While the sodium channel locus SCN10A has been implicated by genome-wide association studies as a modulator of cardiac electrophysiology, the role of its gene product Nav1.8 as a modulator of cardiac ion currents is unknown.
We determined the electrophysiological and pharmacological properties of Nav1.8 in heterologous cell systems and assessed the antiarrhythmic effect of Nav1.8 block on isolated mouse and rabbit ventricular cardiomyocytes.
Methods and results
We first demonstrated that Scn10a transcripts are identified in mouse heart and that the blocker A-803467 is highly specific for Nav1.8 current over that of Nav1.5, the canonical cardiac sodium channel encoded by SCN5A. We then showed that low concentrations of A-803467 selectively block “late” sodium current and shorten action potentials in mouse and rabbit cardiomyocytes. Exaggerated late sodium current is known to mediate arrhythmogenic early afterdepolarizations in heart, and these were similarly suppressed by low concentrations of A-803467.
SCN10A expression contributes to late sodium current in heart, and represents a new target for antiarrhythmic intervention.
SCN10A; sodium channels; heart; afterdepolarizations; arrhythmia
The Kir 6.1 Katp channel is believed to play an important role in ventricular repolarization as determined from both functional and genetic studies of the potassium inwardly-rectifying channel, subfamily J, member 8 (KCNJ8)-S422L missense mutation in patients with J-wave syndromes. Although Kir6.1 is also present in atrial tissue, it is unknown whether this channel modulates atrial repolarization and hence whether the S422L mutation portends a greater risk of atrial arrhythmias. This study sought to examine whether there was an increased frequency of the KCNJ8-S422L mutation among patients with atrial fibrillation (AF) and early repolarization (ER) as a possible novel susceptibility gene for AF.
Methods and results
A total of 325 lone AF probands were identified from the Vanderbilt AF Registry, a collection of clinical data and DNA from consented, consecutively enrolled participants. The coding regions of KCNJ8 were sequenced, and the patient's presenting electrocardiogram (ECG) was reviewed by two independent physicians for ER abnormalities. The KCNJ8-S422L mutation was identified in two AF probands while no other candidate gene variants were identified in these cases. Twenty-two (7%) patients were found to have ER on the ECG, including the two probands carrying the S422L variant. In one small AF kindred, the S422L variant co-segregated with AF and ER.
The KCNJ8-S422L variant is associated with both increased AF susceptibility and ER indicating a role for Kir 6.1 Katp channel in both ventricular and atrial repolarization.
Atrial fibrillation; Early repolarization; KCNJ8; Kir6.1; Mutation
Routine integration of genotype data into drug decision-making could improve patient safety, particularly if many relevant genetic variants can be assayed simultaneously before target drug prescribing. The frequency of pharmacogenetic prescribing opportunities and the potential adverse events (AE) mitigated are unknown. We examined the frequency with which 56 medications with known outcomes influenced by variant alleles were prescribed in a cohort of 52,942 medical home patients at Vanderbilt University Medical Center. Within a five-year window, we estimated that 64.8% (95% CI: 64.4%-65.2%) of individuals were exposed to at least one medication with an established pharmacogenetic association. Using previously published results for six medications with well-characterized, severe genetically-linked AEs, we estimated that 398 events (95% CI, 225 - 583) could have been prevented with an effective preemptive genotyping program. Our results suggest that multiplexed, preemptive genotyping may represent an efficient alternative approach to current single use (“reactive”) methods and may improve safety.
During the past few years, the development of effective, empirical technologies for treatment of cardiac arrhythmias has exceeded the pace at which detailed knowledge of the underlying biology has accumulated. As a result, although some clinical arrhythmias can be cured with techniques such as catheter ablation, drug treatment and prediction of the risk of sudden death remain fairly primitive. The identification of key candidate genes for monogenic arrhythmia syndromes shows that to bring basic biology to the clinic is a powerful approach. Increasingly sophisticated experimental models and methods of measurement, including stem cell-based models of human cardiac arrhythmias, are being deployed to study how perturbations in several biologic pathways can result in an arrhythmia-prone heart. The biology of arrhythmia is largely quantifiable, which allows for systematic analysis that could transform treatment strategies that are often still empirical into management based on molecular evidence.
