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Circulation. Author manuscript; available in PMC Sep 13, 2012.
Published in final edited form as:
PMCID: PMC3211046

Defects in ankyrin-based membrane protein targeting pathways underlie atrial fibrillation

Shane R. Cunha, Ph.D.,1,5 Thomas J. Hund, Ph.D.,1,2,4 Seyed Hashemi, M.D.,6 Niels Voigt, M.D.,8 Na Li, M.D.,10 Patrick Wright, B.S.,1 Olha Koval, Ph.D.,6 Jingdong Li, M.D.,1 Hjalti Gudmundsson, M.D.,6 Richard J. Gumina, M.D., Ph.D.,1,2 Matthias Karck, M.D.,9 Jean-Jacques Schott, Ph.D.,11 Vincent Probst, M.D., Ph.D.,11 Herve Le Marec, M.D., Ph.D.,11 Mark E. Anderson, M.D., Ph.D.,6,7 Dobromir Dobrev, M.D.,8 Xander HT Wehrens, M.D., Ph.D.,10 and Peter J Mohler, Ph.D.1,2,3



Atrial fibrillation (AF) is the most common cardiac arrhythmia, affecting over two million patients in the US alone. Despite decades of research, surprisingly little is known regarding the molecular pathways underlying the pathogenesis of AF. ANK2 encodes ankyrin-B, a multifunctional adapter molecule implicated in membrane targeting of ion channels, transporters, and signaling molecules in excitable cells.

Methods and Results

Here, we report early-onset AF in patients harboring loss-of-function mutations in ANK2. In mice, we show that ankyrin-B-deficiency results in atrial electrophysiological dysfunction and increased susceptibility to AF. Moreover, ankyrin-B+/− atrial myocytes display shortened action potentials, consistent with human AF. Ankyrin-B is expressed in atrial myocytes, and we demonstrate its requirement for the membrane targeting and function of a subgroup of voltage-gated Ca2+ channels (Cav1.3) responsible for low-voltage activated L-type Ca2+current. Ankyrin-B directly associates with Cav1.3, and this interaction is regulated by a short, highly-conserved motif specific to Cav1.3. Moreover, loss of ankyrin-B in atrial myocytes results in decreased Cav1.3 expression, membrane localization, and function sufficient to produce shortened atrial action potentials and arrhythmias. Finally, we demonstrate reduced ankyrin-B expression in atrial samples of patients with documented AF, further supporting an association between ankyrin-B and AF.


These findings support that reduced ankyrin-B expression or mutations in ANK2 are associated with atrial fibrillation. Additionally, our data demonstrate a novel pathway for ankyrin-B-dependent regulation of Cav1.3 channel membrane targeting and regulation in atrial myocytes.

Keywords: Ankyrin, atrial fibrillation, calcium channel, ion channel targeting

Atrial fibrillation (AF), which is characterized by rapid and irregular activation of the atrium, is the most common sustained arrhythmia found in clinical practice, affecting ~2.3 million people in the US alone.1 The prevalence of AF increases with age with ~2-6% of people over the age of 65 exhibiting AF.2, 3 Given the demographics of the US, it is predicted that there will be a five-fold increase in the prevalence of AF by 2050.4 While AF is frequently associated with other cardiac disorders including coronary artery disease, mitral valve disease, congenital heart disease, and congestive heart failure, ~30% of all AF cases are described as lone AF where patients exhibit no previous cardiac pathology.5 The majority of monogenic AF cases described to date are linked to mutations that affect potassium channels, although these cases represent only a small fraction of total monogenic AF cases.6

Ankyrin-B (AnkB, encoded by ANK2) is an adapter protein expressed in a number of excitable cells including neurons, cardiomyocytes, and pancreatic beta cells.7 Given that AnkB targets select ion channels, transporters, and signaling molecules to specific membrane domains, it is not surprising that dysfunction in the AnkB cellular pathway has been linked to neuronal defects, ventricular arrhythmia, and neonatal diabetes.8-10

