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ORIGINAL CONTRIBUTION
Expression and function of Kv1.1 potassium channels in human
atria from patients with atrial fibrillation
Edward Glasscock
1
•Niels Voigt
2,3
•Mark D. McCauley
4
•Qiang Sun
2,3
•
Na Li
4
•David Y. Chiang
4
•Xiao-Bo Zhou
3
•Cristina E. Molina
2
•Dierk Thomas
3
•
Constanze Schmidt
3
•Darlene G. Skapura
4
•Jeffrey L. Noebels
5,6,7
•
Dobromir Dobrev
2,3
•Xander H. T. Wehrens
4
Received: 19 December 2014 / Revised: 2 July 2015 / Accepted: 3 July 2015 / Published online: 11 July 2015
ÓSpringer-Verlag Berlin Heidelberg 2015
Abstract Voltage-gated Kv1.1 channels encoded by the
Kcna1 gene are traditionally regarded as being neural-
specific with no known expression or intrinsic functional
role in the heart. However, recent studies in mice reveal
low-level Kv1.1 expression in heart and cardiac abnor-
malities associated with Kv1.1-deficiency suggesting that
the channel may have a previously unrecognized cardiac
role. Therefore, this study tests the hypothesis that Kv1.1
channels are associated with arrhythmogenesis and con-
tribute to intrinsic cardiac function. In intra-atrial burst
pacing experiments, Kcna1-null mice exhibited increased
susceptibility to atrial fibrillation (AF). The atria of Kcna1-
null mice showed minimal Kv1 family ion channel
remodeling and fibrosis as measured by qRT-PCR and
Masson’s trichrome histology, respectively. Using RT-
PCR, immunocytochemistry, and immunoblotting, KCNA1
mRNA and protein were detected in isolated mouse car-
diomyocytes and human atria for the first time. Patients
with chronic AF (cAF) showed no changes in KCNA1
mRNA levels relative to controls; however, they exhibited
increases in atrial Kv1.1 protein levels, not seen in
paroxysmal AF patients. Patch-clamp recordings of iso-
lated human atrial myocytes revealed significant dendro-
toxin-K (DTX-K)-sensitive outward current components
that were significantly increased in cAF patients, reflecting
a contribution by Kv1.1 channels. The concomitant
increases in Kv1.1 protein and DTX-K-sensitive currents in
atria of cAF patients suggest that the channel contributes to
the pathological mechanisms of persistent AF. These
findings provide evidence of an intrinsic cardiac role of
Kv1.1 channels and indicate that they may contribute to
atrial repolarization and AF susceptibility.
Keywords Voltage-gated potassium channels Atrial
fibrillation Dendrotoxin-K Kcna1 Kv1.1
Introduction
Atrial fibrillation (AF) is the most common sustained car-
diac arrhythmia and associated with significant cardiovas-
cular morbidity and mortality, but its underlying molecular
basis remains only partially understood [47]. During AF,
atrial cells fire rapidly at rates up to tenfold faster than
normal, producing uncoordinated atrial activity and irreg-
ular ventricular contraction, which can lead to blood clot
formation and stroke [21]. Alteration of ion channel
Electronic supplementary material The online version of this
article (doi:10.1007/s00395-015-0505-6) contains supplementary
material, which is available to authorized users.
&Edward Glasscock
aglas1@lsuhsc.edu
1
Department of Cellular Biology and Anatomy, Louisiana
State University Health Sciences Center, 1501 Kings
Highway, P.O. Box 33932, Shreveport, LA 71130-393, USA
2
Faculty of Medicine, Institute of Pharmacology, University
Duisburg-Essen, Essen, Germany
3
Division of Experimental Cardiology, Medical Faculty
Mannheim, University of Heidelberg, Mannheim, Germany
4
Department of Molecular Physiology and Biophysics, and
Medicine/Cardiology, Cardiovascular Research Institute,
Baylor College of Medicine, Houston, TX, USA
5
Departments of Neurology, Baylor College of Medicine,
Houston, TX, USA
6
Departments of Neuroscience, Baylor College of Medicine,
Houston, TX, USA
7
Departments of Molecular and Human Genetics, Baylor
College of Medicine, Houston, TX, USA
123
Basic Res Cardiol (2015) 110:47
DOI 10.1007/s00395-015-0505-6
function by atrial remodeling or genetic mutation can
provide a pro-fibrillatory electrophysiological substrate
conducive to AF [13,22]. A variety of conditions can cause
ion channel remodeling predisposing to AF, including
congestive heart failure and acute myocardial infarction
[23]. In addition, AF itself can cause electrical remodeling
that promotes persistent fibrillation and thereby auto-per-
petuates the arrhythmia. The underlying mechanisms for
this transition likely involve altered channel expression and
function [22,23,50]. Although AF is primarily a sporadic
condition, population-based studies and rare familial kin-
dreds have shown that it has a significant genetic compo-
nent [19,35]. In families with monogenic AF subtypes, the
majority of genes implicated encode subunits of voltage-
gated potassium and sodium channels [19].
Here Kv1.1 voltage-gated potassium channels were
investigated for a role in AF. Kv1.1 channels, encoded by
the Kcna1 gene, exhibit widespread expression throughout
the brain and peripheral nervous system and their dys-
function leads to neurological diseases including epilepsy
and episodic ataxia type 1 [29]. Kv1.1 channels have tra-
ditionally been regarded as predominantly neural-specific
with no known expression or function in the heart. How-
ever, mice lacking Kv1.1 channels exhibit atrioventricular
cardiac conduction abnormalities and bradyarrhythmia
phenotypes that appear to emanate from seizures and
abnormal vagal activity [10,11]. In addition, these previ-
ous studies detected low levels of Kcna1 mRNA and pro-
tein in mouse heart suggesting that Kv1.1 channels may
also contribute to the intrinsic function of the heart [11]. If
so, alteration of Kv1.1 channel function could lead to
independent dual arrhythmia phenotypes in brain and heart.
In this work, a combination of electrophysiological
techniques and molecular analyses was used to evaluate the
contribution of Kv1.1 channels to basal cardiac function
and potential arrhythmia development. Two main
hypotheses were tested: (1) that Kv1.1 channel perturbation
in mice causes arrhythmia susceptibility, and (2) that
dysregulation of Kv1.1 channels in the human heart may be
important for arrhythmogenesis. Our experiments show
that the absence of Kv1.1 channels predisposes the mouse
heart to AF without drastic remodeling of related K?
channel subunits and fibrotic structural changes. Expres-
sion analyses in isolated mouse myocytes demonstrate the
presence of Kv1.1 mRNA and protein in heart apart from
neural tissue. Molecular analyses detect the first clear
evidence of Kv1.1 expression in human atria, and show
that Kv1.1 channels exhibit expression changes in patients
with chronic AF suggestive of pathophysiological channel
remodeling. In addition, patch-clamp recordings of isolated
human atrial myocytes reveal significant DTX-K-sensitive
components that are doubled in patients with cAF,
indicative of a contribution by Kv1.1 channels. Taken
together this work finds a previously unrecognized cardiac
role for the Kcna1 gene and Kv1.1 channels in regulating
atrial repolarization and arrhythmia susceptibility.