Variable function and expression of drug transporters have been proposed as mechanisms contributing to variable response to drug therapy. Block of the HERG channel, encoding IKr, can lead to serious arrhythmias, and a key drug-blocking site in HERG has been identified on the intracellular face of the pore. We begin to advance the hypothesis that active drug uptake enhances IKr block.
Methods and Results
Reverse transcriptase–polymerase chain reaction identified expression in the human atrium and ventricle of 14 of 31 candidate drug uptake and efflux transporters, including OCTN1 (SLC22A4), a known uptake transporter of the HERG channel blocker quinidine. In situ hybridization and immunostaining localized OCTN1 expression to cardiomyocytes. The IC50 for quinidine block of IKr in CHO cells transfected with HERG alone was significantly higher than cells transfected with HERG + OCTN1 (0.66 ± 0.15 μM versus 0.14 ± 0.06 μM [52% absolute increase in drug block; 95% confidence interval, 0.4–0.64 μM]), and this effect was further potentiated by a common genetic variant of OCTN1, L503F. In the absence of OCTN1, quinidine block could be 91% ± 5% washed out, but with the transporter, washout was incomplete (57% ± 6%). OCTN1 coexpression also facilitated HERG block by flecainide and ibutilide, but not erythromycin.
Coexpression of the organic cation transporter, OCTN1, expressed in human cardiac myocytes, intensifies quinidine-induced HERG block. These findings establish a critical hypothesis that variable drug transporter activity may be a potential risk factor for torsade de pointes.
antiarrhythmia agents; arrhythmia; pharmacology; pharmacokinetics; ion channels
Female gender is a risk factor for long QT-related arrhythmias, but the underlying mechanisms remain uncertain. Here, we tested the hypothesis that gender-dependent function of the post-depolarization ‘late’ sodium current (INa-L) contributes.
Methods and results
Studies were conducted in mice in which the canonical cardiac sodium channel Scn5a locus was disrupted, and expression of human wild-type SCN5A cDNA substituted. Baseline QT intervals were similar in male and female mice, but exposure to the sodium channel opener anemone toxin ATX-II elicited polymorphic ventricular tachycardia in 0/9 males vs. 6/9 females. Ventricular INa-L and action potential durations were increased in myocytes isolated from female mice compared with those from males before and especially after treatment with ATX-II. Further, ATX-II elicited potentially arrhythmogenic early afterdepolarizations in myocytes from 0/5 male mice and 3/5 female mice.
These data identify variable late INa as a modulator of gender-dependent arrhythmia susceptibility.
Mouse; Late sodium current; Gender; Arrhythmias
We genotyped 326 “frequently medicated” individuals of European-descent in Vanderbilt’s biorepository linked to de-identified electronic medical records, BioVU, on the ADME Core Panel to assess quality and performance of the assay. We compared quality control metrics and determined the extent of direct and indirect marker overlap between the ADME Core Panel and the Illumina Omni1-Quad. We found the quality of the ADME Core Panel data to be high, with exceptions in select copy number variants (CNVs) and markers in certain genes (notably CYP2D6). Most of the common variants on the ADME panel are genotyped by the Omni1, but absent rare variants and CNVs could not be accurately tagged by single markers. Finally, our frequently medicated study population did not convincingly differ in allele frequency from reference populations, suggesting that heterogeneous clinical samples (with respect to medications) follow similar allele frequency distributions in pharmacogenetics genes as their appropriate reference populations.
ADME; pharmacogenomics; pharmacogenetics; BioVU; biorepository; CYP2D6; personalized medicine; precision medicine
Although multiple lines of evidence suggest variable expression of the cardiac sodium channel gene SCN5A plays a role in susceptibility to arrhythmia, little is known about its transcriptional regulation.
