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. Author manuscript; available in PMC: 2026 Oct 6.
Published before final editing as: Can J Cardiol. 2026 Jun 9:S0828-282X(26)00521-0. doi: 10.1016/j.cjca.2026.05.023

Identification of a Novel Genetic Variant responsible for Familial Atrial Fibrillation

Rebecca Martínez-Moreno 1, Alexandra Pérez-Serra 1, Gopi Shah 2, Elisabet Roura 1, Albert Rigat Pujolàs 1, Anna Iglesias 1, Bernat del Olmo 1, Kui Hong 3, John D Rioux 4,5, Fabiana S Scornik 1,6,7, Guillermo J Pérez 1,6,7, Robert Roberts 8, Ramon Brugada 1,6,7,9
PMCID: PMC13635781  NIHMSID: NIHMS2208562  PMID: 42263934

Abstract

Background:

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Although familial AF (FAF) frequently follows an autosomal dominant inheritance pattern, the genetic mechanisms remain incompletely defined. We sought to identify the causal variant within a previously established linkage region on chromosome 10q22–q24.

Methods:

Additional recombination events and fine mapping reduced the original linkage interval to 0.8 Mb. Targeted sequencing of the locus was performed in affected and unaffected family members. Patient-specific induced pluripotent stem cells (hiPSCs) were differentiated into atrial cardiomyocytes (hiPSC-aCMs) to assess gene expression and ion channel function.

Results:

Refinement of the locus identified three genes (KCNMA1; DLG5; and POLR3A). A novel 15-kb tandem duplication within intron 1 of KCNMA1 segregated with disease in six families and was absent in controls and population databases. Patient-derived hiPSC-aCMs demonstrated reduced KCNMA1 and POLR3A mRNA and protein expression, accompanied by decreased paxilline-sensitive outward current.

Conclusion:

We identify a rare intronic structural variant in KCNMA1 associated with familial AF. The variant segregates with disease and is associated with reduced KCNMA1 expression and BK channel activity in patient-specific cardiomyocytes. These findings implicate the 10q22–q24 locus and highlight the potential contribution of non-coding structural variation to inherited atrial arrhythmia.

Keywords: Atrial Fibrillation, hiPSC, KCNMA1, linkage analysis, cardiac channelopathies

1. Introduction

Atrial fibrillation (AF) is the most common cardiac arrhythmia, with a prevalence of 2% in the general population and 12% in individuals older than 80 years1,2. It is characterized by uncoordinated atrial electrical activity leading to rapid and irregular heartbeat3–5,3–5. Symptoms include fatigue, dizziness, dyspnea, reduced exercise tolerance, chest pain, and syncope4. AF is associated with increased cardiovascular mortality due to stroke, myocardial infarction, and heart failure6. Its presentation is heterogeneous, ranging from highly symptomatic cases to incidental diagnoses. Based on episode duration, AF is classified as paroxysmal (<7 days, self-terminating), persistent (>7 days), or permanent5,6.

Despite extensive investigation, the molecular mechanisms underlying AF onset and maintenance remain incompletely understood1,6. Established risk factors include age, ethnicity, and sex1,5. The familial form (FAF) is less common7, but linkage and candidate gene studies have identified multiple genetic loci associated with the disease8,9. Following our initial identification of a locus on chromosome 10q22–q248, additional genetic contributors have been described. Variants in RYR2, SCN5A, and its regulatory β subunits have been linked to AF1,6. Potassium channel genes including KCNQ1, KCNE1, KCNH2, and KCNJ2 have also been implicated, with most variants producing gain-of-function effects that shorten action potential duration or promote early afterdepolarizations9–12. The role of the large-conductance calcium-activated potassium channel KCa1.1 (BK, MaxiK, Slo1), encoded by KCNMA1 within the 10q22–q24 locus, has been less clear. Early studies failed to detect KCNMA1 transcripts in the heart. However, Pineda et al.13 demonstrated KCa1.1 expression in mouse and human atrial cardiomyocytes at RNA and protein levels. Additional studies suggest roles in mitochondrial function and protection against ischemia-reperfusion injury14–16. KCa1.1 expression has also been reported in cardiac fibroblasts17, telocytes18, coronary artery smooth muscle19 and intracardiac neurons,20,21, and accumulating evidence links it to heart rate regulation16,22–24. Together, these findings suggest a potential role for KCa1.1 in AF pathophysiology.