Here, we demonstrate a critical role for AnkB in atrial function. Individuals harboring loss-of-function mutations in ANK2 developed early-onset AF. AnkB deficient mice phenocopy ANK2-based human disease and displayed striking susceptibility to AF. Primary atrial myocytes from AnkB deficient mice displayed shortened action potentials, a hallmark of AF and this decrease was associated with loss of L-type Ca2+ current (ICa,L). Our data revealed that Cav1.3 is a novel AnkB-binding partner and the structural requirements for AnkB/Cav1.3 association were mapped. Specifically, a unique C-terminal motif in CaV1.3 was found to be necessary and sufficient for ankyrin-binding. In AnkB+/− atrial myocytes, Cav1.3 protein expression and membrane function were reduced. Moreover, we demonstrated that AnkB levels are reduced in human AF, providing an association between AnkB and the pathogenesis of AF. Together, our data describe a novel mechanism for Cav1.3 membrane targeting in primary atrial cardiomyocytes and reveal a potential molecular mechanism underlying ankyrin-associated AF.



Myocytes were washed with phosphate-buffered saline (PBS, pH 7.4) and fixed in warm 2% paraformaldehyde (37°C). Cells were blocked/permeabilized in PBS containing 0.075% Triton X-100 and 3% fish oil gelatin (Sigma), and incubated in primary antibody overnight at 4° C. Following washes (PBS +0.1% Triton X-100), cells were incubated in secondary antibody (Alexa 488, 568; and ToPro-3AM 633) for 8 hours at 4°C and mounted using Vectashield (Vector). Images were collected on Zeiss 510 Meta confocal microscope (40 power oil 1.40 NA, pinhole equals 1.0 Airy disc) using Zeiss Imaging software. Images were imported into Adobe Photoshop for cropping and linear contrast adjustment. Imaging experiments were performed at least three times for each experimental protocol, and >20 myocytes were examined from each experimental set.


Protein lysates were isolated from atria and ventricles of WT and heterozygous ankyrin-B mice as described.11 Antibodies and tissues are described in the online-only Data Supplement.

Cellular electrophysiology

Membrane currents and action potentials were measured with an Axon 200B patch-clamp amplifier controlled by a personal computer using a Digidata 1320A acquisition board driven by pClamp 8.0 software (Axon Instruments). Electrophysiological recordings were only obtained from Ca2+-tolerant, rod-shaped atrial cells. We used perforated (amphotericin B) patch for calcium current and action potential studies. All experiments were conducted at room temperature, unless otherwise noted. Other currents were measured as described.12 Recording pipettes, fabricated from borosilicate glass, had resistance of 2–4 MΩ, when filled with recording solution. All solutions were adjusted to 275–295 mOsm.


For human data, differences between group means were compared by unpaired Student’s t-test. Frequency-data were analyzed with Fisher’s exact test. The Kolmogorov-Smirnov test was used to test whether data follow a normal distribution. Assumption of equal variance per group was confirmed using Bartlett’s test. Data are mean±SEM. P<0.05 was considered statistically significant. For non-human data, all values are presented as mean ± SEM. P values were assessed with a paired Student’s t test (two-tailed) or ANOVA, as appropriate, for continuous data. The Bonferroni test was used for post-hoc testing. The null hypothesis was rejected for P < 0.05.

Human tissue samples

Human tissue experimental protocols were approved by the ethics committee of the Medical Faculty Mannheim, University of Heidelberg (#2011-216N-MA). Each patient gave written informed consent. During routine cannulation procedures in patients undergoing open-heart surgery for cardiac bypass grafting and valve replacement, respectively, the tip of the right atrial appendage was removed and immediately snap-frozen in liquid nitrogen. Appendages were obtained from 12 sinus rhythm (SR) and 10 paroxysmal atrial fibrillation (pAF, Supplemental Table 2 in the on-line only Data Supplement) patients. The AF group included patients in sinus rhythm at surgery with a history of at least one episode of self-terminating AF lasting less than 7 days (pAF patients).

Additional Methods are presented in the online-only Data Supplement.


