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The two-pore domain potassium channel, K2P3.1 (TASK-1) modulates background conductance in isolated human atrial cardiomyocytes and has been proposed as a potential drug target for atrial fibrillation (AF). TASK-1 knockout mice have a predominantly ventricular phenotype however, and effects of TASK-1 inactivation on atrial structure and function have yet to be demonstrated in vivo. The extent to which genetic variation in KCNK3, that encodes TASK-1, might be a determinant of susceptibility to AF is also unknown. To address these questions, we first evaluated the effects of transient knockdown of the zebrafish kcnk3a and kcnk3b genes and cardiac phenotypes were evaluated using videomicroscopy. Combined kcnk3a and kcnk3b knockdown in 72 hour post fertilization embryos resulted in lower heart rate (p<0.001), marked increase in atrial diameter (p<0.001), and mild increase in end-diastolic ventricular diameter (p=0.01) when compared with control-injected embryos. We next performed genetic screening of KCNK3 in two independent AF cohorts (373 subjects) and identified three novel KCNK3 variants. Two of these variants, present in one proband with familial AF, were located at adjacent nucleotides in the Kozak sequence and reduced expression of an engineered reporter. A third missense variant, V123L, in a patient with lone AF, reduced resting membrane potential and altered pH sensitivity in patch-clamp experiments, with structural modeling predicting instability in the vicinity of the TASK-1 pore. These in vitro data suggest that the double Kozak variants and V123L will have loss-of-function effects on ITASK. Cardiac action potential modeling predicted that reduced ITASK prolongs atrial action potential duration, and that this is potentiated by reciprocal changes in activity of other ion channel currents. Our findings demonstrate the functional importance of ITASK in the atrium and suggest that inactivation of TASK-1 may have diverse effects on atrial size and electroph

Both shortening and prolongation of the QTc interval have been associated with atrial fibrillation (AF). We investigated whether 8 single nucleotide polymorphisms (SNPs) at loci previously shown to affect QTc interval duration were associated with lone AF.

Right precordial (V1-3) elevated electrode placement ECG (EEP-ECG) is often used in the diagnosis of Brugada syndrome (BrS). However, the specificity of this has only been studied in smaller studies in Asian populations. We aimed to study this in a larger European population.

The clinical diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) is often challenging due to phenotypic variation, reduced/age-related penetrance, and lack of a diagnostic test. A single report has suggested quantitative myocardial immunoanalysis for the desmosomal protein plakoglobin as a diagnostic test with high sensitivity and specificity. We performed immunohistochemistry for plakoglobin and a control protein on myocardial biopsies with fibrofatty replacements from 50 consecutive, unrelated patients. The clinical, genetic, and immunohistochemical data were evaluated by independent observers in a blinded manner. The immunohistochemical and clinical diagnoses were compared and the sensitivity, specificity, and predictive values calculated. Our analysis showed 37 samples (74%) with a reduced immunosignal for plakoglobin. Of the 34 patients with a clinical diagnosis of ARVC, 29 displayed a reduced plakoglobin signal. Of the 14 patients with a clinical diagnosis other than ARVC, 6 displayed a reduced signal. Two patients were excluded from further analysis. A sensitivity of 85%, a specificity of 57%, a positive predictive value of 83%, and a negative predictive value of 62% were found. In conclusion, immunohistochemical analysis for plakoglobin, applied as a diagnostic test for ARVC, seems associated with a relatively high sensitivity, but limited specificity, and although additional validation is required, we advocate caution in basing clinical decision-making on the proposed diagnostic test.

AimsGenetic factors may be important in the development of atrial fibrillation (AF) in the young. KCNA5 encodes the potassium channel a-subunit K(V)1.5, which underlies the voltage-gated atrial-specific potassium current I(Kur). KCNAB2 encodes K(V)ß2, a ß-subunit of K(V)1.5, which increases I(Kur). Three studies have identified loss-of-function mutations in KCNA5 in patients with idiopathic AF. We hypothesized that early-onset lone AF is associated with high prevalence of genetic variants in KCNA5 and KCNAB2.Methods and resultsThe coding sequences of KCNA5 and KCNAB2 were sequenced in 307 patients with mean age of 33 years at the onset of lone AF, and in 216 healthy controls. We identified six novel non-synonymous mutations [E48G, Y155C, A305T (twice), D322H, D469E, and P488S] in KCNA5 in seven patients. None were present in controls. We identified a significantly higher frequency of rare deleterious variants in KCNA5 in the patients than in controls. The mutations were analysed with confocal microscopy and whole-cell patch-clamp techniques. The mutant proteins Y155C, D469E, and P488S displayed decreased surface expression and loss-of-function in patch-clamp studies, whereas E48G, A305T, and D322H showed preserved surface expression and gain-of-function for K(V)1.5.ConclusionThis study is the first to present gain-of-function mutations in KCNA5 in patients with early-onset lone AF. We identified three gain-of-function and three loss-of-function mutations. We report a high prevalence of variants in KCNA5 in these patients. This supports the hypothesis that both increased and decreased potassium currents enhance AF susceptibility.