Methods
Animals and genotyping
Kcna1
-/-
mice carry null alleles of the Kcna1 gene
resulting from targeted deletion of the open reading frame,
as described [36]. The mice are maintained on a Tac:N:-
NIHS-BC background. Animals were housed at 22 °C, fed
ad libitum, and submitted to a 12 h light/dark cycle. For
surgeries, mice were anaesthetized using 1.5–2 % isoflu-
rane in 95 % O
2
. Animals were euthanized for expression
and tissue analysis using inhaled isoflurane overdose. All
procedures were performed in accordance with the guide-
lines of the National Institutes of Health, as approved by
the Animal Care and Use Committee of Baylor College of
Medicine.
Genomic DNA was isolated by enzymatic digestion of
tail clips using Direct-PCR Lysis Reagent (Viagen Biotech,
Los Angeles, CA, USA). The genotypes of Kcna1 mice
were determined by performing PCR amplification of
genomic DNA using three allele-specific primers: a
mutant-specific primer (50-CCTTCTATCGCCTTCTT
GACG-30), a wild-type-specific primer (50-GCCTCTGA
CAGTGACCTCAGC-30), and a common primer (50-GC
TTCAGGTTCGCCACTCCCC-30). The PCR yielded
amplicons of *337 bp for the wild-type allele and
*475 bp for the null allele.
Intracardiac electrophysiology in mice
In vivo electrophysiology studies were performed in
knockout and wild-type mice of both sexes, as per prior
established protocols [15,37]. Atrial and ventricular
intracardiac electrograms were recorded simultaneously
using a 1.1F octapolar catheter (EPR-800, Millar Instru-
ments, Houston, TX, USA) inserted via the right jugular
vein. Surface ECG and intracardiac electrophysiology
parameters were assessed at baseline. Right atrial pacing
was performed using 2-ms current pulses at 800 lA
delivered by an external stimulator (STG-3008, Multi
Channel Systems, Reutlingen, Germany). AF inducibility
was determined using an overdriving pacing protocol, and
AF was defined as the occurrence of rapid and fragmented
atrial electrograms with irregular AV-nodal conduction and
ventricular rhythm for at least 1 s. To be counted as AF
positive, a mouse had to exhibit AF in response to at least
two out of three pacing trials. For Kcna1-null mice, the
mean AF duration was determined by calculating the
47 Page 2 of 15 Basic Res Cardiol (2015) 110:47
123
average elapsed time of all observed AF episodes. The
stimulation protocols used for intracardiac burst pacing are
summarized in Supplemental Table S1.
Mouse RNA extraction, cDNA synthesis, and real-
time PCR
Similarly aged Kv1.1-knockout mice (n=5;
112 ±5 days old) and wild-type control mice (n=5;
121 ±5 days old) were euthanized using isoflurane
inhalation and their left atria quickly excised. The tissue
was immediately placed in ice-cold TRIzol reagent,
homogenized, and the total RNA extracted according to the
manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA).
Following resuspension in water, RNA samples were
DNase treated using the DNA-free Kit (Applied Biosys-
tems, Carlsbad, CA, USA). The quantity of total RNA was
measured and the quality checked by agarose gel
electrophoresis.
First-strand cDNA was synthesized from 250 ng of total
RNA using the Phusion RT-PCR Kit with oligo(dT) primers
(ThermoScientific, Waltham, MA, USA). Before proceeding to
real-time PCR, the quality of cDNA and the tissue genotype
was verified by 40 cycles of PCR amplification using primers
specific for the GAPDH and Kcna1 genes, followed by agarose
gel electrophoresis. A 982-bp GAPDH-specific band was
amplified using the following primers: forward, 50-
TGAAGGTCGGTGTGAACGGATTTGGC-30; reverse, 50-
ATGTAGGCCATGAGGTCCACCAC-30. A 710-bp Kcna1-
specific band was amplified using the following primers: for-
ward, 50-GCATCGACAACACCACAGTC-3; reverse, 50-
CGGCGGCTGAGGTCACTGTCAGAGGCTAAGT-30.The
lack of GAPDH PCR products in –RT controls confirmed the
absence of genomic DNA contamination, while the presence or
absence of Kcna1 amplicons confirmed the tissue genotype and
verified the absence of Kcna1 mRNA in knockouts.
To quantify the relative gene expression patterns in left
atria of knockout and control animals, real-time RT-PCR of
first strand cDNA was performed with TaqMan gene
expression assays using the 7500 Real-Time PCR System
(Applied Biosystems). TaqMan gene expression assays
were designed and preoptimized by Applied Biosystems
for the detection of Kcna1 (Mm00439977_s1), Kcna2
(Mm00434584_s1), Kcna3 (Mm00434599_s1), Kcna4
(Mm00445241_s1), Kcna5 (Mm00524346_s1), Kcna6
(Mm00496625_s1), Kcna7 (Mm01197268_m1), Kcnab1
(Mm00440017_m1), Kcnab2 (Mm01260263_m1), Kcnab3
(Mm01337146_m1), col6a1 (Mm00487160_m1), and
Hprt1 (Mm00446968_m1). Each assay consisted of an
unlabeled gene-specific PCR primer pair and a TaqMan
probe with a fluorescent FAM dye label and a minor
groove binder moiety on the 50end and a nonfluorescent
quencher on the 30end. Individual PCRs were performed in
triplicate using 100 ng of cDNA and TaqMan Gene
Expression Master Mix (Applied Biosystems). No template
and –RT reactions were included as negative controls to
verify the absence of contamination leading to unwanted
PCR amplification and detection. The reactions were
cycled at 50 °C for 2 min, 95 °C for 10 min, and then 40
cycles of 95 °C for 15 s and 60 °C for 1 min. Spectral data
were collected and analyzed with SDS 1.3 software (Ap-
plied Biosystems) using a manual threshold of 0.1, auto-
matic baseline detection, and automatic outlier removal.
The data were analyzed using the threshold cycle (C
t
)
relative quantification method [16]. Hprt1 was used as a
reference gene for normalization since it showed stable and
reliable expression in our samples. GAPDH was also
evaluated as a reference gene but its expression was more
variable than Hprt1, so it was excluded from the analysis.
The C
t
values were averaged and then used to calculate the
expression levels relative to Hprt1 using the formula 2DCt
X 100, where 2DCtcorresponds to the difference in the
threshold cycle of the gene of interest versus Hprt1. In two
instances, once for Kcna6 and once for Kcnab3 (both in wild-
type samples), the assays failed to amplify across the entire
triplicate, even when the experiment was repeated, likely due
to extremely low levels of gene expression. In both cases, a
value of zero was imputed for the 2DCtmeasurement for the
given samples. In rare instances where triplicates produced
only one successful replicate, the C
t
value used for data
analysis was derived from the single working reaction,
assuming that the measurement was similar to the other
biological replicates. Values for Kcna1 levels in knockout
tissue were imputed as zero since the TaqMan assay used for
detection is uninformative in knockouts; the Kcna1 assay is
targeted to the 50-UTR of the gene which is still present in
knockouts despite the absence of the open reading frame. As
stated above, all knockout tissues were verified to be nega-
tive for Kcna1 mRNA using 40 cycles of RT-PCR followed
by agarose gel electrophoresis.