Ankyrin-B dysfunction is associated with human atrial fibrillation

We identified a high incidence of AF in ANK2 mutation-positive probands. Probands harboring ANK2 loss-of-function alleles displayed early onset AF, commonly progressing to permanent AF.9, 13, 14 In one large kindred (74 members), 13 of 25 ANK2 loss-of-function variant carriers (phenotypes previously mapped to ANK2, Zmax=7.059) displayed AF (mean onset 40 ±18 years, 5 paroxysmal, 8 permanent), whereas non-carriers were asymptomatic. Only two individuals were non-penetrant for atrial phenotypes. The others had AF, AF with sinus node dysfunction, or sinus node dysfunction alone. In an unrelated large family, 20/36 individuals were positive for the ANK2 disease allele (maximal LOD score was for marker D4S1616; linkage Zmax=5.9, θ=0), and 3 of 20 disease allele carriers displayed AF (mean onset 48 ±12 years, two paroxysmal, 1 permanent), while thirteen displayed sinus node disease requiring pacemaker implantation (mean age for implantation 30±18 years12). Only four individuals were non-penetrant for atrial phenotypes. In addition to AF, prior work demonstrates that ANK2 variant carriers may display prolonged ventricular QT intervals, which predisposes individuals to ventricular arrhythmias (examples of cardiac phenotypes are presented in Supplemental Table 1 in the on-line only Data Supplement).9, 13 As AF and long QT syndrome (LQTS) are often associated with opposing action potential characteristics (i.e., AF with shortened atrial action potentials; LQTS with prolonged ventricular action potentials), we hypothesized that AnkB might uniquely affect atrial ion channels resulting in an increased susceptibility to AF.

Mice deficient for ankyrin-B expression display atrial dysfunction

Previous studies have demonstrated that human ANK2 gene mutations linked with arrhythmia behave as loss-of-function when analyzed in primary cardiomyocytes.9, 12-14 Therefore, we assessed the role of AnkB deficiency in atria of mice heterozygous for a null mutation in AnkB (AnkB+/− mice, as AnkB−/− mice die at birth). Continuous telemetry ECG recordings from conscious AnkB+/− mice revealed atrial arrhythmias also present in human carriers of ANK2 variants, including spontaneous bradycardia with erratic atrial activity, lack of discrete P waves and variable ventricular response (Figure 1A-D).

Figure 1
Ankyrin-deficient mice display atrial dysfunction and atrial fibrillation (AF) inducibility

We next tested the susceptibility to atrial arrhythmias in AnkB+/− mice following atrial burst pacing.15 Intracardiac electrograms were recorded simultaneously with the surface ECG to confirm the nature of abnormal atrial electrical activities. Periods of pacing-induced AF were observed in the majority (~75%) of AnkB+/− mice (11/15 mice; Figure 1E-F). In contrast, a significantly lower fraction of WT mice demonstrated AF inducibility using this protocol (2/9 WT mice, p<0.05 vs. AnkB+/−; Figure 1F). AnkB+/− atrial episodes showed characteristic absence of P waves and RR interval variability.15 Moreover, atrial fibrillation/tachycardia was confirmed by the presence of rapid and irregular A waves on the atrioventricular electrogram (Figure 1E). Together, the electrophysiological data in human ANK2 mutation carriers and AnkB+/− mice clearly demonstrate that AnkB dysfunction is detrimental for normal atrial electrical activity.

Ankyrin-B is expressed in atria

We used immunoblot to identify AnkB expression patterns in human atria. AnkB was expressed in all human heart chambers at similar levels (Figure 2A). As expected, voltage-gated calcium channel Cav1.2 was most abundant in the right and left ventricle, whereas Cav1.3 was enriched in right and left atria compared with ventricle16 (Figure 2A). At the level of the single atrial cardiomyocyte, AnkB was present at both the peripheral sarcolemma (yellow arrows) as well as the M-line (Figure 2B, white arrows). These data demonstrate that AnkB is expressed in atrial tissue and localized at select membrane domains in isolated primary atrial cells.

Figure 2
Ankyrin-B+/− atrial myocytes display reduced APD and decreased ICa,L

Ankyrin-B+/− atrial myocytes display aberrant atrial electrical activity

To define the role of AnkB in atria, we evaluated action potentials from isolated WT and AnkB+/− atrial cardiomyocytes. Notably, AnkB+/− atrial myocytes displayed significantly reduced action potential durations (measured at 90% of repolarization, APD90) compared with WT atrial cardiomyocytes (Figure 2C-D). In fact, APD90 was reduced ~25% in AnkB+/− atrial cardiomyocytes at both room (Figure 2C-D, n=7 WT, n=8 AnkB+/−, p<0.05) and physiological temperature (Supplemental Figure 1 in the on-line only Data Supplement, n=6 WT, n=5 AnkB+/−, p<0.05), a cellular phenotype consistent with that observed in AF patients.17 In contrast, prior studies revealed that AnkB+/− ventricular cardiomyocytes do not display significant differences in APD90 compared to WT.9