BACKGROUND: Three distinct genetic loci on chromosomes 1q21, 4q25, and 16q22 have been associated with atrial fibrillation (AF) in genome-wide association studies (GWAS). Five additional loci have been associated primarily with the PR interval and subsequently with AF. We aimed to investigate if 8 single nucleotide polymorphisms (SNPs), representing the 8 genomic loci previously linked with AF in genome-wide association studies, were associated with early-onset lone AF. METHODS: We included 209 patients with early-onset lone AF, and a control group consisting of 534 individuals free of AF. The 8 SNPs were genotyped using TaqMan assays (Applied Biosystems, Foster City, CA). RESULTS: Three SNPs were found to be significantly associated with early-onset lone AF: rs2200733 closest to PITX2 (odds ratio [OR], 1.62; 95% confidence interval [CI], 1.16-2.27; P = 0.004), rs3807989 near to CAV1 (OR 1.35; 95% CI, 1.06-1.72; P = 0.015), and rs11047543 near to SOX5 (OR 1.70; 95% CI, 1.18-2.44; P = 0.004). When correcting for multiple testing, rs2200733 and rs11047543 were still significantly associated with AF. CONCLUSIONS: Three SNPs, rs2200733 (4q25), rs3807989 (7p31), and rs11047543 (12p12), were associated with early-onset lone AF. All 3 SNPs are positioned close to genes that in previous studies have been demonstrated to be important for cardiac morphology/development, thereby suggesting a link between these SNPs and structural heart disease. Our results however, indicate that variants in these 3 loci are associated with AF through mechanisms that do not involve major structural abnormalities in the heart.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia. The cardiac sodium channel, Na(V)1.5, plays a pivotal role in setting the conduction velocity and the initial depolarization of the cardiac myocytes. We hypothesized that early-onset lone AF was associated with genetic variation in SCN5A. METHODS AND RESULTS: The coding sequence of SCN5A was sequenced in 192 patients with early-onset lone AF. Eight nonsynonymous mutations (T220I, R340Q, T1304M, F1596I, R1626H, D1819N, R1897W, and V1951M) and 2 rare variants (S216L in 2 patients and F2004L) were identified. Of 11 genopositive probands, 6 (3.2% of the total population) had a variant previously associated with long QT syndrome type 3 (LQTS3). The prevalence of LQTS3-associated variants in the patients with lone AF was much higher than expected, compared with the prevalence in recent exome data (minor allele frequency, 1.6% versus 0.3%; P=0.003), mainly representing the general population. The functional effects of the mutations were analyzed by whole cell patch clamp in HEK293 cells; for 5 of the mutations previously associated with LQTS3, patch-clamp experiments showed an increased sustained sodium current, suggesting a mechanistic overlap between LQTS3 and early-onset lone AF. In 9 of 10 identified mutations and rare variants, we observed compromised biophysical properties affecting the transient peak current. CONCLUSIONS: In a cohort of patients with early-onset lone AF, we identified a high prevalence of SCN5A mutations previously associated with LQTS3. Functional investigations of the mutations revealed both compromised transient peak current and increased sustained current.

BACKGROUND: The use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted to compare melting curves and sequencing results from wgaDNA derived from DBS samples with gDNA derived from whole blood.METHODS: gDNA was extracted from whole blood obtained from 10 patients with lone atrial fibrillation (mean age 22.3 years). From their newborn DBS samples, stored at -24°C, genomic DNA was extracted and whole-genome amplified in triplicates. Using high resolution melting curve analysis and direct sequencing in both wgaDNA and gDNA samples, all coding regions and adjacent intron regions of the genes SCN5A and KCNA5 were investigated.RESULTS: Altered melting curves was present in 85 of wgaDNA samples and 81 of gDNA samples. Sequence analysis identified a total of 31 variants in the 10 wgaDNA samples. The same 31 variants were found in the exact same pattern of samples in the gDNA group. There was no false positive or negative sequence variation in the wgaDNA group.CONCLUSIONS: The use of DNA amplified in triplicates from DBS samples is reliable and can be used both for high resolution curve melting analysis as well as direct sequence analysis. DBS samples therefore can serve as an alternative to whole blood in sequence analysis.