We used in silico and in vitro experiments to identify possible non-coding sequences important for transcriptional regulation of SCN5A. The results were extended to mice in which a putative regulatory region was deleted.
Methods and Results
We identified 92 non-coding regions highly conserved (>70%) between human and mouse SCN5A orthologs. Three conserved non-coding sequences (CNS) showed significant (>5-fold) activity in luciferase assays. Further in vitro studies indicated one, CNS28 in intron 1, as potential regulatory region. Using Recombinase-Mediated Cassette Exchange (RMCE), we generated mice in which a 435 bp region encompassing CNS28 was removed. Animals homozygous for the deletion showed significant increases in SCN5A transcripts, NaV1.5 protein abundance, and sodium current measured in isolated ventricular myocytes. ECGs revealed a significantly shorter QRS (10.7±0.2ms in controls vs. 9.7±0.2ms in knockouts) indicating more rapid ventricular conduction. In vitro analysis of CNS28 identified a short 3′ segment within this region required for regulatory activity and including an E-box motif. Deletion of this segment reduced reporter activity to 3.6±0.3% of baseline in CHO cells and 16±3% in myocytes (both P<0.05), and mutation of individual sites in the E-box restored activity to 62±4% and 57±2% of baseline in CHO cells and myocytes, respectively (both P<0.05).
These findings establish that regulation of cardiac sodium channel expression modulates channel function in vivo, and identify a non-coding region underlying this regulation.
Gene Expression Regulation; Sodium Channels; Mice; Transgenic
To identify novel genetic loci influencing interindividual variation in red blood cell (RBC) traits in African-Americans, we conducted a genome-wide association study (GWAS) in 2315 individuals, divided into discovery (n = 1904) and replication (n = 411) cohorts. The traits included hemoglobin concentration (HGB), hematocrit (HCT), RBC count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Patients were participants in the electronic MEdical Records and GEnomics (eMERGE) network and underwent genotyping of ~1.2 million single-nucleotide polymorphisms on the Illumina Human1M-Duo array. Association analyses were performed adjusting for age, sex, site, and population stratification. Three loci previously associated with resistance to malaria—HBB (11p15.4), HBA1/HBA2 (16p13.3), and G6PD (Xq28)—were associated (P ≤ 1 × 10−6) with RBC traits in the discovery cohort. The loci replicated in the replication cohort (P ≤ 0.02), and were significant at a genome-wide significance level (P < 5 × 10−8) in the combined cohort. The proportions of variance in RBC traits explained by significant variants at these loci were as follows: rs7120391 (near HBB) 1.3% of MCHC, rs9924561 (near HBA1/A2) 5.5% of MCV, 6.9% of MCH and 2.9% of MCHC, and rs1050828 (in G6PD) 2.4% of RBC count, 2.9% of MCV, and 1.4% of MCH, respectively. We were not able to replicate loci identified by a previous GWAS of RBC traits in a European ancestry cohort of similar sample size, suggesting that the genetic architecture of RBC traits differs by race. In conclusion, genetic variants that confer resistance to malaria are associated with RBC traits in African-Americans.
red blood cell (RBC) traits; genome-wide association study; African-Americans; natural selection; informatics; electronic medical record
Genome-wide association (GWA) studies have identified numerous common polymorphisms associated with atrial fibrillation (AF). The 3 loci most strongly associated with AF occur at chromosome 4q25 (near PITX2), 16q22 (in ZFHX3), and 1q21 (in KCNN3).
To evaluate if timing of AF recurrence after direct current cardioversion (DCCV) is modulated by common AF susceptibility alleles.
208 patients (age 65±11 years, 77% men) with persistent AF underwent successful DCCV and were prospectively evaluated at 3, 6 and 12 months for AF recurrence. Four single nucleotide polymorphisms (SNPs): rs2200733 and rs10033464 at 4q25; rs7193343 in ZFHX3 and rs13376333 in KCNN3 were genotyped.
