Enhanced heterogeneity of myocardial conduction and severe cardiac
electrical instability in annexin A7-deficient mice
Jan W. Schrickela,⁎, Klara Brixiusb, Claudia Herrc, Christoph S. Clemenc, Philipp Sassed,
Kathrin Reetzb, Christian Grohéa, Rainer Meyere, Klaus Tiemanna, Rolf Schröderf,
Wilhelm Blochg, Georg Nickeniga, Bernd K. Fleischmannd, Angelika A. Noegelc,
Robert H.G. Schwingerb,1, Thorsten Lewaltera,1
aDepartment of Medicine-Cardiology, University of Bonn, Germany
bLaboratory of Muscle Research and Molecular Cardiology, Department of Internal Medicine III, University of Cologne, Germany
cInstitute of Biochemistry I and Center for Molecular Medicine Cologne, Medical Faculty, University of Cologne, Germany
dInstitute of Physiology I, Life and Brain Center, University of Bonn, Germany
eInstitute of Physiology II, University of Bonn, Germany
fInstitute of Neuropathology, University Erlangen-Nürnberg, Germany
gDepartment of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany
Received 20 September 2006; received in revised form 12 June 2007; accepted 3 July 2007
Available online 6 July 2007
Time for primary review 27 days
Objectives: Annexin A7 is involved in cardiomyocyte membrane organization and Ca2+-dependent signalling processes. We investigated the
impact of annexin A7 on cardiac electrophysiological properties using an annexin A7-deficient mouse strain (annexin A7−/−).
Methods: Nineteen adult annexin A7−/−and 14 wild-type mice were examined electrophysiologically in vivo by transvenous catheterization.
Hearts were additionally perfused by the Langendorff method and epicardial activation mapping was performed.
Results: The susceptibility to induction of atrial fibrillation was elevated in annexin A7−/−mice. Ten deficient animals showed atrial
fibrillation (AF) episodes ≥1 min and sustained AF ≥30 min was observed in 4 annexin A7−/−mice, but in none of the wild-type mice. The
incidence of ventricular tachycardia (VT) was higher in annexin A7−/−mice and VT duration was prolonged. Epicardial mapping showed
elevated anisotropy and inhomogeneity of conduction, leading to conduction blocks in the deficient mice. Besides alterations of intracellular
calcium homeostasis, electron microscopy showed a homogeneous, electron-dense material that filled the myocardial intercellular
compartments and accumulated at the basement membranes. This led to expansion of the extracellular spaces, which was the most probable
substrate factor responsible for the disturbances of electrical communication.
Conclusions: Annexin A7 deficiency causes severe electrical instability in the murine heart, including conduction disturbances and
anisotropy of impulse propagation, which is accompanied by disturbed calcium handling and intercellular deposits.
© 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Arrhythmia (mechanisms); Cell communication; Conduction; Extracellular matrix; Annexin A7
Annexin proteins are characterized by Ca2+-dependent
phospholipid binding to natural and artificial membranes .
A variable N-terminal domain confers functional diversity to
for Ca2+- and phospholipid-binding . This protein family is
involved in regulation of endo- and exocytotic processes ,
aggregation of chromaffin granules , and modulation and
formation of ion channels . Annexin A7 (synexin, anxA7)
was the first annexin to be identified. It promotes chromaffin
granule aggregation and membrane fusion processes 
Cardiovascular Research 76 (2007) 257–268
⁎Corresponding author. Tel.: +49 228 287 5507; fax: +49 228 287 4053.
E-mail address: firstname.lastname@example.org (J.W. Schrickel).
1Both senior authors contributed equally to this work.
0008-6363/$ - see front matter © 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
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and has been reported to act as a Ca2+-activated GTPase,
. AnxA7 influences cellular Ca2+homeostasis and forms
voltage-gated Ca2+-channels . It is an abundant protein in
murine and human myocardial tissue . Herr et al. created
anxA7-deficient mice (anxA7−/−) by homologous recombina-
velocity of Ca2+-waves in cultured primary astroglial cells
[10,12]. Frequency-dependent cell shortening and the positive
cardiomyocytes, which was attributable to disturbances of the
excitation-contraction coupling .
to also influence electrophysiological properties of the heart
. We therefore evaluated cardiac electrophysiological
susceptibility to atrial fibrillation (AF) and ventricular tachy-
cardia (VT) caused by disturbances of myocardial conductive
The generation of anxA7−/−mice has been previously
described . Isogeneic 129SV-wild-type littermates (WT)
were used as controls. Mice were handled according to the
animal protection law stated in the German civil code and the
investigations were approved by the district government of
and Use of Laboratory Animals published by the US National
Institute of Health and the Position of the American Heart
Association on Research Animal Use (AHA, Nov. 11, 1984).
Thirty-three mice (14 WT, 19 anxA7−/−, 12.7±0. 5 weeks,
25–32 g) were electrophysiologically examined using an in
vivo single catheter technique as described before .
Briefly, preparation, catheterization, and EPI were performed
under inhalation anaesthesia (induction period 2.5 vol.%,
maintenance 1.2 vol.% isoflurane in 70%N2O/30%O2).