Here, we investigated the cellular mechanisms underlying FAF in a family exhibiting autosomal dominant inheritance. This family, along with two others, was included in our original 1997 study8. Subsequent identification of three additional families narrowed the locus to 0.8 Mb. Direct sequencing identified a 15-kb tandem duplication in intron 1 of KCNMA1 that segregated with disease and was absent in controls and population databases (DGV, gnoAD-SV v4.1 and TOPMed SV (dbVar accession nstd229)). To investigate functional consequences, we generated patient-specific human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs), enriched toward an atrial phenotype using retinoic acid (hiPSC-aCMs)25,26, providing a patient-specific model to study arrhythmogenic mechanisms2,5.

2. Materials and methods

Family history.

The study was approved by the local ethical committee. The original family consisted of 34 known members of three generations, twelve of them affected with the disease (Figure 1). The index case (IV-9) was diagnosed (age 22), with paroxysmal AF, which evolved to chronic AF over the years. The rest of the members suffered from long-standing, persistent AF. Five additional families from the same geographic region were collected. Out of the 105 members, 41 of them were patients with AF.

Figure 1.

Figure 1.

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Top: pedigrees of the original families with Familial Atrial Fibrillation. Bottom, additional pedigrees identified from the same geographical region. Circles denote female members and squares denote male members. Filled symbols are clinically affected members, and symbols with a slash represent deceased members. A skin biopsy was obtained from individual IV-9 in family 22–001

Genomic DNA.

Genomic DNA was obtained from venous whole blood with the Chemagic Magnetic Separation Module I (MSM I) from Perkin Elmer.

Microsatellite analysis.

In the three additional families, microsatellite analyses were performed to determine whether they shared the same locus. The samples were PCR-amplified with fluorescently tagged microsatellite and run on an ABI PRISM® 3100 Genetic Analyzer using the GeneScan™ 500 LIZ® Size Standard.

SNP analysis.

To further define the genetic crossovers in the genetic locus, SNP fine mapping was performed using MassArray technology (Sequenom) following manufacturer instructions.

Locus sequencing analysis.

Illumina's Nextera Rapid Capture custom enrichment kit (NRC) was used for DNA library preparation. DNA samples were fragmented and uniquely barcoded for paired-end sequencing using NRC index 1 and index 2 primers. Final DNA libraries were normalized to 2–3nM and sequenced on the Illumina HiSeq2500 sequencer using a 100-base paired-end double index read format.

Data analysis.

DNA reads were mapped to an annotated reference sequence (Hg19) to determine the extent of variation from this reference. Our laboratory has developed an in-house pipeline for secondary data analysis (see supplementary methods).

Sanger sequencing.

Primer 3 v3.1 was used for specific primer design. Rare variants were amplified and sequenced by Sanger technology (see supplementary methods).

Generation of hiPSC lines

This study was approved by the Advisory Committee for Human Tissue and Cell Donation and Use at the Instituto de Salud Carlos III (P11/2015). Written informed consent was obtained from the participants included in the study.

We collected a skin biopsy of individual IV-9 (ESi108-A, from now on referred to as patient), from which fibroblasts were obtained. Reprogramming and characterization of induced pluripotent stem cells (hiPSCs) was performed as previously published27. Home-grown hiPSCs derived from a healthy donor (IDIBGIi001-A) were used as control 28.

Cardiomyocyte differentiation

hiPSC were differentiated into atrial cardiomyocytes (hiPSC-aCM) following a 3-step protocol. First, differentiation into cardiomyocytes was performed as previously described29, supplemented with 1 mM Retinoic Acid (Sigma-Aldrich-Merck, Saint Louis, MO, USA) from day 5 to day 826. Second, glucose-free medium (SILAC, Gibco-Thermo Fisher, Waltham, MA USA) was used on days 10 to 16 and cells were then re-plated. Third, from days 17 to 30 cells were fed daily with differentiation medium supplemented with 10 μg/mL insulin, 1 μM dexamethasone and 0.1 μM 3,3′,5-triiodo-L-thyronine. On day 30, cells were collected for protein or RNA extraction or disaggregated to perform electrophysiological studies.