Mouse histology
Whole hearts from 4- to 6-month-old mice were excised,
submerged in 1 M KCl for 45–60 s, rinsed briefly in PBS,
and then fixed in 10 % neutral buffered formalin for 24 h.
Following fixation, hearts were embedded in paraffin,
longitudinally sectioned (5 lm), and stained with Masson’s
trichrome for fibrosis. The percentage of fibrotic atrial
tissue was quantified with Adobe Photoshop using previ-
ously described methods [8]. In brief, images were adjusted
using the selective color command to intensify the contrast
between reds and blues. The color range function was then
used to generate histograms which counted the pixel area
for each of the color components. The fraction of tissue
fibrosis was then calculated by dividing the sum of the cyan
Basic Res Cardiol (2015) 110:47 Page 3 of 15 47
123
and blue pixel areas by the total pixel area. For each ani-
mal, percentage fibrosis was estimated by averaging mea-
surements from left and right atria in similarly oriented
single longitudinal sections. Fibrosis percentages appeared
similar between left and right atria for each genotype. For
one knockout and one control animal, fibrosis was mea-
sured in only the left or right atria due to the loss of tissue
during processing.
Cardiomyocyte isolation and expression analyses
C57BL/6 mice (3 months old) were used for cell isolation
as described previously [15]. In brief, following isoflurane
anesthesia, the heart was removed from mice. After dis-
secting, the heart was cannulated through the aorta and
perfused on a Langendorff apparatus with 0 Ca
2?
Tyrode,
then 0 Ca
2?
Tyrode containing 0.02 mg/mL Liberase TH
Research Grade (Roche Applied Science, Penzberg, Ger-
many) for 10–15 min at 37 °C. After digestion, atria and
ventricle were separated and placed in KB solution
(90 mmol/L KCl, 30 mmol/L K
2
HPO
4
, 5 mmol/L MgSO
4
,
5 mmol/L pyruvic acid, 5 mmol/L b-hydroxybutyric acid,
5 mmol/L creatine, 20 mmol/L taurine, 10 mmol/L glu-
cose, 0.5 mmol/L EGTA, 5 mmol/L HEPES; pH 7.2), then
minced thoroughly and agitated gently, and filtered through
a 210 lm mesh. The isolated myocytes were spun down at
800 rpm for 5 min. After removing supernatant, the cells
were used immediately for expression analysis or stored at
-80 °C for biochemical studies later.
Total RNA was isolated using Direct-zolTM RNA
MiniPrep (Zymo Research, Irvine, CA, USA) according to
the manufacturer’s instructions. In column DNase I digestion
was performed to remove DNA contamination. First-stand
cDNA was synthesized from 300 ng of total RNA using
iScript
TM
cDNA Synthesis Kit (Bio-Rad, Hercules, CA,
USA). PCR was then performed as described above in
‘‘ Mouse RNA extraction, cDNA synthesis, and real-time
PCR’’ using the same primers and amplification protocol.
Quantitative real-time PCR (qRT-PCR) was performed as
previously reported with modifications [28]. Briefly, qRT-
PCR was performed in triplicates with PerfeCTa
Ò
SYBR
Ò
Green FastMix (Quanta BioSciences, Inc., Gaithersburg,
MD, USA) in 96-well plates using Mastercycler ep realplex
(Eppendorf, Hamburg, Germany). The following program
was run to amplify the products: (1) 95 °C for 5 min, (2) 45
cycles of 95 °C for 5 s, 60 °C for 10 s, 72 °C for 1 s, (3)
melting curve ramp from 65 °Cto95 °C at 0.1 °C per sec-
ond. mRNA expression levels were compared using the
relative CT (cycle number) method after normalization to
Rpl7. Primer sequences for Kcna1 were the following: for-
ward, 50-GAGAATGCGGACGAGGCTTC-30and reverse,
50-CCGGAGATGTTGATTACTACGC-30. For Rpl7, the
primers had the following sequence: forward, 50GAAGCT
CATCTATGAGAAGGC-30and reverse, 50-AAGACGAA
GGAGCTGCAGAAC-30.
For immunocytochemistry, isolated myocytes were
added to cover glasses coated with 20 lg/ml laminin in
PBS for 30 min before fixation with 4 % formalin for
15 min. After washing with PBS 3 times, the myocytes
were permeabilized with 0.1 % Triton X in PBS for
10 min, washed another 3 times with PBS, and blocked
with 1 % normal goat serum (NGS) in PBS for 1 h. After
blocking, the samples were incubated overnight at 4 °C
with antibodies against Kv1.1 (K20/78, NeuroMab, Davis,
CA, USA; 1:100) and JPH2 (custom-made [26]; 1:100)
diluted in 1 % NGS and 1 % bovine serum albumin (BSA)
in PBS. Next day, the cover glasses were washed with PBS
3 times and incubated with Alexa Fluor
Ò
568 Goat Anti-
Mouse IgG and Alexa Fluor
Ò
488 Goat Anti-Rabbit IgG
(#A11004, Invitrogen, Carlsbad, CA, USA; 1:2000 for
both) in 1 % NGS/BSA in PBS for 1 h at room tempera-
ture. Afterward, the cover glasses were washed with PBS 3
times and mounted on glass slide with Vectashield media
with DAPI (#H-1200, Vector Laboratories, Burlingame,
CA, USA). Fluorescence images were taken with the Zeiss
LSM510 confocal microscope (Jena, Germany).
Human tissue samples
Right atrial appendages were obtained from 32 patients in
sinus rhythm (SR), 14 in paroxysmal AF (pAF) and 23 in
chronic AF (cAF), undergoing open-heart surgery
(Table 1). Experimental protocols were approved by the
ethics committee of the Medical Faculty Mannheim,
University of Heidelberg (#2011-216 N-MA) and per-
formed in accordance with the Declaration of Helsinki.
Each patient gave written informed consent. After excision,
atrial appendages were used for either myocyte isolation (6
SR, 6 cAF) or snap-frozen in liquid-nitrogen for bio-
chemical studies (26 SR, 14 pAF, 17 cAF patients).
Human RNA and protein biochemistry
RNA isolation, reverse-transcription and real-time PCR
were performed as described [43]. For initial detection of
KCNA1 mRNA, cDNA was PCR-amplified using 35 cycles
and primers specific for the KCNA1 gene, followed by
agarose gel electrophoresis. A 232-bp KCNA1-specific
band was amplified using the following primers: forward,
50-CATCGTGGAAACGCTGTGTAT-3; reverse, 50AAC
CCTTACCAAGCGGATGAC-30. Commercial primers
were used for real-time PCR detection of human KCNA1
mRNA expression in patients (Hs00264798_s1, Life
Technologies, Foster City, CA, USA). KCNA1 mRNA
expression was normalized to HPRT1 (Hs01003267_m1;
Life Technologies).