Atrial myocyte depolarization and repolarization is regulated by the synchronized activities of membrane-associated ion channels, transporters, and pumps. To define the molecular basis of action potential shortening in AnkB+/− atrial myocytes, we investigated the activities of a number of critical atrial myocyte currents. We observed a significantly reduced ICa,L in AnkB+/− atrial myocytes (Figure 2E-F, n=7 WT, n=8 AnkB+/−, p<0.05) in contrast to AnkB+/− ventricular myocytes that display normal ICa,L and APD.9 We observed no difference in INa (Figure 2G-H, n=7 WT, n=8 AnkB+/−, N.S.), total IK, or Ito between WT and AnkB+/− atrial myocytes (n=7 WT, n=8 AnkB+/−; N.S.; not shown). However, we did observe a significant decrease in INCX, consistent with previous findings in AnkB-deficient ventricular myocytes (Supplemental Figure 2 in the online-only Data Supplement, n=8 WT, n=8 AnkB+/−, p<0.05).18 Thus, atrial AnkB+/− myocytes display decreased APD and reduced ICa,L. Both phenotypes are unique to AnkB+/− atrial cells compared with ventricular myocytes.

Reduced Cav1.3 expression in ankyrin-B+/− atria

In atrial myocytes, ICa,L is comprised of the combined activities of Cav1.2 (α1C) and Cav1.3 (α1D). Based on reduced ICa,L AnkB+/− in AnkB+− atrial cardiomyocytes, we examined the expression and localization of both Cav1.2 and Cav1.3 in WT and AnkB+/− mouse atria. Consistent with electrophysiological data, we observed a significant decrease in Cav1.3 expression in AnkB+/− atria (reduced ~30%, n=3, p<0.05; Figure 3A-B), whereas Cav1.2 expression was unchanged between genotypes (Figure 3A-B, p=N.S.). Furthermore, in agreement with our earlier studies in ventricle9, 19, Nav1.5 expression levels were not altered in AnkB+/− atrial cells (Figure 3A). Immunofluorescence of individual atrial myocytes using channel-specific antibodies demonstrated that Cav1.3, but not Cav1.2, Cav3.1, or Nav1.5 membrane expression was reduced (Figure 3C-F). In fact, we observed a ~40% decrease in membrane staining intensity for Cav1.3 in AnkB+/− atrial cardiomyocytes compared with WT cells (Figure 3E, p<0.05; n=5 WT, n=6 AnkB+/−).

Figure 3
Reduced Cav1.3 expression and targeting in ankyrin-B+/− atria

As a final test to assess AnkB function in Cav1.3 membrane targeting, we evaluated Cav1.3 membrane activity in primary cardiac fibroblasts from WT and AnkB−/− (homozygous null) mice (harvested before death of AnkB−/− neonatal mice). Primary fibroblasts were chosen to allow recording of ICa,L from exogenously expressed Cav1.3 without confounding native current from endogenous Ca2+ channels (note that WT and AnkB−/− primary fibroblasts display little endogenous ICa, open circles in Figure 3G). As expected, WT fibroblasts expressing Cav1.3 displayed significantly greater ICa,L compared with non-transfected cells (Figure 3G, n=9 WT, n=7 AnkB+/− cells, p<0.05). In contrast, AnkB−/− primary cells expressing Cav1.3 showed >50% reduction in ICa,L (Figure 3G; n=9 WT, n=7 AnkB+/− cells, p<0.05) and reduced Cav1.3 membrane immunofluorescence (Figure 3H) compared to WT cells, despite similar Cav1.3 protein expression in both cell types (based on immunoblot). These phenotypes were directly related to AnkB expression, as exogenous AnkB expression restored ICa,L levels in AnkB−/− cells (Figure 3I-J, n=8, p<0.05). Notably, in contrast to Cav1.3, we observed no functional difference in Cav1.2 membrane expression between wild-type and ankyrin-B-deficient cells (Supplemental Figure 3A-B in the online-only Data Supplement, n=5 WT, n=6 AnkB+/−; N.S.).