RNA extraction and reverse transcription quantitative PCR (RT-qPCR)

mRNA was extracted using a protocol based on the QIAzol Lysis Reagent (QIAGEN, Hilden, Germany). Cells were collected in QIAzol, incubated 10’ with 100% chloroform, and centrifuged 15’ at 4°C. The superior transparent phase was transferred to another tube and incubated for 10’ with 100% isopropanol. Samples were centrifuged 10’ at 4°C, and pellet was washed 5 times with 70% ethanol. Pellet was incubated at 55°C until ethanol had evaporated and dissolved in nuclease-free H2O. Extracted RNA was quantified using NANODROP-1000 (Thermo Fisher), and retro-transcribed to cDNA using the QuantiTect Reverse Transcription Kit (QIAGEN) according to the manufacturer’s specifications.

Real-time quantitative PCR (qPCR) in hiPSC-CM

Expression of pluripotency transcription factors (Nanog, Sox-2, Oct-4), cardiac gene markers (NKX2–5, TNNT2, ACTN2), atrial markers (MYH6, MYL7), ventricular markers (MYH7, MYL2), and of the genes located in the region of interest (DLG5, POLR3A, KCNMA1) was quantified using the TaqMan probes listed in Supplemental Table S1. PPIA was used as a housekeeping gene. Commercially available total RNA from human adult atrium (R1234126 and R1234127, Biochain, Newark, CA, USA) was used as a control in the assessment of atrial and ventricular expression. Data analysis was performed with the ThermoFisher Connect tool (https://www.thermofisher.com/es/es/home/digital-science/thermo-fisher-connect.html). Relative gene expression was calculated by the 2^−ΔCt method using PPIA as the housekeeping gene.where ΔCt = Cttarget - CtPPIA. When evaluating atrial versus ventricular identity, expression values were additionally normalized to TNNT2 to correct for potential differences in cardiomyocytes yield between differentiations.

Western blot

Western blot experiments were performed, developed, and analyzed as previously described (Martinez-Moreno, 2024). PVDF membranes were incubated with the antibodies listed in Supplemental Table S2. A total of 6 independent biological replicates (differentiation rounds) for each cell line were divided into 3 (for KCNMA1 and POLR3A) and 2 (for DGL5) independent western blot experiments.

Protein values were quantified by densitometry, normalized to total protein per lane measured from stain-free gels and, where appropriate, to TNNT2 expression. To reduce variability between blots, densitometric values were further normalized by the sum of all values within the same blot before group comparisons. Western blot membranes were cut horizontally prior to antibody incubation to allow probing for multiple targets on the same membrane. As a result, full-length blots and molecular weight markers are not visible on all membrane fragments. Protein bands were identified based on their expected molecular weight and antibody specificity. For each experiment, all replicates used for quantification are provided as uncropped images in the Supplementary Material.

Electrophysiological studies

Cells were disaggregated on day 30 of differentiation to obtain single cells for electrophysiological studies. KCa1.1 current was studied at room temperature using the whole-cell patch clamp configuration. Solution composition (in mmol/l): Bath: 140 NaCl, 3 KCl, 10 HEPES, 1.8 CaCl2 and 1.2 MgCl2, pH 7.4 (NaOH). Pipette: 110 KCl, 3.8 EGTA, 10 HEPES, 10 NaCl, 3.3 CaCl2 and 2 ATP-Mg2+, pH 7.2 (KOH). Bath and pipette solution osmolality was adjusted with glucose to around 297 and 281 mOsm, respectively. When indicated, 100 nM paxilline was added to the bath solution by gravity perfusion.

Statistical analysis

Statistical analyses were performed using OriginPro, Version 2022b. OriginLab Corporation, Northampton, MA, USA. Technical replicate measurements from the same biological sample were averaged before statistical analysis, so that each independent differentiation round contributed a single value to group comparisons. Data distribution was assessed for normality using the Kolmogorov–Smirnov test. Comparisons between two independent groups were performed using a two-tailed unpaired Student’s t test. Data were considered statistically significant when p < 0.05. The number of biological replicates or recorded cells for each experiment is indicated in the corresponding figures or legends.