The jugular vein was dissected from surrounding tissue and
a 2-French octapolar mouse electrophysiological catheter
[eight 0.5 mm circular electrodes; electrode-pair spacing
0.5 mm (Ciber Mouse, NuMed Inc., NY, USA)] was posi-
tioned transvenously on atrial and ventricular level. A surface
6-lead ECG was monitored continuously and standard ECG
parameters were analyzed under stable baseline conditions
(Fig. 1A). QTc and JTc (JT: interval from the S-wave meeting
the isoelectrical line to the end of the T-wave) were calculated
as described previously [14,15]. All data were amplified,
filtered, sampled at 4 kHz and digitally stored (LabSystem,
C.R. Bard Inc., New Jersey, USA).
EPI included transvenous atrial and ventricular recording
and stimulation as previously described . In brief, bipolar
electrograms were obtained from each intracardiac electrode-
pair. Pacing threshold currents at 1 ms were 0.76±0.41 mA
at atrial and 0.69±0.49 mA at ventricular level. Twice pacing
threshold rectangular stimulus pulses were administered by a
modified multi-programmable stimulator (Model 5328;
Medtronic, MN, USA). Performing fixed-rate and extra-
stimulus pacing, sinus node recovery time (SNRT), Wenck-
ebach periodicity (WBP), 2:1 AV conduction, and atrial and
AV nodal refractory periods (ARP, AVNRP) were evaluated.
SNRT was defined as maximum return cycle length (CL)
after 10 s fixed-rate pacing (S1S1: 120 ms, 100 ms and
80 ms). ARP and AVNRP were evaluated by programmed
atrial stimulation (8 stimuli fixed rate at S1S1 cycle length:
120 ms, 110 ms, and 100 ms; one short coupled extrastimulus
with a 10 ms-stepwise S1S2-reduction). AVNRP was defined
as longest S1S2 with loss of AV-nodal conduction, ARP as
longest S1S2 with absent atrial response. Ventricular
refractory period (VRP) was evaluated similar to ARP.
2.2. Arrhythmia induction
Atrial and ventricular burst stimulations were performed
for 5 s (S1S1: 50 ms–10 ms, 10 ms stepwise reduction;
stimulus amplitudes 1.0 and 2.0 mA) [14,15]. Ventricular
vulnerability was additionally tested by extrastimulus pacing
(S1S1: 120 ms, 100 ms, and 80 ms followed by up to 3 extra
beats). AF was defined as rapid and fragmented atrial elec-
trograms with irregular AV-nodal conduction for ≥1 s .
Number of AF episodes, mean ventricular heart-rate during
AF and AF duration were analyzed. VT was defined as ≥4
ventricular premature ventricular beats. Number of inducible
VT episodes/animal, VT duration and tachycardia-CL were
2.3. Langendorff-perfused hearts and epicardial mapping
For analyses of myocardial conduction velocities and
characteristics, hearts were Langendorff-perfused, and epi-
cardial activation mapping (EAM) using a 128-electrode
Fig. 1. A: ECG-parameters analyzed in sinus-rhythm. PQ: onset of P-wave to beginning of Q-wave; QRS beginning of Q to the point of S-wave returning to
isoelectrical line; QT: onset of the Q-wave to returning-point of T to baseline. J-point: point of S-wave intersecting with isoelectrical line. B: Alterations in
standard-ECG-parameters: P-wave (⁎: P=0.008) and QRS-durations (⁎⁎: P=0.03) were significantly shortened in the anxA7−/−-mice as compared to WT. C:
Induction of atrial fibrillation (AF) by atrial burst stimulation (stimulation-cycle-length: 20 ms at 1 mA amplitude) in anxA7−/−. This episode lasted 14.8 s before
spontaneous conversion into normofrequent sinus-rhythm (lower traces). D: AF is characterized by irregular ventricular conduction (surface lead III: Surf) and
fragmented, polymorphic high frequency atrial activation (intracardiac electrograms on His-bundle and atrial level). E: Analyses of AF-inducibility among the
of multiple inducible animals (≥4 episodes; 10 testings) was significantly greater in the deficient animals as compared to WT (⁎: P=0.04). F: The probability of
inducingAF episodeswas significantlyhigherin the deficientmice(total anxA7−/−andWT-groups:⁎: P=0.001;subcohortsof inducibleanimals:⁎⁎:P=0.015).
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array was performed. Thirty atrial and ventricular areas of 15
anxA7−/−and 8 atrial and ventricular areas of 4 WT mice
were analyzed. Hearts were excorporated and dissected
from surrounding tissue in ice-cold Krebs-Henseleit buffer.