OBJECTIVE: Genome wide association studies have shown an association between rs2200733 at 4q25 and atrial fibrillation (AF). In this case-control study we investigate the association of rs2200733 and lone AF in young patients.METHODS: We included 196 young patients with lone AF and the first episode before the age of 40 years. We analyzed the single nucleotide polymorphism (SNP) rs2200733 for the lone AF patients and compared them to a control group of 176 age matched healthy individuals.RESULTS: No significant differences, in neither genotype distribution, nor minor allele frequencies were found between the lone AF patients and the healthy controls.CONCLUSION: Our results suggest that the rs220733 is not a risk factor for AF in patients with no other cardiovascular disease and with early onset of the arrhythmia.

OBJECTIVES: The aim of this study was to screen lone atrial fibrillation (AF) patients for mutations in the genes KCNJ2, KCNJ3 and KCNJ5, all encoding potassium channels. Furthermore, we wanted to replicate the prior association of two single-nucleotide polymorphisms (SNPs) in KCNJ5, C171T (rs6590357) and G810T (rs7118824), with lone AF in Han Chinese.METHODS: We sequenced the coding region and splice site of KCNJ2, KCNJ3 and KCNJ5 in 187 early-onset lone-AF patients screening for mutations and counting SNP frequencies for the two noted SNPs in KCNJ5.RESULTS: No mutations were found in KCNJ2, KCNJ3 or KCNJ5. Both genotype distribution and allele frequencies of the SNPs rs6590357 and rs7118824 significantly differed between the AF and control group (p(genotype) = 0.0067, p(allele) = 0.0021 and p(genotype) = 0.014, p(allele) = 0.0101, respectively). On allele level, the OR for lone AF for rs6590357 was 1.77 (95% CI 1.16-2.73, p = 0.009) and for rs7118824 it was 1.71 (95% CI 1.13-2.57, p = 0.01) in a model adjusted for age and gender.CONCLUSIONS: Our findings indicate that rs6590357 and rs7118824 in KCNJ5 are associated with early-onset lone AF in Caucasians. No mutations were found in the exon or splice site of KCNJ2, KCNJ3 or KCNJ5.

Growing evidence exists that living with an ICD can lead to fear and avoidance behaviour including the avoidance of physical activity. It has been suggested that psychological stress can increase the risk of shock and predict death. Small studies have indicated a beneficial effect arising from exercise training and psychological intervention, therefore a large-scale rehabilitation programme was set up.

Systemic inflammation is associated with atrial fibrillation (AF) and inflammatory processes are involved in the pathophysiology of AF. We hypothesized that genetic polymorphisms, which determine the rate of inflammatory cytokines, are associated with increased risk of AF.

Aims The aim of this study was to screen KCNN3 encoding the small-conductance calcium-activated K+ channel (SK3) in lone atrial fibrillation patients. Atrial fibrillation (AF) is the most common cardiac arrhythmia. A genome-wide association study has recently associated an intronic single-nucleotide polymorphism (SNP) in KCNN3 with lone AF. Methods and results We sequenced the coding region and splice junctions of KCNN3 in 209 early-onset lone AF patients, screening for variations. A group of 208 healthy blood donors with normal ECGs and without cardiac symptoms were used as controls. All patients and controls were of Danish ethnicity. No mutations were found in the coding regions or splice sites of KCNN3. We found one known exonic synonymous SNP (rs1131820) in KCNN3 that was associated with AF. Both the genotype distribution and allele frequencies of SNP rs1131820 were significantly different between the AF cases and controls (PGenotype= 0.047 and PAllele= 0.027). Being a homozygous carrier of the major allele (GG) vs. the minor allele (AA) of rs1131820 was associated with an odds ratio of 2.85 (95% CI 1.13–7.18, P = 0.026) for lone AF. Conclusions In this study of 209 young lone AF patients, we found no mutations in the exons or splice sites of KCNN3, but we found an association between the synonymous SNP rs1131820 in KCNN3 and lone AF., Aims The aim of this study was to screen KCNN3 encoding the small-conductance calcium-activated K(+) channel (SK3) in lone atrial fibrillation patients. Atrial fibrillation (AF) is the most common cardiac arrhythmia. A genome-wide association study has recently associated an intronic single-nucleotide polymorphism (SNP) in KCNN3 with lone AF. Methods and results We sequenced the coding region and splice junctions of KCNN3 in 209 early-onset lone AF patients, screening for variations. A group of 208 healthy blood donors with normal ECGs and without cardiac symptoms were used as controls. All patients and controls were of Danish ethnicity. No mutations were found in the coding regions or splice sites of KCNN3. We found one known exonic synonymous SNP (rs1131820) in KCNN3 that was associated with AF. Both the genotype distribution and allele frequencies of SNP rs1131820 were significantly different between the AF cases and controls (P(Genotype)= 0.047 and P(Allele)= 0.027). Being a homozygous carrier of the major allele (GG) vs. the minor allele (AA) of rs1131820 was associated with an odds ratio of 2.85 (95% CI 1.13-7.18, P = 0.026) for lone AF. Conclusions In this study of 209 young lone AF patients, we found no mutations in the exons or splice sites of KCNN3, but we found an association between the synonymous SNP rs1131820 in KCNN3 and lone AF.

