Original article
Augmented potassium current is a shared phenotype for two genetic defects associated with familial atrial fibrillation

https://doi.org/10.1016/j.yjmcc.2009.07.020Get rights and content

Abstract

Mutations in multiple genes have been implicated in familial atrial fibrillation (AF), but the underlying mechanisms, and thus implications for therapy, remain ill-defined. Among 231 participants in the Vanderbilt AF Registry, we found a mutation in KCNQ1 (encoding the α-subunit of slow delayed rectifier potassium current [IKs]) and separately a mutation in natriuretic peptide precursor A (NPPA) gene (encoding atrial natriuretic peptide, ANP), both segregating with early onset lone AF in different kindreds. The functional effects of these mutations yielded strikingly similar IKs “gain-of-function.” In Chinese Hamster Ovary (CHO) cells, coexpression of mutant KCNQ1 with its ancillary subunit KCNE1 generated ∼ 3-fold larger currents that activated much faster than wild-type (WT)-IKs. Application of the WT NPPA peptide fragment produced similar changes in WT-IKs, and these were exaggerated with the mutant NPPA S64R peptide fragment. Anantin, a competitive ANP receptor antagonist, completely inhibited the changes in IKs gating observed with NPPA S64R. Computational simulations identified accelerated transitions into open states as the mechanism for variant IKs gating. Incorporating these IKs changes into computed human atrial action potentials (AP) resulted in 37% shortening (120 vs. 192 ms at 300 ms cycle length), reflecting loss of the phase II dome which is dependent on L-type calcium channel current. We found striking functional similarities due to mutations in KCNQ1 and NPPA genes which led to IKs “gain-of-function”, atrial AP shortening, and consequently altered calcium current as a common mechanism between diverse familial AF syndromes.

Introduction

Atrial fibrillation (AF) is the most common cardiac arrhythmia affecting ∼ 2% of the US population and resulting in substantial morbidity and mortality [1], [2], [3], [4]. While most AF is associated with other cardiac or systemic disorders, 10–30% of individuals with AF have no evidence of structural heart disease (so called idiopathic or ‘lone’ AF) [5], [6]. There is now accumulating evidence that genetic factors play a role in the pathogenesis of lone AF [7], [8]. Identification of specific electrophysiologic abnormalities in genetically-defined AF holds the promise of moving therapy from the current empiric approach to one that is mechanism-based.

The role of gene variants in the pathogenesis of AF has only recently begun to be appreciated [7], [9]. KCNQ1, the first disease gene identified for familial AF [10], encodes the pore-forming α-subunit of the potassium channel that conducts the slow component of the delayed rectifier potassium current (IKs) in the atrium and ventricle. Functional analysis of the S140G mutant revealed a gain-of-function, which contrasts with the dominant negative or loss-of-function effects of KCNQ1 mutations previously associated with the congenital long QT syndrome [11]. Similarly, small kindreds with AF and mutations in other potassium channel genes, including KCNE2 [12], KCNJ2 [13], and KCNA5 [14], and in genes encoding sodium channel α- and β-subunits have been reported [15], [16].

To date, familial AF has been linked to mutations in potassium and sodium channels that are predicted to either shorten or lengthen the duration of the cardiac action potential (AP). However, a recent report has identified a novel molecular genetic basis for AF with the identification of a mutation in the natriuretic peptide precursor A gene (NPPA), which encodes atrial natriuretic peptide (ANP) [17]. This mutation segregated with familial AF, thereby uncovering an unexpected association between a defect in a circulating hormone and susceptibility to AF. Although the precise mechanism through which the mutant ANP leads to the development AF is not completely clear, a shortening of the atrial AP duration was demonstrated in an isolated rat heart model.

The three coding exons of NPPA transcribe a 151 amino acid peptide called preproANP (Fig. 1) [18], [19], [20]. This peptide then undergoes dual modification involving a signal peptidase and the enzyme corin to produce ANP, the mature 28 amino acid carboxy terminal end, and a 98 amino acid N-terminus [21], [22]. The N-terminal peptide undergoes further degradation to produce a 16 amino acid peptide called long acting natriuretic peptide (LANP), a vessel dilator, and kaliuretic hormone [23], [24]. These additional peptides generated from preproANP have been shown to have biological activity similar to that of the mature ANP [23], [24], [25].

Here we report genetic analyses of KCNQ1 and NPPA in a large cohort of individuals with lone AF or AF associated with heart disease, as well as in population controls. We identified mutations in both genes that cosegregated with AF in separate kindreds. In vitro studies demonstrated strikingly similar gain-of-function defects associated with the mutant forms. Computational simulations of atrial APs incorporating wild-type (WT) or mutant IKs demonstrated a mutation-dependent change in AP configuration – AP shortening due to loss of the AP “dome” with premature stimuli – known to increase AF susceptibility [26], [27].

Section snippets

Study subjects

Subjects prospectively enrolled between November 2002 and October 2005 in the Vanderbilt AF Registry, which comprises clinical and genetic databases, were studied. Individuals enrolled in the Registry were greater than 18 years old with an ECG-confirmed diagnosis of AF. Subjects were excluded if AF was diagnosed in the setting of recent cardiac surgery or if they were unable to give informed consent or report for follow-up. The study protocol was approved by the Institutional Review Board of

Mutation screening

During the 3-year enrollment period, 245 patients with AF were approached and 231 (94%) subjects agreed to participate in the study. This study cohort included 98 patients (42%) with lone AF and 133 with AF associated with heart disease. The majority of subjects were Caucasian (94%), and 5% were African-American. AF was diagnosed at a mean age of 50 ± 14 years.

In the study cohort of 231 individuals with AF, screening for KCNQ1 mutations in genomic DNA identified a unique sequence variant in a

Discussion

In this study we identified mutations in KCNQ1 (IAP54–56) and NPPA (S64R) in moderate-sized Caucasian kindreds with early onset familial AF and normal QT intervals. Although both these genes have previously been linked with familial AF, this is the first study to demonstrate that augmented potassium current is a shared phenotype across these diverse genetic defects associated with familial AF. IKs gain-of-function mutations have previously been reported for potassium channel defects, but the

Funding sources

This work was supported in part by NIH grants HL75266, HL092217 and AHA award 0940116N to Dr. Darbar and HL65962 to Dr. Roden.

Acknowledgment

We are deeply indebted to Dr. Al George for his insight and guidance in the preparation of this manuscript.

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