47 Page 4 of 15 Basic Res Cardiol (2015) 110:47
123
Protein levels of Kv1.1 (1:1000; rabbit polyclonal anti-
Kv1.1; ARP34923_P050; Aviva Systems Biology, San
Diego, CA, USA) were quantified by Western blotting and
normalized to GAPDH (1:200,000; HyTest, Turku, Fin-
land) as described [43]. Peroxidase-conjugated goat anti-
rabbit (1:5000; Sigma-Aldrich, St. Louis, MO, USA) and
goat anti-mouse (1:50,000; Sigma-Aldrich) were used as
secondary antibodies and visualized by chemifluorescense
(GE Healthcare, Chalifont St. Giles, UK). AIDA Image
Analyzer Software (raytest, Straubenhardt, Germany) was
used for analysis.
For immunocytochemistry, cells (from 3 patients in SR;
8 cells/patient) were fixed with paraformaldehyde (PFA;
2 %) for 15 min. After centrifugation at 750 rpm for
5 min, PFA was removed, the myocytes were washed, and
PFA was neutralized with Glycin (0.1 M). Cells were then
washed 3 times with phosphate-buffered saline (PBS) and
permeabilized for 5 min with Triton X-100 (0.5 %) diluted
in PBS. Cells were rinsed 3 times with PBS and then
blocked with 1 % BSA in PBS. Myocytes were then
labeled with the primary antibody (rabbit polyclonal anti-
Kv1.1 (1:1000); ARP34923_P050; Aviva Systems
Table 1 Patient characteristics SR PAF CAF
Patients, n32 14 23
Gender, m/f 24/8 10/4 15/8
Age, years 64.09 ±11.2 66.9 ±10.1 67.78 ±9.41
Body mass index, kg/m
2
28.09 ±5.79 29.54 ±7.53 29.23 ±4.21
CAD, n(%) 17 (53) 4 (29) 8 (35)
AVD, n(%) 8 (25) 6 (43) 6 (26)
MVD, n(%) 0 (0) 1 (7) 5 (22)
CAD ?AVD, n(%) 7 (22) 4 (29) 5 (22)
Hypertension, n(%) 28 (88) 14 (100) 22 (96)
Diabetes, n(%) 7 (22) 4 (29) 8 (35)
Hyperlipidemia, n(%) 29 (91) 12 (86) 17 (74)
LVEF
Normal, n(%) 16 (50) 6 (43) 11 (48)
Mildly reduced, n(%) 10 (31) 2 (14) 3 (13)
Moderately reduced, n(%) 4 (13) 4 (29) 6 (26)
Severely reduced, n(%) 1 (3) 2 (14) 3 (13)
b-Blockers, n(%) 20 (62) 14 (100) 19 (83)
Digitalis, n(%) 2 (6) 2 (14) 7 (30)
Amiodarone, n(%)
#
0 (0) 0 (0) 1 (7)
Other AADs, n(%) 0 (0) 0 (0) 0 (0)
ACE inhibitors, n(%) 19 (59) 8 (57) 12 (52)
AT1 blockers, n(%) 6 (19) 2 (14) 3 (13)
Dihydropyridines, n(%) 8 (25) 4 (29) 3 (13)
Diuretics, n(%) 9 (28) 10 (71) 11 (48)
Nitrates, n(%) 1 (3) 2 (14) 3 (13)
Lipid-lowering drugs, n(%) 22 (69) 11 (79) 14 (61)
Biguanides, n(%)
#
2 (7) 2 (18) 1 (7)
Sulfonylurea derivatives, n(%)
#
1 (4) 0 (0) 0 (0)
Insulin, n(%)
#
0 (0) 3 (27) 2 (14)
OAC, n(%)
#
0 (0) 5 (45)** 13 (93)***
Antiplatelet drugs, n(%)
#
23 (88) 8 (73) 6 (43)*
AAD antiarrhythmic drug, ACE angiotensin converting enzyme, AT angiotensin receptor, AVD aortic valve
disease, CAD coronary artery disease, CAF chronic atrial fibrillation, LVEF left ventricular ejection fraction
(normal, C55 %; mild impairment, 45–54 %; moderate impairment, 30–44 %; severe impairment,\30 %),
MVD mitral valve disease, OAC oral anticoagulation, PAF paroxysmal atrial fibrillation, SR sinus rhythm
*P\0.05, ** P\0.01, *** P\0.001 versus SR from Fisher exact test followed by Bonferroni multiple
comparisons procedure for categorical variables
#
Data were not available for 6 SR, 3 pAF and 9 cAF patients
Basic Res Cardiol (2015) 110:47 Page 5 of 15 47
123
Biology, San Diego, CA, USA), which was diluted in PBS
containing 1 % BSA. After an overnight incubation at
4°C, the myocytes were washed 3 times using 1 % BSA in
PBS and then incubated with Fluorescein (FITC, excitation
488 nm, emission 520 nm) anti-rabbit IgG to reveal the
Kv1.1 staining. After 3 successive 5-min washes using 1 %
BSA in PBS, the cells were mounted with Mowiol med-
ium. Images were acquired using a CAIRN Spinning Disk
confocal microscope.
Patch-clamp experiments
Atrial myocytes were isolated using a standard protocol
and were suspended in storage solution (mmol/L: KCl 20,
KH
2
PO4 10, glucose 10, K-glutamate 70, b-hydroxybu-
tyrate 10, taurine 10, EGTA 10, albumin 1, pH =7.4) [44].
Membrane currents were measured in whole-cell ruptured-
patch configuration using voltage clamp. pClamp-Software
V10.2 (Molecular Devices, Sunnyvale, USA) was used for
data acquisition and analysis. Borosilicate glass micro-
electrodes had tip resistances of 2–5 MXwhen filled with
pipette solution (mmol/L: EGTA 0.02, GTP-Tris 0.1,
HEPES 10, K-aspartate 92, KCl 48, Mg-ATP 1, Na
2
-ATP
4; pH =7.2). Seal resistances were 4–8 GX. Series resis-
tance and cell capacitance were compensated. The series
resistance was kept below 10 MXand was compensated by
at least 50 %. With a current amplitude of 1 nA, the
potential for error amounted to \5 mV. To control for
myocyte-size variability, currents are expressed as densi-
ties (pA/pF). Myocytes were superfused at 37 °Cwitha
bath solution containing (mmol/L): CaCl
2
2, glucose 10,
HEPES 10, KCl 4, MgCl
2
1, NaCl 140, probenecid 2;
pH =7.4. I
Ca,L
was blocked by adding CdCl
2
(0.3 mmol/
L) to the bath solution. Drugs were applied via a rapid-
solution exchange system (ALA Scientific Instruments,
Farmingdale, USA). Dendrotoxin-K (10 nmol/L; Alomone
Labs, Jerusalem, Israel) was used to block Kv1.1 currents
[31].
Statistical analysis
Differences between group means for continuous data were
compared by unpaired two-tailed Student’s ttest. Differ-
ences between mean mRNA levels in mouse ventricular
and atrial myocytes were compared using a paired ttest.
Categorical data were analyzed with Fisher’s exact test.
Data are mean ±SEM. P\0.05 was considered statisti-
cally significant. In statistical comparisons of myocyte
electrophysiology data, patients may contribute more than
one observation to each sub-analysis suggesting that
observations are not necessarily independent. Within-pa-
tient correlations were not taken into account in statistical
comparisons due to the low sample size.