The final study cohort consisted of 184 patients. In 162 (88%) patients sinus rhythm was restored with DCCV, of which 108 (67%) had AF recurrence at a median of 60 (29 – 176) days. In multivariable analysis the presence of any common SNP (rs2200733, rs10033464) at the 4q25 locus was an independent predictor of AF recurrence, (hazard ratio [HR]: 2.1, 95% confidence interval [CI]:1.21–3.30, P=0.008). Furthermore, rs2200733 exhibited a graded allelic dose response for early AF recurrence (homozygous variants: 7 [4–56] days, heterozygous: 54 [28–135] days and wild type: 64 [29–180] days, P=0.03).
To our knowledge, this is the first study to evaluate whether genomic markers can predict timing of AF recurrence in patients undergoing elective DCCV. Our findings show that a common polymorphism on chromosome 4q25 (rs2200733) is an independent predictor of AF recurrence after DCCV and point to a potential role of stratification by genotype.
atrial fibrillation; direct current cardioversion; genetic predictors; recurrence
Atrial fibrillation (AF) is the most-common sustained arrhythmia observed in clinical practice, but response to therapy is highly variable between patients. Current drug therapies to suppress AF are incompletely and unpredictably effective and carry substantial risk of proarrhythmia and noncardiac toxicities. The limited success of therapy for AF is partially the result of heterogeneity of the underlying substrate, interindividual differences in disease mechanisms, and our inability to predict response to therapies in individual patients. In this Review, we discuss the evidence that variability in response to drug therapy is also conditioned by the underlying genetic substrate for AF. Increased susceptibility to AF is mediated through diverse genetic mechanisms, including modulation of the atrial action-potential duration, conduction slowing, and impaired cell-to-cell communication, as well as novel mechanisms, such as regulation of signalling proteins important in the pathogenesis of AF. However, the translation of genetic data to the care of the patients with AF has been limited because of poor understanding of the underlying mechanisms associated with common AF-susceptibility loci, a dearth of prospective, adequately powered studies, and the challenges associated with determining efficacy of antiarrhythmic drugs. What is apparent, however, is the need for appropriately designed, genotype-directed clinical trials.
Atrial fibrillation (AF) is the most common cardiac arrhythmia and carries a significant risk of stroke and heart failure. The molecular etiologies of AF are poorly understood, leaving patients with limited therapeutic options. AF has been recognized as an inherited disease in almost 30% of patient cases. However, few genetic loci have been identified and the mechanisms linking genetic variants to AF susceptibility remain unclear. By sequencing 193 probands with lone AF, we identified a Q76E variant within the coding sequence of the bone morphogenetic protein (BMP) antagonist gremlin-2 (GREM2) that increases its inhibitory activity. Functional modeling in zebrafish revealed that, through regulation of BMP signaling, GREM2 is required for cardiac laterality and atrial differentiation during embryonic development. GREM2 overactivity results in slower cardiac contraction rates in zebrafish, and induction of previously identified AF candidate genes encoding connexin-40, sarcolipin and atrial natriuretic peptide in differentiated mouse embryonic stem cells. By live heart imaging in zebrafish overexpressing wild-type or variant GREM2, we found abnormal contraction velocity specifically in atrial cardiomyocytes. These results implicate, for the first time, regulators of BMP signaling in human AF, providing mechanistic insights into the pathogenesis of the disease and identifying potential new therapeutic targets.
Randomized studies have shown that ventricular rate-control is an acceptable treatment strategy in patients with atrial fibrillation (AF). However, identification of patients who will adequately respond to rate control therapy remains a challenge.
In this study, we evaluated the impact of two common β1-adrenergic receptor (β1-ADR) polymorphisms (G389R and S49G) on response to ventricular rate control therapy in patients with AF.
We studied 543 subjects (63% men; age 61.8 ± 14) prospectively enrolled in the Vanderbilt AF registry and managed with rate control strategy. A ‘responder’ displayed adequate ventricular rate control based on the AFFIRM criteria: 1. Average heart rate (HR) at rest ≤80 bpm and 2. Maximum HR during 6-min walk test ≤110 bpm or average HR during 24-hr Holter ≤100 bpm.