Ankyrin-B directly associates with Cav1.3

Based on electrophysiological data from primary myocytes and fibroblasts showing reduced Cav1.3 in AnkB-deficient atrial myocytes, we tested for a direct interaction between AnkB and Cav1.3. Full-length Cav1.3 has five major cytoplasmic domains including an amino terminal domain, three cytoplasmic segments that connect the four major transmembrane regions (DI-DIV), and a large C-terminal domain (Figure 4A). We generated radiolabeled purified protein of each Cav1.3 cytoplasmic region (N1, L1-L3, C1-C4). Due to the extensive length of the C-terminal domain, this region was further subdivided into four segments for direct binding experiments (Figure 4A, C1-C4). We observed significant association between GST-labeled AnkB membrane-binding domain and only one intracellular region of Cav1.3, the C4 region of the C-terminus (residues 2014-2203; Figure 4B). This interaction was specific to AnkB, as it was not observed for the similar ankyrin-G (AnkG) membrane-binding domain or GST alone (Figure 4B). These data support a direct and specific interaction between AnkB membrane-binding domain and the C-terminal domain of Cav1.3.

Figure 4
Ankyrin-B directly associates with Cav1.3. A, Schematic representation of rat CaV1.3 and intracellular domains for in vitro binding assays

While our functional data suggested a specific interaction of AnkB with Cav1.3, Cav1.2 and Cav1.3 share significant sequence similarity in the distal C-terminal regions (residues 2014-2203 of Cav1.3; Figure 4C). We therefore tested for the interaction of the AnkB membrane-binding domain with both Cav1.2 and Cav1.3 C-terminal domains (residues 1958-2159 of Cav1.2). Consistent with our previous imaging, biochemical, and functional data, we observed no interaction of AnkB or AnkG with the Cav1.2 C-terminal domain (Figure 4D).

The Cav1.3, but not Cav1.2 C-terminal domain contains a short motif resembling the ankyrin-binding sequence found in voltage-gated Nav channels (i.e. Nav1.2, Nav1.5) and inwardly rectifying K+ channels (i.e. Kir6.2; Figure 4E).8, 20, 21 Importantly, this sequence is conserved across Cav1.3 orthologues (Figure 4F). We tested the requirement of this motif for AnkB-binding using a mutant C-terminal domain lacking these residues (see Figure 4G). While Cav1.3 C-terminal domain associated with AnkB (but not AnkG), Cav1.3 C4 ΔABD lacking the putative ankyrin-binding domain failed to associate with AnkB (Figure 4H). Based on these results, we tested whether residues 2175-2198 were sufficient for ankyrin-binding using biotinylated peptides. We observed an association of full-length endogenous AnkB from human atrial lysates with an immobilized peptide containing the AnkB-binding domain in Cav1.3 (Figure 4I-J). We observed no interaction with a peptide of the corresponding region in Cav1.2, or with streptavidin beads alone (Figure 4J). Together, our data demonstrate that Cav1.3 residues 2175-2198 are necessary and sufficient for direct AnkB association.

To test whether Cav1.3 membrane expression requires a direct interaction with AnkB, we measured ICa,L in primary cardiac fibroblasts expressing wild-type Cav1.3 and a mutant Cav1.3 lacking the ankyrin-binding motif. As expected, wild-type Cav1.3 expression resulted in robust ICa,L in primary cells (Figure 4K, n=7, p<0.05). In contrast, Cav1.3Δ2174 lacking the AnkB-binding motif displayed greater than 50% reduction in membrane activity compared to the WT channel (Figure 4K, n=7, p<0.05). These data support the requirement of a direct AnkB interaction for Cav1.3 membrane targeting. However, these data also suggest that an ankyrin-independent pathway(s) also plays a critical role in Cav1.3 membrane delivery, as Cav1.3 lacking ankyrin-binding still displayed a greatly reduced, but detectable current.