3. Results

3.1. Genetic screening

In 1997, the study performed by our group on three independent families affected with AF enabled us to link the disease to the gene locus 10q22–q248. Three additional families were identified from the same geographical area in the following years (Figure 1). Haplotype analyses with known markers showed that most affected individuals shared the full chromosomal locus described in the original publication.

However, in family 22–006, there was a recombination in chromosomal marker D10s607. In addition, in family 22–011 there was one individual (RB3043) with an additional recombination in chromosomal marker D10S1752. This narrowed the region to 1.3 cM from the original 11 cM. Fine mapping with a SNP Mass Array system (Sequenom), narrowed the locus to 796 Kb with a final haplotype bounded by SNPs rs42455 and rs1149745.

The locus contains three genes: KCNMA1, DLG5 and POLR3A which were completely sequenced in several affected individuals with non-exonic variants detected. Subsequently, the whole genetic locus was fully sequenced in 28 members of the family (11 controls and 17 affected). A total of 14 single nucleotide variants (SNV) with MAF < 0.1% shared among all affected individuals were found (Supplemental Table S3).

Of the fourteen variants, two had not been previously reported: one intergenic and one intronic DLG5 variant. Both were identified in a group of control DNAs. Additionally, a 15 Kb tandem duplication located in intron 1 of the KCNMA1 gene was identified among all patients and in none of the controls. (Figure 3).

Figure 3.

Figure 3.

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Schematic representation of the locus region where the tandem intronic duplication within the KCNMA1 gene was identified.

3.2. Potential effect of the tandem duplication on the locus regulatory elements

Extensive promoter–enhancer contacts have been observed across the 5′ portion of the KCNMA1 locus (exons 1–4; see orange arcs Figure 4), consistent with a regulatory landscape that is sensitive to alterations in local chromatin architecture. The tandem duplication is predicted to introduce an additional CTCF-binding site, a key architectural protein implicated in loop formation, boundary definition, and the modulation of topologically associating domains (TADs)

Figure 4.

Figure 4.

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Schematic representation of the locus region and its regulatory regions

The presence of an ectopic CTCF site could modify local chromatin looping patterns and potentially alter enhancer–promoter contact frequencies (for example, involving the proximal enhancer GH10J077415), a scenario that may be compatible with reduced transcriptional output of KCNMA1.

3.3. Patient and control hiPSC successfully differentiate into atrial-like CMs

In this study, we used hiPSC-aCMs as a cellular model. We differentiated hiPSC from an AF patient and a healthy non-related control towards atrial cardiomyocytes using retinoic acid, as previously reported26,30. We generated and characterized the hiPSC from both the patient and the control. We next aimed to confirm that the generated hiPSC-aCMs showed characteristics of cardiomyocyte-type cells at both the RNA and protein level, with an enrichment of atrial cardiac markers which would serve as a more reliable cellular model for the study of AF.

We first confirmed by qPCR that the expression levels of the studied pluripotency transcription factors (Nanog, Sox-2, Oct-4) in the generated hiPSC-aCMs from the patient and the control were severely decreased, compared with their respective hiPSC lines (Figure 5 and Supplemental Table S4).

Figure 5. Expression of pluripotency transcription factors is significantly decreased in both the patient and the control derived hiPSC-aCMs, compared to their respective hiPSCs.

Figure 5

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Dot-plot with superimposed box-plot depicts the distribution of the relative expression of Nanog (left), Sox-2 (middle), and Oct-4 (right) for 1 sample of hiPSCs and 6 rounds of differentiation of each individual hiPSC-aCM

In addition, Figure 6A and Supplemental Table S5 shows that, compared to the hiPSCs, the studied cardiac gene markers (NKX2–5, TNNT2, ACTN2) were significantly expressed in the hiPSC-aCMs. At the protein level, while both the patient’s and the control’s differentiated hiPSC-aCMs expressed the corresponding translated proteins (alpha-actinin-2, troponin T, homeobox protein Nkx-2.5), we could not detect them in their respective hiPSC parental lines (Figure 5B).

Figure 6. Cardiac marker expression is increased in patient and control derived hiPSC-aCMs, compared to their respective hiPSCs.