295 (54.3%) met the AFFIRM criteria. Baseline clinical characteristics were similar in responders and non-responders except for mean resting heart rate (76 ± 20 vs. 70 ± 15; P <0.01) and smoking (6% vs. 1%; P <0.01). Multiple clinical variables (age, gender, HTN) failed to predict response to rate-control therapy. By contrast, carriers of Gly variant at 389 were more likely to respond favorably to rate control therapy; 60% vs. 51% in Arg389Arg genotype, P = 0.04. This association persisted after correction for multiple clinical factors (OR 1.42, 95% CI 1.00–2.03, P<0.05). Among responders, subjects carrying the Gly389 variant required the lowest doses of rate-control meds; atenolol 92 mg vs. 68 mg; carvedilol 44 mg vs. 20 mg; metoprolol 80 mg vs. 72mg; diltiazem 212 mg vs. 180 mg and verapamil 276 mg vs. 200 mg respectively (P < 0.01 for all comparisons).
We have identified a common β1-ADR polymorphism, G389R that is associated with adequate response to rate control therapy in AF patients. Gly389 is a ‘loss of function’ variant, consequently, for the same adrenergic stimulation it produces reduced levels of adenyl cyclase and hence attenuates the β-adrenergic cascade. Mechanistically, the effect of rate-control drugs will be synergistic with that of the Gly389 variant, which could possibly explain our findings. These findings represent a step forward in development of a long-term strategy of selecting treatment options in AF based on genotype.
atrial fibrillation; genomics; beta-adrenergic receptor; polymorphisms; rate control
The degree of QT prolongation by drug is highly variable and related to risk for polymorphic ventricular tachycardia due to drugs.
to determine factors that affect the degree of QT prolongation by drugs.
QT and QTc were measured before and after administration of the QT-prolonging drug ibutilide in 253 normal volunteers aged 18-40 years. Drug effect on QTc prolongation was defined as ΔQTc = QTc after drug minus QTc before drug.
Ibutilide prolonged QT from 396 ± 31 ms to 418 ± 39 ms (P<0.001) and QTc from 406 ± 15 ms to 446 ± 33 ms (P<0.001). The ΔQTc did not correlate with baseline QTc (Pearson correlation 0.016, P = 0.8). Post-drug QTc was correlated weakly with pre-drug QTc (Pearson correlation 0.484, p<0.001), and strongly with ΔQTc (Pearson correlation 0.882, P < 0.001). ΔQTc was identical for men and women (39 ± 29 ms vs. 39 ± 27 ms, P = 0.9), but displayed significant differences among body mass index categories (P<0.001). Overweight (48 ± 27 ms) and obese (61 ± 31 ms) subjects had significantly more QT prolongation by drug than normal (31 ± 25 ms) or underweight (24 ± 12 ms) subjects.
QT prolongation by ibutilide does not correlate to baseline QTc, and does not differ between men and women. Overweight and obese subjects have greater drug effect on QTc than subjects with normal or low body mass index. These findings have implications for drug-induced long QT syndrome.
Electrocardiography; Obesity; Ion channels; Pharmacology; QT interval
This study tested the hypothesis that two common polymorphisms in the chromosome 4q25 region that have been associated with atrial fibrillation (AF) contribute to the variable penetrance of familial AF.
Although mutations in ion channels, gap junction proteins, and signaling molecules have been described for Mendelian forms of AF, penetrance is highly variable. Recent studies have consistently identified 2 common single nucleotide polymorphisms (SNPs) in the chromosome 4q25 region as independent AF susceptibility alleles.
We studied 11 families in which AF was present in ≥2 individuals who also shared a candidate gene mutation. These mutations were identified in all subjects with familial lone AF (n=33) as well as apparently unaffected family members (age >50 yrs with no AF; n=17).