Results
Increased susceptibility to pacing-induced AF
in Kcna1-null mice
In our previous studies using video electroencephalography
(EEG) combined with ECG, unequivocal spontaneous AF
was not identified in the ambulatory ECGs of Kcna1-null
mice [11]. To test for AF inducibility, Kcna1-null mice
(age 4.0 ±0.3 months) and WT controls (age
4.1 ±0.3 months) were subjected to intracardiac right
atrial burst pacing stimulation. Simultaneous surface elec-
trocardiograms (ECG) and intracardiac atrial and ventric-
ular electrograms were recorded to monitor the occurrence
of atrial arrhythmias (Fig. 1a). AF was characterized by the
combination of a lack of P waves combined with the
presence of irregular RR intervals in lead I of the surface
ECG (Fig. 1b). Burst pacing induced AF more frequently
in Kcna1-null mice (40 %, 5 of 12) than WT mice (0 %, 0
of 10; P\0.05; Fig. 1c). The average duration of AF
episodes in Kcna1-null mice was 8.0 ±2.9 s, and normal
sinus rhythm always resumed spontaneously. Experiments
using ventricular burst pacing protocols did not show
obvious differences in ventricular arrhythmia susceptibility
between genotypes.
Baseline recordings during anesthesia did not reveal any
significant differences in surface ECG characteristics
between Kcna1-null and WT animals, except for slight but
significant shortening of the QRS interval in null mice
(6.9 ±0.2 ms in Kcna1-null mice versus 7.8 ±0.4 ms in
WT; P\0.05; Table 2). Cardiac conduction system
properties also appeared unaltered, since no significant
differences were found in the sinus node recovery time,
right atrial effective refractory period, and atrioventricular
node effective refractory period (Table 2). The lack of
obvious baseline changes in atrioventricular node function
and sinus cycle length in Kcna1-null mice suggests a
possible neural origin for the increased frequency of AV
conduction blocks found previously in this model; how-
ever, autonomic inputs remain intact in our preparations
and could mask latent cardiac-intrinsic differences. Heart
mass was compared between genotypes to rule out any
effects due to cardiac hypertrophy or remodeling. Hearts of
Kcna1-null mice tended to be larger as measured by
absolute heart mass and heart mass-to-body mass ratio, but
this trend did not reach significance (Table 2).
Minimal Kv1 channel and fibrotic structural
remodeling in atria of Kcna1-null mice
Next, mRNA expression analysis was used to determine
whether the increased AF susceptibility in Kcna1-null mice
47 Page 6 of 15 Basic Res Cardiol (2015) 110:47
123
was potentially caused by compensatory expression chan-
ges in related Kv1.x potassium channel a-subunits, such as
Kcna5 which has been linked to AF [25,52]. Real-time
PCR was performed on atrial tissue from Kcna1-null and
WT mice to compare mRNA expression levels for the
genes Kcna1-7and Kcnab1-3, which encode the Kv1.1-
Kv1.7 pore-forming a-subunits and their associated
Kvbeta1-3 accessory b-subunits, respectively. In WT mice,
measurable expression levels were detected for all seven a-
subunits and all three b-subunits. Among the a-subunits,
Kcna5 was expressed at the highest level followed by
Kcna4 and Kcna7, while Kcna1,Kcna2,Kcna3, and Kcna6
were expressed at similarly low levels (Kcna5 Kc-
na4 [Kcna7 [Kcna1 &Kcna2 &Kcna3 &Kcna6).
There were no substantial compensatory changes in gene
expression levels for the Kv1.2–Kv1.7 a-subunits in Kc-
na1-null mice (age 3.7 ±0.2 months; Fig. 2a). Among the
b-subunits, all were expressed at similarly low levels, and
the Kcnab1 subunit showed a modest but significant 31 %
decrease (P\0.05). However, the overall lack of major
Kv1.x channel subunit remodeling suggests that the
observed increase in AF susceptibility is likely directly
related to Kv1.1 deficiency and not attributable to com-
pensatory dysregulation of related Kv1 subunits.
Since fibrotic structural remodeling can provide a sub-
strate conducive to AF, the hearts of Kcna1-null mice were
examined for evidence of increased fibrillar collagen
deposits. First, transcript levels of the collagen, type VI,
alpha 1 (col6a1) gene were measured as an indicator of
fibrosis. Col6a1 expression levels were significantly
increased by more than 50 % in Kcna1-null atria relative to
WT controls (Fig. 2b; P\0.05). To determine whether
this increase in collagen mRNA correlated with an increase
WT
e
cn
edi
c
n
i
FA
10 12
*
100%
80%
60%
40%
20%
0%
KO
WT
Kcna1-null
(AF onset)
Kcna1-null
(AF termination)
Surface
Atrial
Ventricular
A
BC
77 87 99 100 100 95 109 98 88 93 80 95 92
200 ms
Fig. 1 Mice lacking Kv1.1
channels are vulnerable to
pacing-induced AF.
aRepresentative simultaneous
recordings of surface ECG (lead
I) and intracardiac atrial and
ventricular electrograms after
burst pacing in wild type (WT)
and Kcna1-null (KO) mice. In
the KO animal shown, pacing
produced AF (AF onset) that
lasted about 13 s before
returning to normal sinus
rhythm (AF termination).
bDuring inducible AF in KO
animals, the surface ECG
exhibited absent P waves and
irregular RR intervals, which
are shown labeled in
milliseconds. cKO mice
showed significantly higher
incidence of pacing-induced AF
than WT controls. *P\0.05
Table 2 Baseline ECG parameters in WT and Kcna1-null mice
WT (n=10) KO (n=12) P
Age, months 4.0 ±0.3 4.1 ±0.3 0.68
Heart mass, g
a
160 ±11 173 ±12 0.48
Heart:body ratio, mg/g
a
4.8 ±0.2 5.3 ±0.3 0.23
PQ, ms 27.0 ±0.6 27.0 ±1.2 1.00
QRS, ms 7.8 ±0.4 6.9 ±0.2 0.04
QT, ms 34.0 ±1.6 34.8 ±1.3 0.69
QTc, ms 57.2 ±1.6 57.6 ±1.7 0.88
RR, ms 96.8 ±2.1 98.3 ±2.6 0.67
SCL, ms 96.7 ±2.0 98.3 ±2.5 0.65
AV, ms 33.4 ±0.9 33.8 ±0.9 0.73
SNRT, ms 122.0 ±6.2 125.5 ±8.4 0.75
RA ERP, ms 28.7 ±2.0 31.7 ±2.1 0.34
AV ERP, ms 40.1 ±1.9 41.5 ±3.2 0.75
Data are expressed as mean ±SEM. Student’s ttest used to compare
intragroup differences. Pvalues between WT and KO mice are shown
SCL sinus cycle length, AV atria-to-ventricle conduction time, SNRT
sinus node recovery time, RA ERP right atrial effective refractory
period, AV ERP atrioventricular node effective refractory period
a
Data derived from 9 KO and 6 WT animals
Basic Res Cardiol (2015) 110:47 Page 7 of 15 47
123
in fibrosis, Masson’s trichrome staining of atria was per-
formed to label collagen fibers (Fig. 2c). Quantification of
the percentage of atrial fibrotic tissue showed that Kcna1-
null hearts (1.9 ±0.7 %) tended to have about twice as
much collagen as age-matched wild-type controls
(1.0 ±0.6 %), but the results were highly variable
between animals and did not reach statistical significance
(Fig. 2d; P=0.35). Thus, atrial arrhythmogenesis in Kc-
na1-null hearts is not clearly correlated with increased
atrial fibrosis.