Figure 6

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A) Dot-plot with superimposed box-plot depicts the distribution of the relative expression of ACTN2 (left), TNNT2 (middle), and NKX2–5 (right) for 1 sample of hiPSCs and 6 differentiation rounds of each individual hiPSC-aCM. B) Western blot detection of proteins ACTN2 (left, up), TNNT2 (left, middle), and NKX2–5 (left, bottom) and corresponding total protein blotted membrane (right) from the hiPSC-aCM of the individuals under study. Dot-plot with superimposed box-plot (bottom) depicts the distribution of the ACTN2 (left), TNNT2 (middle), and NKX2–5 (right) protein expression for triplicates of six differentiation rounds of each individual’s hiPSC-aCM, normalized by total protein expression.

We assessed whether mRNA from the differentiated hiPSC-aCM overexpressed atrial markers (MYH6 and MYL7, encoding for the myosin heavy chain 6 and the myosin light chain 7 respectively) in comparison to ventricular markers (MYH7 and MYL2, encoding for the myosin heavy chain 7 and the myosin light chain 2 respectively). Since none of the established differentiation protocols yield a pure population of cardiomyocytes, we used the TNNT2 values to normalize the data obtained for the atrial and ventricular markers expression in the qPCR experiments. In addition, we used RNA from atria as a control. As seen in Figure 7A and Supplemental Table S6, hiPSC-aCMs derived from both the patient and the control highly expressed the studied atrial markers, in comparison with the ventricular ones. Furthermore, we found that this increase is more pronounced in the hiPSC-aCMs in comparison with the atrium sample, especially the MYH6/MYH7 ratio.

Figure 7. Atrial markers are upregulated in patient and control derived hiPSC-aCMs, compared to ventricular markers.

Figure 7

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A) Dot-plot with superimposed box-plot depicts the distribution of the relative expression of MYH6/MYH7 ratio (left), and MYL7/MYL2 ratio (right) for 1 sample of atrium and 6 differentiation rounds of each individual hiPSC-aCM. B) Western blot detection of proteins TNNT2 (left, up), MYL7 (left, middle), and MYL2 (left, bottom) and corresponding total protein blotted membrane (right) from the hiPSC-aCM of the individuals under study. Dot-plot with superimposed box-plot (bottom) depicts the distribution of the MYL7/MYL2 ratio for triplicates of six differentiation rounds of each individual’s hiPSC-aCM, normalized by total protein and TNNT2 expression.

At the protein level, we analyzed the expression of myosin light chain 7 (MYL7) and myosin light chain 2 (MYL2). Similar to the qPCR experiments, data were normalized to TNNT2 expression, and atrial tissue protein was used as a control. Figure 7B further confirms that differentiated hiPSC-aCMs from both the patient and the control show increased expression of the atrial isoform MYL7 relative to the ventricular isoform MYL2.

3.4. hiPSC-aCMs derived from the patient show less expression of both KCNMA1 and POLR3A compared to the control

We characterized by qPCR and western blot the expression of the genes POLR3A, DLG5, and KCNMA1, present in the 10q22–q24 locus, and their respective proteins (RNA polymerase III subunit A, Discs Large MAGUK Scaffold Protein 5, and calcium-activated potassium channel subfamily M alpha 1 or KCa1.1), in hiPSC-aCM derived from a patient diagnosed with familial atrial fibrillation who carries the intronic duplication in intron 1 of KCNMA1.

As shown in Figure 8A and Supplemental Table S7, both POLR3A and KCNMA1 relative mRNA expression is significantly decreased in hiPSC-aCM derived from the patient, compared to the control. The relative expression of DLG5 is similar in both individuals. Western blot analysis further confirmed the results obtained by qPCR. As shown in Figure 8B, KCa1.1 and POLR3A expression in the patient-derived hiPSC-aCMs is significantly lower than in the control’s hiPSC-aCMs. DLG5 is not affected at the protein level (Supplemental Figure S1).

Figure 8. Decreased POLR3A and KCNMA1 expression in patient-derived hiPSC-aCMs.

Figure 8.

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(A) Dot plot with superimposed box plot showing relative mRNA expression of DLG5 (left), POLR3A (middle), and KCNMA1 (right) across six independent differentiation rounds for each hiPSC-aCM line.