INTRODUCTION: Structural right atrial abnormalities have been described in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). However, little is known about electrocardiographic signs of atrial involvement in ARVC because no systematic studies have been conducted. METHODS: P-wave-triggered signal-averaged orthogonal electrocardiogram from 40 ARVC patients (46 ± 15 years, 16 females) was compared with recordings from age- and sex-matched healthy control subjects for assessment of P-wave duration and morphology. P-wave morphology was classified with regard to the P-wave polarity in leads X, Y, and Z. RESULTS: P-wave duration was longer in patients (135 ± 18 vs 124 ± 12 milliseconds; P = .003). Two typical P-wave morphologies were identified in the controls: positive in X and Y and negative (45%) or biphasic (55%) in Z. In patients with ARVC , typical P waves were seen in only 60%, whereas 15 patients (37%) had atypical P-wave positive in all 3 leads (P <.0001). The presence of atypical P waves in the ARVC group was not associated with the presence of either structural or functional right ventricular abnormality. CONCLUSIONS: Patients with ARVC commonly demonstrate deteriorated atrial activation expressed either as prolonged P-wave duration or abnormal P-wave morphology. The P-wave abnormalities were not secondary to right ventricular dilatation. These findings show that atrial involvement is common in ARVC and may represent yet another manifestation of the disease to be considered for inclusion in ARVC diagnostic workup.

INTRODUCTION: Structural right atrial abnormalities have been described in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). However, little is known about electrocardiographic signs of atrial involvement in ARVC because no systematic studies have been conducted. METHODS: P-wave-triggered signal-averaged orthogonal electrocardiogram from 40 ARVC patients (46 ± 15 years, 16 females) was compared with recordings from age- and sex-matched healthy control subjects for assessment of P-wave duration and morphology. P-wave morphology was classified with regard to the P-wave polarity in leads X, Y, and Z. RESULTS: P-wave duration was longer in patients (135 ± 18 vs 124 ± 12 milliseconds; P = .003). Two typical P-wave morphologies were identified in the controls: positive in X and Y and negative (45%) or biphasic (55%) in Z. In patients with ARVC , typical P waves were seen in only 60%, whereas 15 patients (37%) had atypical P-wave positive in all 3 leads (P < .0001). The presence of atypical P waves in the ARVC group was not associated with the presence of either structural or functional right ventricular abnormality. CONCLUSIONS: Patients with ARVC commonly demonstrate deteriorated atrial activation expressed either as prolonged P-wave duration or abnormal P-wave morphology. The P-wave abnormalities were not secondary to right ventricular dilatation. These findings show that atrial involvement is common in ARVC and may represent yet another manifestation of the disease to be considered for inclusion in ARVC diagnostic workup.

Udgivelsesdato: 2010, BACKGROUND: Sinus node dysfunction (SND) is a heterogeneous disorder of unknown etiology characterized by a variety of supraventricular arrhythmias with symptoms of syncope, palpitations, and dizziness. The mechanism underlying the abnormal rhythm is incompletely understood. OBJECTIVE: Because vagal stimulation and acetylcholine (ACh) affect the function of pacemaker cells, we hypothesized that genetic variation in the genes encoding the ACh-activated K(+) channels, the KACh channels, could be involved in the pathogenesis of SND. METHODS AND RESULTS: We screened 184 patients listed in the pacemaker registry of the Copenhagen University Hospital aged <60 years at pacemaker implantation for SND in the period 1982-2005. Forty-three patients fulfilled the following inclusion criteria: documented sinus arrest, asystole, or extreme sinus bradycardia. The coding sequences of KCNJ3 and KCNJ5, encoding the main subunits of the KACh channels, were re-sequenced. We identified several known single nucleotide polymorphisms in KCNJ3 and KCNJ5, but no mutations in either of the genes. CONCLUSIONS: Genetic variation in KCNJ3 and KCNJ5 encoding the subunits of the KACh channels is apparently not involved in the pathogenesis of SND.