Kv1.1 channels are expressed in isolated mouse
cardiomyoctes
To verify that Kcna1 is expressed in cardiomyocytes and
not only intracardiac neurons, individual atrial and ven-
tricular myocytes were isolated from mouse heart and
analyzed for Kv1.1 mRNA and protein. RT-PCR revealed
the unequivocal presence of Kv1.1 mRNA in isolated atrial
myocytes, but ventricular transcripts were below the limit
of detection (Fig. 3a). Using the more sensitive technique
of real-time PCR to quantify relative mRNA levels, Kv1.1
transcripts were detected in both atrial and ventricular
cells, but the abundance in atrial myocytes was about ten-
fold higher (Fig. 3b; P=0.07). To determine whether
Kcna1 expression could be detected at the protein level in
individual mouse myocytes, immunocytochemistry was
performed. Kv1.1 immunoreactivity was observed in both
atrial and ventricular myocytes (Fig. 3c, d); however, the
Kv1.1-positive ventricular staining appeared much less
intense overall. Importantly, Kv1.1 immunoreactivity was
absent in cardiomyocytes from Kcna1-null mice demon-
strating the specificity of the antibody labeling (Fig. 3c, d).
Kv1.1 channels are expressed in human heart
and upregulated in chronic AF
The increased AF susceptibility in the Kcna1-null mouse
model suggests that Kv1.1 channels may play a role in AF
pathophysiology in patients. However, the expression of
KCNA1 has never been demonstrated in human heart nor
associated with arrhythmia in humans. Using RT-PCR,
KCNA1 mRNA was detected in human atrial samples
confirming its presence in human cardiac tissue for the first
time (Fig. 4a). Since KCNA1 transcripts were measurable
in human atria, mRNA levels of KCNA1 were quantified in
patients with paroxysmal (pAF) and chronic AF (cAF) to
look for disease-associated alterations. Using real-time RT-
PCR, KCNA1 mRNA expression levels were found to be
significantly increased in pAF patients but not in cAF
individuals relative to controls in sinus rhythm (SR)
(Fig. 4b; P\0.0005).
Since transcription levels do not always accurately
reflect the amount of translated protein, western blots were
AB
*
col6a1
CD
Percentage fibrosis
WT KO
ns
WT
KO
*
0
1
2
3
4
0
50
100
150
WT KO
40
30
20
6
4
2
0
WT
KO
2
-Δct
versus Hprt1 (X100)
2
-Δct
versus Hprt1 (X100)
-subunits -subunits
Fig. 2 Kcna1-null mice exhibit
minimal Kv1.x channel
remodeling and atrial fibrosis.
aReal-time PCR expression
analysis of Kv1.x potassium
channel a- and b-subunit genes
in atria from KO and control
WT mice (n=5 mice per
genotype). bReal-time PCR
analysis of col6a1 mRNA levels
(n=5 mice per genotype).
cRepresentative samples of
atria from WT and KO animals
stained with Masson’s
trichrome to visualize fibrosis
which appears bluish. Images
were chosen out of sections
from 5 WT mice and 5 KO
mice. dQuantification of the
percentage of atrial fibrosis
between genotypes. *P\0.05;
ns not significant
47 Page 8 of 15 Basic Res Cardiol (2015) 110:47
123
performed to measure Kv1.1 subunit abundance in patients
and controls. Immunoblots of atrial tissue revealed the
presence of detectable amounts of Kv1.1 protein in human
heart for the first time (Fig. 4c, d). Quantification of Kv1.1
subunit levels revealed no change in Kv1.1 protein levels
in pAF patients (P =0.804) but an unexpected and sig-
nificant 75 % increase in Kv1.1 protein levels in cAF
patients compared to SR controls (Fig. 4c, d; P\0.05).
Kv1.1 protein appeared similarly increased in cAF patients
with and without valvular heart disease (Supplementary
Figure 1). To verify that human Kv1.1 protein expression
was associated with cardiomyocytes and not with other cell
types, immunocytochemistry of isolated human atrial
myocytes was performed revealing significant myocyte-
Fig. 3 Kcna1 expression in
mouse cardiomyocytes. aRT-
PCR detection of Kcna1 mRNA
in isolated atrial (A) and
ventricular (V) myocytes from
three mice labeled 1-3. Brain
tissue (B) was used as a positive
control, while no cDNA (N) was
used as a negative control.
bQuantitative RT-PCR shows
that Kcna1 mRNA levels
(normalized to Rpl7) are about
tenfold more abundant in atrial
myocytes than in ventricular
(n =3 mice; P=0.07, paired
ttest). Immunostaining of
isolated atrial (c) and ventricular
(d) myocytes isolated from
adult mice and co-stained with
antibodies against Kv1.1 and
Junctophilin-2 (Jph2), a t-tubule
protein. The gain was increased
in the ventricular images
relative to the atrial images to
allow visualization of Kv1.1
immunoreactivity, which is
much less intense in ventricular
myocytes. The boxed regions in
the overlay images are shown
magnified 500 % in the bottom
panels. Representative images
were chosen out of 15 cells (7
atrial and 8 ventricular
myocytes) from 2 WT mice and
8 cells (5 atrial and 3 ventricular
myocytes) from 1 KO mouse.
Scale bars 20 lm
Basic Res Cardiol (2015) 110:47 Page 9 of 15 47
123
specific Kv1.1 immunoreactivity (Fig. 5). The substantial
upregulation of Kv1.1 subunits in the atria of cAF patients,
but not pAF, suggests that the channels undergo expression
remodeling that may contribute to AF pathophysiology and
be a potential determinant of the transition to maintained
AF.
Kv1.1 subunits form functional channels in human
atrial myocytes
Having detected Kv1.1 subunits in human atria, patch-
clamp recordings were performed utilizing a Kv1.1-speci-
fic inhibitor, dendrotoxin-K (DTX-K), to determine whe-
ther the channels make a functional contribution to human
cardiac currents [31]. Following enzymatic isolation of
human myocytes from right atrial appendages of patients in
SR, whole-cell voltage clamp recordings were performed
to measure outward membrane currents using a voltage
step protocol composed of brief one-second depolarizations
from -60 mV to ?50 mV at 0.1 Hz to activate outward
K
?
currents (Fig. 6a). In the presence of DTX-K to
selectively block Kv1.1 subunits, the peak and late outward
current amplitudes were significantly reduced by about 20
and 10 %, respectively (Fig. 6b, c; P\0.01 and 0.05,
respectively). This DTX-K-sensitive component of the
outward current is indicative of a small but significant
contribution by Kv1.1-containing channels to the total
repolarizing K
?
current in human atrial myocytes. Thus,
Kv1.1 channels are not only present in human heart, but
they are also functional and contribute to atrial
repolarization.