Mutations were identified in SCN5A (n=6); NPPA (n=2); KCNQ1 (n=1); KCNA5 (n=1) and NKX2.5 (n=1). In genetic association analyses, un-stratified and stratified according to age of onset of AF and unaffected age > 50 yrs, there was a highly statistically significant association between the presence of both common (rs2200733, rs10033464) as well as rare variants and AF (un-stratified P=1×10−8; stratified [age of onset <50 yrs and unaffected age >50 yrs], P=7.6×10−5) (un-stratified P<0.0001; stratified [age of onset <50 yrs and unaffected age >50 yrs], P<0.0001). Genetic association analyses showed that the presence of common 4q25 risk alleles predicted whether carriers of rare mutations developed AF (P = 2.2×10−4).
Common AF-associated 4q25 polymorphisms modify the clinical expression of latent cardiac ion channel and signaling molecule gene mutations associated with familial AF. These findings support the idea that the genetic architecture of AF is complex and includes both rare and common genetic variants.
Patients vary in their responses to drug therapy, and some of that variability is genetically-determined. This review outlines general approaches used to identify genetic variation that influences drug response. Examples from specific therapeutic areas are presented: cholesterol management, arrhythmias, heart failure, hypertension, warfarin anticoagulation, and anti-platelet agents. A brief view of potential pathways to implementation is presented.
genetics; pharmacogenetics; pharmacogenomics; drug therapy
Atrial fibrillation (AF) is the most common sustained arrhythmia affecting over 700,000 individuals in Japan and 2.2 million in the USA. The proper management of patients with AF is critical due to the well-documented association with heart failure and stroke. A strategy to better define the emergency department (ED) management, admission decisions, and spectrum of risk from low to high is needed.
Methods and subjects
The Atrial Fibrillation and Flutter Outcomes & Risk Determination investigation is a prospective, observational cohort study to develop a multivariable clinical prediction rule that accurately estimates risk for adverse outcomes in patients presenting to the ED with symptomatic AF. We will enroll 430 patients at 2 sites who present to the ED with symptomatic AF defined as a new or established diagnosis of AF or atrial flutter that require ED evaluation for a complaint thought related to their rhythm disturbance. The study’s endpoint is to develop an accurate, objective, internally validated, reliable clinical prediction rule to risk-stratify ED patients presenting with AF exacerbations. The rule will incorporate patient history and examination findings and laboratory studies obtained upon ED presentation, as well as trends over the first 2 hours of care. This investigation’s primary outcome is the incidence of any AF-related adverse event at 5 days and 30 days. We expect to complete the study by the end of 2014. The study was registered at Clinicaltrials.gov NCT01138644.
Atrial fibrillation; Atrial flutter; Prognosis; Emergency care
Atrial fibrillation (AF) is more common in those with obstructive sleep apnea (OSA) than in unaffected individuals and recurs more frequently in the presence of severe OSA after electrical cardioversion and AF ablation. However, it is unknown whether severity of OSA influences the efficacy of anti-arrhythmic drug (AAD) therapy in patients with OSA and AF. This study examined the impact of OSA severity on treatment of symptomatic AF with AADs. We studied 61 patients (62 ± 15 years; 21 women) treated with AADs for symptomatic AF who had overnight polysomnography. Rhythm control was prospectively defined as successful if a patient remained on the same AAD therapy for a minimum of 6 months with ≥75% reduction in symptomatic AF burden. Twenty-four patients (40%) had severe OSA. Thirty patients (49%) were rhythm controlled with AADs. Non-responders to AADs were more likely to have severe OSA than milder disease (52% vs 23%; p < 0.05); those with severe OSA were less likely to respond to AADs than participants with non-severe OSA (39% vs 70%; p = 0.02). Non-responders had higher apnea-hypopnea indices than responders (34 ± 25 vs 22 ± 18 events/hour; p = 0.05), but there were no differences between these groups in minimum oxygen saturation or % time spent in REM sleep. In conclusion, patients with severe OSA are less likely to respond to AAD therapy for AF than those with milder forms of OSA.
obstructive sleep apnea; atrial fibrillation; anti-arrhythmic drugs