación riesgo-beneficio del tratamiento anticoagulante constituye un auténtico reto. Son necesarios ensayos clínicos bien diseñados para establecer el uso racional del tratamiento antitrombótico. Abstract: Atrial fibrillation (AF) represents an important social and healthcare problem. There is wide variability in the prevalence of this arrhythmia in studies analysing patients on haemodialysis (HD). Objective: To investigate the prevalence, clinical profile and therapeutic management of patients with AF on HD in Andalusia. Methods: We asked the public healthcare system of Andalusia to provide us with the number of patients who were being treated with HD. We asked attending nephrologists from all hospital and outpatient centres in 5 of the 8 Andalusian provinces to perform an electrocardiogram and to fill out a questionnaire on patients selected by simple random sampling. Results: A total of 2,348 patients were being treated with HD in the 5 provinces included in the study. The estimated sample size was 285 patients. We obtained an electrocardiogram and information from 252 patients (88.4%); mean age 65.3 ± 16 years; 40.9% women. Sixty-three patients (25%) had AF. Of these, 36 (14.3%) had AF in the recorded ECG and in the rest it had been documented previously. In the multivariate analysis, older age (OR: 1.071; 95% CI: 1.036-1.107; P = 0.000) and greater time on HD (OR: 1.009; 95% CI: 1.004-1.014; P = 0.000) were independently associated with the presence of AF. Of the patients with AF, 41.3% were on anticoagulant treatment at the time of the study; and 41.2% were on antiplatelet agents. Conclusions: AF in dialysis units is an important finding. Establishing the risk-benefit ratio of anticoagulant treatment constitutes a real challenge. Well-designed clinical trials are pivotal in order to define the rational use of antithrombotic drugs. Palabras clave: Insuficiencia renal crónica terminal, Hemodiálisis, Fibrilación auricular, Tratamiento antitrombótico, Keywords: End-stage chronic kidney failure, Haemodialysis, Atrial fibrillation, Antithrombotic therap