Kv1.1-associated currents are increased in cAF
patients
To determine if the observed cAF-associated increase in
Kv1.1 protein levels corresponds with an increase in
Kv1.1-associated currents, the DTX-K-sensitive compo-
nent was then measured in atrial myocytes isolated from
patients with cAF. Membrane capacitance was not signif-
icantly different between SR and cAF myocytes suggesting
similar cell sizes in each group (P=0.59; Supplementary
Figure 2). Myocytes from patients with cAF showed a
slight reduction in K
?
-current amplitudes, which did not
reach the level of statistical significance (Fig. 6a). How-
ever, both the peak and late components of the DTX-K-
sensitive current showed a significant two- to three-fold
increase compared to SR (Fig. 6b–d; P\0.05 and 0.01 at
peak and late current levels, respectively). To further
characterize our patient cohort, I
Ca,L
was measured in
myocytes from SR and cAF patients as a major hallmark of
electrical remodeling. Consistent with previous reports,
I
Ca,L
was significantly reduced by about 50 % in cAF
myocytes compared to SR (Supplementary Figure 3) [46].
The substantial increase in DTX-K-sensitive current in cAF
patients, coupled with increased KCNA1 expression,
0
5
10
15
76
9
SR pAF
0.0
0.5
1.0
1.5
2.0
2.5
15 14
Relative to SR
(KCNA1/HPRT1)
500 bp
1500 bp
KCNA1
A
ns
CD
*
Kv1.1 - 56 kDa
GAPDH - 34 kDa
SR SR cAF cAF
Relative to SR
(norm. to GAPDH)
SR cAF
0.0
0.5
1.0
1.5
2.0
2.5
911
*
Kv1.1 - 56 kDa
GAPDH - 34 kDa
SR SR pAF pAF
R
S
o
t
e
vi
tal
e
R
)
H
D
P
AGo
t
.m
ron(
ns
SR pAF cAF
B
Fig. 4 KCNA1 mRNA and
protein expression in human
atria. aRT-PCR detection of
KCNA1 mRNA in human atria.
bmRNA levels (mean ±SEM)
of KCNA1 in patients with
chronic AF (cAF) and
paroxysmal AF (pAF) relative
to sinus rhythm (SR).
Representative Western blots of
Kv1.1 protein with
corresponding densitometric
quantification (mean ±SEM)
of protein levels in patients with
pAF (c) and cAF (d) relative to
SR. *P\0.05 vs. SR; ns not
significant. Numbers in bars
indicate atrial samples
47 Page 10 of 15 Basic Res Cardiol (2015) 110:47
123
suggests that Kv1.1 channels undergo molecular and
electrical remodeling, which may contribute to the path-
omechanisms of persistent AF.
Discussion
Traditionally, Kv1.1 channels have been regarded as brain-
specific channels with little to no cardiac expression but
our evidence suggests that this paradigm needs to be
reconsidered. Here RT-PCR, western blotting, and
immunocytochemistry demonstrate the presence of Kv1.1
channels in human atria; the first time they have been
reported in human heart. Prior studies found Kv1.1 tran-
scripts in mouse and rat hearts using Northern blotting but
the abundance was so low that they were thought to result
from sample contamination by neural tissues [18,30].
More recently, newer RT-PCR-based methods with higher
sensitivity reproducibly detected Kv1.1 transcripts in
mouse heart, especially in nodal regions where the gene
appears to be expressed at relatively high levels [11,12,14,
20]. This work verifies the presence of Kv1.1 mRNA in
mouse atria, albeit at apparently low abundance. Impor-
tantly, Kv1.1 mRNA was detected in isolated mouse car-
diomyocytes for the first time in this study, demonstrating
that Kv1.1 expression in cardiac tissue cannot be solely
attributed to contamination by neural cells. Interestingly,
Kv1.1 transcripts showed higher expression in atrial myo-
cytes than ventricular, similar to what has been reported for
Kv1.5 subunits [39]. Furthermore, at the protein level, this
study provides the first evidence of Kv1.1 immunoreac-
tivity in individual cardiomyocytes. In mice, the Kv1.1-
positive labeling in atrial myocytes appeared predomi-
nantly intracellular, whereas the weaker staining in ven-
tricular myocytes showed clustering in a pattern consistent
with transverse tubules, as revealed by co-labeling with the
t-tubule marker protein Junctophilin-2. The Kv1.1
immunoreactivity in human atrial myocytes was similar to
that seen in mice with a predominantly intracellular
staining pattern, as well as striations consistent with
localization at Z-lines. The mostly intracellular Kv1.1
staining pattern in atrial myocytes may reflect a relative
abundance of the channel in intracellular pools awaiting
membrane insertion in response to some physiological
trigger. For example, atrial Kv1.5 channels are dynamically
recruited to the membrane from submembranous pools in
response to high sheer stress or cholesterol depletion [1,3].
However, further experimentation will be required to
determine whether Kv1.1 channels undergo similar traf-
ficking mechanisms.
Our identification of DTX-K-sensitive outward currents
in human atrial myocytes suggests that Kv1.1 channels
make a previously unrecognized contribution to membrane
repolarization during the atrial action potential. Biochem-
ical and electrophysiological studies have repeatedly
demonstrated the exquisitely strict specificity of DTX-K
for inhibition of only Kv1.1 subunits [31,48,49]. The two
primary voltage-gated potassium currents in human atrial
myocytes are the fast transient outward current (I
to,f
) and
the ultra-rapid delayed rectifier potassium current (I
Kur
),
which are encoded by Kv4.3 and Kv1.5 a-subunits,
respectively. Since DTX-K reduced the peak and late
outward currents, we predict that Kv1.1 channels make a
small but significant contribution to I
to,f
and I
Kur
. Of note,
Fig. 5 Kv1.1 immunoreactivity in isolated human atrial cardiomy-
ocytes. Representative confocal images of Kv1.1-positive immuno-
staining (a,b) and a negative control (c). Kv1.1 immunoreactivity
showed a strong fluorescent signal throughout the cell and a tight
striated pattern which overlapped with the Z-line. In negative
controls, primary antibody was omitted and cells were labeled only
with the secondary antibody revealing an absence of nonspecific,
background staining
Basic Res Cardiol (2015) 110:47 Page 11 of 15 47
123
human atrial myocytes exhibit a substantial late outward
I
Kur
current component of unknown molecular origin and
composition that is characterized as being non-inactivating,
insensitive to the Kv1.5-blocker AVE0118, and increased
in cAF myocytes relative to SR [6,41]. The DTX-K-sen-
sitive late currents recorded here fit the profile of this
unidentified non-inactivating, AVE0118-insensitive current
component of I
Kur
, suggesting that it may be encoded by
Kv1.1 channels. In addition to I
to,f
and I
Kur
, human atrial
myocytes also exhibit a rapidly activating and inactivating
current (I
Kr
) and a slowly activating current (I
Ks
), mediated
by hERG1 and KCNQ1 channels, respectively, but they are
0
1
2
3
4
DTX-K sensitive
current (pA/pF)
"late""peak"
*
**
0
2
4
6
8
10
***
***
"late""peak"
0
2
4
6
8
10
**
Current amplitude
(pA/pF)
"late""peak"
*
500 ms
0 pA/pF
5 pA/pF
500 ms
0 pA/pF
5 pA/pF
DTX-K sensitive current
A
B
C
500 ms
0 pA/pF
4 pA/pF
DTX-K
(10 nM)
Control
500 ms
0 pA/pF
4 pA/pF
SR cAF
DTX-K
(10 nM)
Control
”peak“
“late“ ”peak“
“late“
-60 mV
50 mV
500 ms
f = 0.1 Hz
n=9/3 n=11/3
Control
DTX-K
SR (n=9/3)
cAF (n=11/3)
SR cAF
D
Fig. 6 Dendrotoxin-K sensitive
outward current in isolated
human atrial myocytes.