(B) Representative Western blots of POLR3A (top), KCa1.1 (middle), and TNNT2 (bottom), with corresponding total protein staining. Quantification (bottom) shows protein levels of POLR3A (left), and KCa1.1 (right), normalized to total protein and TNNT2. P < 0.05 vs control hiPSC-aCM, n=6.

3.5. Paxilline-sensitive outward current is decreased in patient-derived hiPSC-aCM compared to an unrelated control

Since we detected a decrease in KCa1.1 total protein expression in hiPSC-aCM derived from the patient, in comparison with the control, we evaluated the cell membrane expression of the ion channel.

Using the whole-cell patch clamp technique, we recorded outward currents from hiPSC-aCM derived from the patient and from one unrelated control. In agreement with what we observed at the mRNA expression levels, we found a decrease in the paxilline-sensitive outward current in the patient hiPSC-aCMs (Figure 9).

Figure 9. Paxilline-sensitive outward current is decreased in patient-derived hiPSC-aCMs with respect to an unrelated control.

Figure 9

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A: Traces show representative currents from control (top) and patient (bottom) hiPSC-aCMs in the absence and in the presence of 100 nM paxilline. Currents were obtained by whole-cell patch clamp. A step protocol from −20 mV to 100 mV at 5 mV intervals was applied. B: Graph depicts paxilline sensitive peak outward currents measured at the end of the traces obtained by a pulse protocol as in A. n= 3 (control) and 4 (AFib), where n refers to number of cells measured, coming from different differentiation rounds.

4. Discussion

We previously established linkage between AF and the 10q22–q24 locus in three families8 Inclusion of three additional families narrowed this region to 0.8 Mb, containing a fragment of KCNMA1, DLG5, and POLR3A. Within this interval, we identified a novel tandem duplication in intron 1 of KCNMA1 that segregates with disease and is absent from controls, including large population structural variant databases (DGV, gnomAD-SV v4.1, and TOPMed SV (dbVar accession nstd229)). In patient-derived hiPSC-aCMs, this variant is associated with reduced KCNMA1 and POLR3A expression and decreased paxilline-sensitive current, supporting a functional effect at the cellular level. Although we cannot rule out that the duplication had a regulatory effect on distant genes, our findings point at KCNMA1 as the most likely candidate gene.

KCNMA1 encodes the α-subunit of the large-conductance calcium- and voltage-activated potassium channel (KCa1.1, BK, MaxiK, Slo1)31 a channel widely expressed in excitable tissues where it limits hyperexcitability by promoting membrane hyperpolarization and reducing L-type Ca2+ channel activity31. Although early studies did not detect cardiac expression 32,33, subsequent work identified mitochondrial BK channels (mitoBK) in guinea pig and rat cardiomyocytes14,32, and the DEC splice variant has been linked to cardioprotection 15. Pineda et al. demonstrated a weak striation pattern with relatively stronger expression in the intercalated disk of human right atrial tissue and in isolated atrial cardiomyocytes from adult wild-type mice. In addition, they showed that KCa1.1 was present in human atrial cardiomyocyte T-tubules, sarcoplasmic reticulum, sarcolemma, nucleus, and intercalated discs13, suggesting a complex subcellular distribution. Genetic and functional studies in mice, zebrafish, and Drosophila further implicate KCNMA1 in heart rate13,22–24 and ischemia/reperfusion injury 16.

While animal models provide important insight, interspecies differences in atrial electrophysiology limit direct extrapolation to human AF5,6 Computational human atrial models have been developed33,34, but require biological validation. In this context, patient-specific hiPSC-aCMs provide a complementary platform to evaluate the functional consequences of rare variants within their native genetic background. Although hiPSC-aCMs do not fully recapitulate adult atrial physiology, they enable controlled assessment of gene expression and ion channel activity in human cells.

Patient-derived hiPSC-aCMs showed reduced KCNMA1 transcript and protein levels compared with independent control lines. We acknowledge that these differences could reflect not only the AF-linked locus but also variability in genetic background, cell age, batch effects, and cell composition across differentiation rounds. Although isogenic controls would strengthen causal inference, the variant identified here is a 15-kb intronic tandem duplication, making precise CRISPR correction or knock-in technically demanding and beyond the scope of this study. To reduce differentiation-related variability, we analyzed material from at least six independent differentiation rounds and normalized expression data to TNNT2 as a marker of cardiomyocyte content. The reproducibility of the reduction across multiple differentiations argues against a single-batch effect.