Udgivelsesdato: 2009-Oct, AIMS: The aim of this study is to investigate whether protection with rubber or plastic gloves during post-mortem explantation of an implantable cardioverter defibrillator (ICD) offers enough protection for the explanting operator during a worst-case scenario (i.e. ICD shock). METHODS AND RESULTS: We investigated the insulating properties of rubber and plastic gloves (double layer) within the first 60 min exposure (mimicking the maximum time of an explantation procedure) to saline (simulating the effects of body fluids on the gloves). For latex gloves, we measured an increase in voltage up to 68.1 V (P < 0.0001), for neoprene a maximum voltage of 5.3 V (P = 0.245), and for plastic a voltage of 2.3 V within the first hour. If the exposure time to fluid did not exceed 50 min, a double pair of intact gloves made of latex, neoprene, or plastic constituted such a large resistance that the resting voltage over the operating person would not exceed 50 V. CONCLUSION: The use of intact medical gloves made of latex, neoprene, or plastic eliminates the potential electrical risk during explantation of an ICD. Two gloves on each hand offer sufficient protection. We will recommend the use of neoprene gloves.

BACKGROUND: Atrial fibrillation (AF) is the most common clinical arrhythmia and a major cause of cardiovascular morbidity and mortality. Among the gene defects previously associated with AF is a gain of function of the slowly activating delayed rectifier potassium current IKs, secondary to mutations in KCNQ1. Coexpression of KCNE5, the gene encoding the MiRP4 beta-subunit, has been shown to reduce IKs. OBJECTIVE: The purpose of this study was to test the hypothesis that mutations in KCNE5 are associated with AF in a large cohort of patients with AF. METHODS: One-hundred fifty-eight patients with AF were screened for mutations in the coding region of KCNE5. RESULTS: A missense mutation involving substitution of a phenylalanine for leucine at position 65 (L65F) was identified in one patient. This patient did not have a history of familial AF, and neither KCNQ1 nor KCNE2 mutations were found. Transient transfection of Chinese hamster ovary (CHO) cells expressing IKs(KCNQ1+KCNE1) with KCNE5 suppressed the developing and tail currents of IKs in a concentration-dependent manner. Transient transfection with KCNE5-L65F failed to suppress IKs, yielding a current indistinguishable from that recorded in the absence of KCNE5. Developing currents recorded during a test pulse to +60 mV and tail currents recorded upon repolarization to -40 mV both showed a significant concentration-dependent gain of function in IKs with expression of KCNE5-L65F vs KCNE5-WT. CONCLUSION: The results of this study suggest that a missense mutation in KCNE5 may be associated with nonfamilial or acquired forms of AF. The arrhythmogenic mechanism most likely is a gain of function of IKs Udgivelsesdato: 2008/3, BACKGROUND: Atrial fibrillation (AF) is the most common clinical arrhythmia and a major cause of cardiovascular morbidity and mortality. Among the gene defects previously associated with AF is a gain of function of the slowly activating delayed rectifier potassium current IKs, secondary to mutations in KCNQ1. Coexpression of KCNE5, the gene encoding the MiRP4 beta-subunit, has been shown to reduce IKs. OBJECTIVE: The purpose of this study was to test the hypothesis that mutations in KCNE5 are associated with AF in a large cohort of patients with AF. METHODS: One-hundred fifty-eight patients with AF were screened for mutations in the coding region of KCNE5. RESULTS: A missense mutation involving substitution of a phenylalanine for leucine at position 65 (L65F) was identified in one patient. This patient did not have a history of familial AF, and neither KCNQ1 nor KCNE2 mutations were found. Transient transfection of Chinese hamster ovary (CHO) cells expressing IKs(KCNQ1+KCNE1) with KCNE5 suppressed the developing and tail currents of IKs in a concentration-dependent manner. Transient transfection with KCNE5-L65F failed to suppress IKs, yielding a current indistinguishable from that recorded in the absence of KCNE5. Developing currents recorded during a test pulse to +60 mV and tail currents recorded upon repolarization to -40 mV both showed a significant concentration-dependent gain of function in IKs with expression of KCNE5-L65F vs KCNE5-WT. CONCLUSION: The results of this study suggest that a missense mutation in KCNE5 may be associated with nonfamilial or acquired forms of AF. The arrhythmogenic mechanism most likely is a gain of function of IKs.