Loss of ICa,L alters APD90 in ankyrin-B+/− atrial myocytes

Our results identify a direct association of AnkB with Cav1.3 and a striking reduction of Cav1.3 and ICa,L in primary AnkB+/− atrial cardiomyocytes. To test whether observed differences in ICa,L in AnkB+/− atrial myocytes are sufficient to account for dramatic action potential shortening and atrial fibrillation susceptibility, we performed computational modeling of WT and AnkB+/− atrial action potentials (Figure 5). We first incorporated experimentally measured changes in ICa,L (Figure 2), INCX (Supplemental Figure 2 in online-only Data Supplement), as well as reduction in Na/K ATPase expression12, 22 into a physiological and well-validated model of the atrial action potential from Nattel and colleagues.23 Decreasing all three currents was sufficient to reduce the action potential nearly 25%, similar to the observed differences in APDs between AnkB+/− and WT atrial myocytes (Figure 5B, G). We next simulated action potentials in atrial myocytes deficient in NCX, Na/K ATPase, or ICa,L and compared APDs to that in the WT model (Figure 5K). Reduction of ICa,L alone shortened APD by nearly the same amount as observed in the AnkB+/− model, indicating that loss of ICa,L is the dominant mechanism for APD shortening in AnkB+/− myocytes (Figure 5K).

Figure 5
Computational analysis of mechanism for action potential remodeling in ankyrin-B+/− atrial myocytes and reduced atrial ankyrin-B expression in AF patients

Patients with common AF display reduced ankyrin-B expression

Finally, as a first step to further investigate the linkage of AnkB with human AF in the general population, we examined the protein expression of AnkB in the right atria of patients with normal sinus rhythm versus patients with paroxysmal AF. Patients with documented paroxysmal AF displayed striking reductions in AnkB expression compared with individuals in normal sinus rhythm (Figure 6A-B, n=12 patients in sinus rhythm, n=10 pAF; p<0.05). Notably, we also observed a significant reduction in Cav1.3 protein expression levels in pAF samples (Figure 6C-D, n=12 patients in sinus rhythm, n=10 pAF; p<0.05). Together, these data provide additional data that associates reduced ankyrin-B function with human atrial disease.

Figure 6
Decreased ankyrin-B and CaV1.3 protein expression in atria from human pAF patients


AnkB, required for the targeting and stability of critical membrane and submembrane molecules 18, 24, 25 is important for normal membrane excitability in multiple cardiac cell types 26. The multifunctional capacity of ankyrin-B is evident in the strong link between ankyrin dysfunction and a spectrum of cardiac disorders collectively referred to as “ankyrin-B syndrome”. In this study, we identify the L-type calcium channel 1.3 (α1D) as a novel AnkB-binding partner and provide an association between decreased AnkB function and AF. Specifically, we demonstrate early-onset familial AF in human patients with an ANK2 mis-sense mutation. AnkB+/− mice display similar atrial dysfunction including increased incidence of spontaneous AF episodes and enhanced susceptibility to burst-pacing induced AF. At the cellular level, ankyrin-B+/− atrial myocytes exhibit decreased ICa,L and shortened action potential duration, both hallmarks of clinical AF.5, 27 Underlying the reduced ICa,L in AnkB+/− atrial myocytes, there is a selective loss of both protein expression and membrane targeting of CaV1.3, but not CaV1.2. This selective interaction is mediated by a unique motif in the C-terminal domain of CaV1.3 that is both necessary and sufficient for ankyrin-binding. Thus, AnkB-dependent membrane targeting of Cav1.3 is required for normal atrial ICa,L and atrial function. While AnkB haploinsufficiency in atrial myocytes reduces NCX and NKA currents, ankyrin-B-dependent reduction in ICa,L appears primarily responsible for shortened action potential durations as suggested by computational modeling.

There is a strong association between reduced ICa,L and AF.27 While it is unclear whether ICa,L dysfunction is a cause or effect of AF, there are numerous examples of AF linked to reduced ICa,L. Atrial tissues from human AF patients display reduced mRNA expression of L-type Ca2+ channels CaV1.2 and CaV1.3 in addition to an overall decrease in ICA,L.28, 29 In addition, patients with loss-of-function mutations in L-type Ca2+ channel CaV1.2 manifest complex cardiac phenotypes including AF.30 Similarly, atrial myocytes from Cav1.3−/− mice exhibit reduced ICa,L, a depolarizing shift in voltage-dependent ICa,L activation (CaV1.3 activates at more negative potentials than Cav1.2)31, and increased AF susceptibility upon burst-pacing stimulation.31, 32 Given the increased susceptibility to AF in Cav1.3−/− mice and our findings that CaV1.3 membrane targeting is regulated by a direct interaction with AnkB, it will be critical in future experiments to determine the direct role of Cav1.3-dysfunction in human ANK2-related atrial disease.