aAveraged current traces
recorded from human atrial
myocytes from patients in sinus
rhythm (SR) or chronic AF
(cAF). Current traces show
control measurements at
baseline followed by application
of 10 nmol/L dendrotoxin-K
(DTX-K) to specifically block
Kv1.1 channels. The voltage
step protocol used for
recordings is shown at the top.
bTraces show the averaged
current difference representing
the dendrotoxin-K sensitive
component. cAmplitudes of the
peak and late phases of the
outward K
?
currents
(mean ±SEM) before (control)
and after application of DTX-K
in atrial myocytes from SR and
cAF patients. dAmplitudes of
the peak and late phases of the
DTX-K sensitive current
difference (mean ±SEM) in
atrial myocytes from SR and
cAF patients. *P\0.05,
**P\0.01, ***P\0.001 vs.
corresponding means under
control conditions using
Student’s paired (c) and
unpaired (d) t test, respectively.
The nvalues indicate
myocyte/patient numbers
47 Page 12 of 15 Basic Res Cardiol (2015) 110:47
123
much smaller outward potassium currents relative to I
to,f
and I
Kur
[2,24,33,34,40]. Whether Kv1.1 channels
contribute to I
Kr
or I
Ks
cannot be inferred from this work,
since the myocyte isolation protocol largely destroys the
I
Kr
current and the voltage step duration is too short to
accurately discriminate classical I
Ks
. In summary, human
atrial myocytes exhibit significant DTX-K-sensitive peak
and late outward currents that may represent a previously
unrecognized contribution by Kv1.1 channels to I
to,f
and
I
Kur
.
Elucidation of the subunit stoichiometry of the func-
tional cardiac Kv1.1 channel may help resolve the identity
and role of the Kv1.1-associated cardiac current in the
atrial action potential. The kinetics and gating properties of
Kv1-mediated currents are determined by their associated
a- and b-subunit composition. For example, Kv1.4 a-sub-
units and Kvb1 accessory subunits confer fast N-type
inactivation via the presence of ball domains, whereas
Kv1.6 a-subunits possess N-type inactivation prevention
(NIP) domains that can neutralize inactivation gating [27,
32,38]. Studies in brain show that Kv1.1 a-subunits
preferentially form functional heterotetrameric assemblies
with Kv1.2 or Kv1.4 and sometimes Kv1.6 [49]. Future
biochemical analyses will be required to determine which
Kv1 a- and b-subunit combinations exist in heart, and what
their respective functional roles might be.
The significant increase in DTX-K-sensitive currents in
cAF myocytes with concomitant upregulation of Kv1.1
protein levels suggests that Kv1.1 channels may contribute to
electrical remodeling in cAF. The transition from paroxys-
mal to persistent AF is facilitated by atrial remodeling of ion
channels at the level of expression and electrophysiological
properties [9,13,23,42,45]. These AF-related electrical
adaptations generally promote shortening of the atrial action
potential and blunting of the atrial effective refractory per-
iod, which increases vulnerability to AF by providing a pro-
arrhythmogenic substrate prone to premature beats [9,23]. In
AF patients, Kv4.3 channel mRNA and protein are down-
regulated leading to a 60 % decrease in I
to
, which is thought
to indirectly increase the upstroke velocity of the atrial action
potential leading to augmented wave propagation that pro-
motes maintenance of AF [5,51]. Remodeling has also been
reported for the atrial voltage-gated K
?
channels hERG1,
Kv1.5 and KCNQ1, but their expression changes have either
been inconsistent or not correlated with corresponding
changes in atrial currents [23]. The concurrent increase in
Kv1.1 protein expression and Kv1.1-mediated outward
currents provides one of the first examples of cAF-associated
upregulation remodeling of a voltage-gated potassium
channel. Interestingly, we did not find corresponding
increases in KCNA1 mRNA levels in cAF atria suggesting
the influence of post-transcriptional modifications similar to
Kv1.5 channels which also exhibit a discordance between
mRNA and protein levels in cAF patients [4,5]. The aug-
mentation of Kv1.1 protein expression and currents is con-
sistent with the reduced action potential durations
characteristic of AF, but whether these are primary causative
changes or secondary compensations remains to be tested.
The increased incidence of inducible AF in Kv1.1-de-
ficient mice suggests that Kcna1 may be a new candidate
gene for AF susceptibility; however, the overexpression of
Kv1.1 channels in cAF patients appears contradictory to
this notion. Some of this discrepancy could be related to
inter-species differences in atrial physiology and channel
expression between mice and humans [17]. Loss-of-func-
tion mutations in the related voltage-gated potassium
channel KCNA5 unexpectedly lead to AF by an atypical
mechanism involving decreased I
Kur
which leads to a
predisposition for atrial action potential prolongation and
early after-depolarizations [25,52]. Kv1.1-deficiency may
cause similar complex AF-promoting alterations in atrial
function; however, in humans KCNA1 gene variants have
yet to be associated with AF. Determination of whether the
upregulation of Kv1.1 channels in cAF patients is causative
for AF or a compensatory mechanism is necessary to
resolve the role of Kv1.1 channels in AF. If it is com-
pensatory, Kv1.1 currents would be expected to oppose AF
and their absence would be expected to promote AF as seen
in our mouse model. Additional studies will be required to
dissect the exact contribution of Kv1.1 currents to action
potential duration and AF susceptibility in mice and
humans, especially given the complex relationship between
modification of outward potassium currents and action
potential morphology [7].
Acknowledgments The authors thank Ramona Nagel and Katrin
Kupser for excellent technical assistance. This work was supported by
grants from the National Institutes of Health (HL107641 to EG;
HL089598, HL091947, and HL117641 to X.H.T.W.; NS076916 to
J.L.N.), the Muscular Dystrophy Association (X.H.T.W.), American
Heart Association (13EIA14560061 to X.H.T.W.), the Deutsche
Forschungsgemeinschaft (Do 769/1-1-3 to D.D.), the German Federal
Ministry of Education and Research through DZHK (German Centre
for Cardiovascular Research to D.D.), the European Union through
the European Network for Translational Research in Atrial Fibrilla-
tion (EUTRAF, FP7-HEALTH-2010, large-scale integrating project,
Proposal No. 261057 to D.D), and Fondation Leducq (‘Alliance for
CaMKII Signaling in Heart’ to X.H.T.W. and ‘European North-
American Atrial Fibrillation Research Alliance’ to D.D.).
Compliance with ethical standards
Conflicts of interest None.
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