In contrast to our findings, Pineda et al. (2021) reported increased KCNMA1 expression in atrial tissue from patients with AF13. The higher KCNMA1 expression observed by these authors may reflect remodeling-related or compensatory changes associated with chronic AF, as well as differences in tissue composition, not present in our hiPSC-aCM model. Importantly, our findings suggest that deviation from physiological KCNMA1 expression, whether increased or decreased, may be associated with atrial arrhythmogenesis.

Horváth et al. (2020) questioned the reproducibility of BK currents in hiPSC-aCMs35. However, the reduction in paxilline-sensitive current observed here is consistent with the decreased KCNMA1 expression and aligns with prior evidence supporting a role for BK channels in cardiac rhythm regulation13,16,22–24

We observed a large reduction in outward current at voltages above the main physiological range for atrial repolarization, with only modest effects up to about +50 mV. However, it is possible that in intact myocytes, Ca2+ levels and the activation of KCa1.1 channels at physiological voltage might be much greater. Thus, a greater role in intact atrial tissue, particularly in AF, cannot be excluded, but it was not directly tested in the present study and remains to be explored. Whether reduced KCa1.1 function contributes to AF through atrial cardiomyocytes, intracardiac neurons, or other cardiac cell types remains unknown. Importantly, cells carrying the 10q22–q24 locus from six AF families showed reduced KCNMA1 mRNA, KCa1.1 protein, and paxilline-sensitive outward current.

POLR3A, also located within the refined locus, encodes the largest subunit of RNA polymerase III, a ubiquitously expressed enzyme essential for non-coding RNA synthesis. Pathogenic variants in POLR3A have primarily been associated with neurodegenerative disorders such as spastic paraplegia and cerebellar ataxia36,37. Although reduced POLR3A expression was observed in patient-derived cells, its specific contribution to the AF phenotype remains undefined and requires further investigation.

Overall, our data provide genetic and functional evidence implicating the 10q22–q24 locus in familial AF. We identify a rare intronic structural variant in KCNMA1 that segregates with the disease across multiple families and is associated with reduced gene expression and BK channel activity in patient-derived cardiomyocytes. While the precise molecular mechanism linking this duplication to atrial electrophysiology remains to be elucidated, these findings expand the spectrum of ion channel-related mechanisms in inherited AF and highlight the potential contribution of non-coding structural variation to arrhythmia susceptibility.

Supplementary Material

1

Figure 2.

Figure 2.

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Additional recombination detected in two of the families, which narrowed the locus to the final 800 Kb, between STR markers D10S607 (Family 22–006) and D10S1752 (Family 22–011)

Acknowledgments

We would like to thank the patients for their generous sample donations. The CIBERCV is an initiative of the Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness.

Funding Sources:

This work was supported by Fondation Leducq, Doris Duke Clinical Scientist Development Award, NIH R01HL66169, AHA Scientist Development Grant, Obra social “la Caixa”; and Centro Nacional de Investigaciones Cardiovasculares (Grant No. CNIC-03-2008). Elisabet Roura was a recipient of SLT028/23/000317, from Departament de Salut (PERIS) – Generalitat de Catalunya, and the Foundation of the Montreal Heart Institute. This research was made possible in part by infrastructure supported by the Canada Foundation for Innovation (Project numbers 202832 and 202695). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

Footnotes

Conflict of interest: The authors have no conflicts to disclose.

Patient Consent Statements: The authors confirm that a patient consent form(s) has been obtained for this article

Declaration of generative AI and AI-assisted technologies

During the preparation of this work the authors used ChatGPT in order to revise grammar and shorten sections of the article to meet the word limit. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

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Data availability:

All data generated in this study are available in the manuscript and Supplementary Information. Additional data is available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2208562-supplement-1.docx (23.8MB, docx)

Data Availability Statement

All data generated in this study are available in the manuscript and Supplementary Information. Additional data is available from the corresponding author upon reasonable request.

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