An intriguing finding from this study is that despite overlapping expression patterns of the L-type calcium channel alpha-subunits in atrial cells, AnkB preferentially interacts with CaV1.3 and not CaV1.2 in atrial myocytes. A second interesting observation is that CaV1.3 is expressed, albeit to a lesser extent, in membranes of AnkB−/− cells, suggesting that ankyrin-independent mechanisms also contribute to CaV1.3 membrane expression. These observations are consistent with L-type calcium channels interacting with a variety of signaling and scaffolding proteins that influence membrane expression. Some of the molecular mechanisms that regulate L-type calcium channel membrane expression include channel auxiliary subunits (β-subunits)33, signaling molecules (calmodulin)34, and scaffolding/adapter proteins (α-actinin2, A-kinase anchoring protein 79).35, 36


We propose that ankyrin-B dysfunction is associated with AF based on our findings that: 1) a loss-of-function ANK2 mutation is associated with highly penetrant AF; 2) AnkB haplo-insufficient mice exhibit increased susceptibility to AF; 3) AnkB-deficient atrial myocytes display shortened action potentials, a hallmark of clinical AF; 4) AnkB+/− atrial myocytes exhibit reduced expression and membrane targeting of Cav1.3, a L-type calcium channel subunit previously associated with increased AF susceptibility31, 32; and 5) patients with pAF demonstrate reduced AnkB. Collectively, these findings suggest that ankyrin-B dysfunction may account for cases of monogenic AF in the general human population and that ANK2 should be considered for familial AF screening.

Atrial fibrillation (AF) is the most prevalent sustained arrhythmia in clinical practice. In fact, in the US alone, AF is present in >two million individuals. Despite the high incidence of AF in the population, surprisingly little is known regarding the molecular mechanisms underlying this complex disease. Ankyrin proteins target and stabilize proteins at specialized membrane domains. Notably, dysfunction in ankyrin- and ankyrin-associated pathways has been linked with disorders including spherocytosis, spinocerebellar ataxia, diabetes, neurological deficits, and cardiac arrhythmias. Nearly a decade ago, ankyrin-B (ANK2) was discovered as a critical component of heart, and work in humans and mice has implicated ankyrin-B as critical for cardiac function. In fact, human ANK2 loss-of-function variants are associated with potentially fatal ventricular arrhythmias. Here, we demonstrate the importance of ankyrin-B for atrial function and identify an association between ankyrin-B dysfunction and AF. Individuals harboring ANK2-variants display AF and these phenotypes are reproduced in mice deficient in ankyrin-B. Ankyrin-B is expressed in the atria and ankyrin-B+/− myocytes display shortened action potentials, a hallmark of AF, and decreased L-type calcium channel current (ICa,L). We show that Cav1.3, responsible for one component of ICa,L in atria, is a novel ankyrin-binding partner and that Cav1.3 expression/activity is reduced in ankyrin-deficient atrial myocytes. Finally, ankyrin-B is reduced in atrial samples from human AF patients, further supporting the role of ankyrin-B in normal atrial function. Together, our work implicates ankyrin-B as a surprising, yet critical component of atrial excitability, and supports the role of atypical myocyte proteins in disease pathogenesis.

Supplementary Material


The authors acknowledge the Heidelberg Cardiosurgeon Team and excellent technical assistance from Claudia Liebetrau in Mannheim.

Sources of Funding We acknowledge support from the NIH (HL084583, HL083422 to PJM; HL079031, HL 62494, HL70250 to MEA; HL089598, HL091947 to XHTW; HL096805 to TJH; HL092232 to SRC; Pew Scholars Trust (PJM), Gilead Sciences Research Scholars Program (TJH), W.M. Keck Foundation (XHTW), American Heart Association (NL), and Fondation Leducq Award to the Alliance for Calmodulin Kinase Signaling in Heart Disease (PJM, XHW, MEA), and Fondation Leducq Grant to the European-North American Atrial Fibrillation Research Alliance (DD).


Disclosures None

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