After cannulation of the aorta, hearts were retrogradely
perfused in a Langendorff-apparatus (Radnoti Technologies
259 J.W. Schrickel et al. / Cardiovascular Research 76 (2007) 257–268
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Inc., Monrovia, CA, USA) at constant pressure perfusion
(80 mm Hg). The perfusate composition was (in mM): NaCl
110, KCl 4.6, MgSO41.2, CaCl 2, NaH2PO42, NaHCO325,
glucose 8.3, Na-pyruvate 2 and gassed with carbogen (O2
95%, CO25%), pH 7.35–7.45. A perfusate temperature of
37 °C was continuously maintained. The heart was immersed
in a water-jacketed chamber and further fixed on a
moisturized support. Unipolar extracellular electrograms
(128) were recorded from the epicardial surface of the ven-
tricles with regard to a reference electrode positioned in the
distance: 300±7 μm). Fixed-rate and single extrastimulus
stimulation were performed using two adjacent electrodes of
the array. Electrograms were recorded using a 128-channel,
computer-assisted recording system (Multi Channel Systems,
Reutlingen, Germany) with a sampling rate of 25 kHz (25000
samples per second). Data were bandpass filtered (50 Hz),
digitized with 12 bit and a signal range of 20 mV.
Activation maps were calculated from these data using
custom-programmed software (Labview 7.1, National Instru-
electrogram was evaluated and maximal negative dV/dt
activation was defined as timepoint of local activation .
With regard to myocardial fibre orientation, longitudinal and
transversal conduction velocities (CV) were evaluated by cal-
culating latencies between two electrodes, divided by the
interelectrode distance, and anisotropic ratios were calculated
as described before . To obtain an index of local conduc-
tionslowing for each electrode,the activation timedifferences
to neighbouring points were normalized to interelectrode dis-
tance. The largest difference at each site was defined as local
phase delay. The variation coefficient of these phase delays
was usedasinhomogeneity index for inhomogeneity inglobal
conduction, as described before [18,19].
2.4. Electron microscopy
Small pieces of the ventricular myocardium of 4% para-
formaldehyde immersion-fixed hearts (5 anxA7−/−, 5 WT)
PBS for 2 h at 4 °C. After three-times washing in 0.1 M
cacodylate buffer for 10 min, the specimens were dehydrated
araldite. Sections of plastic-embedded specimens were cut
with glass for thin sections and a diamond knife for ultra thin
sections on a Reichert ultramicrotome. The 0.5-μm thin slices
were stained with methylene blue and investigated using a
light microscope (Axiophot, Zeiss, Oberkochen, Germany).
Ultra thin 60 nm sections were examined using an electron
microscope (902A, Leo, Oberkochen, Germany) after con-
trasting with uranyl acetate-lead citrate.
2.5. Measurement of cell shortening
Murine ventricular cardiomyocytes were isolated from
anxA7−/−and WT as described previously  and placed
on laminin-coated glass slides in 12-well dishes covered by
cell medium (M199, Gibco, Karlsruhe, Germany, supple-
mented with vitamins and essential amino acids). Experi-
ments were performed at 32 °C in a pre-warmed Tyrode's
solution (in mM: NaCl 140, KCl 5.8, CaCl21, KH2PO40.5,
NaHPO40.4, MgSO40.9, glucose 11.1, Hepes 10.0, pH 7.1,
Penicillin/Streptomycin 1:100) . The glass slides were
mounted on the stage of a microscope (Diaphot 300, Nikon,
Japan). Cardiomyocytes were electrically stimulated using
field stimulation (pulse duration: 5 ms).
Baseline surface-ECG and intracardiac EPI parameters
AH (ms) †
HV (ms) ‡
SNRT (ms) § (S1S1 120 ms)
ARP (ms) ∥ (S1S1 120 ms)
AVNRP (ms)¶ (S1S1 120 ms)
WBP (ms) #
VRP (ms) ††(S1S1 120 ms)
+/++: rate corrected QT/JT-intervals; †: interval from atrial to His-signal; ‡:
interval from His to first QRS-movement in surface-ECG; §: sinus-node
recovery-time; ∥: atrial refractory period; ¶: AV-nodal refractory period; #:
Wenckebach-periodicity;⁎⁎: ††: ventricular refractory period.
Fig. 2. A: Ventricular burst stimulation in a WT (upper traces, surface ECG: Surf; intracardiac electrograms on His-level: His; stimulation-cycle length: 40 ms at
1 mA) fails to induce a VTand a normofrequent sinus-rhythm persists. Short lasting polymorphic VT (10 beats, lower traces) in a WTafter programmed extrabeat
stimulation (8 beats with constant cycle length S1S1: 120 ms) with single short coupled extrastimulus (S1S2: 25 ms). Note the presence of atrioventricular
dissociation in the intracardiac recording on His-level. B: Induction of a polymorphic VT in anxA7−/−(upper traces) after ventricular burst stimulation (stimulus
cycle length: 40 ms at 1 mA). Programmed stimulation (as performed above but S1S1: 80 ms; S1S2: 40 ms) induces a long lasting, primarily polymorphic VT in
an anxA7−/−mouse. C: The statistical comparison of different stimulation regimes for VT induction shows a tendency towards a higher overall susceptibility
towards VT induction in the mutants (at a total of 19 different testings per animal). The same tendency is present after burst stimulation (burst; 10 testings).
Significantly more anxA7−/−mice were inducible after administration of ventricular programmed stimulation (progr.; 9 testings;⁎: P=0.02) D: Evaluation of the
number of episodes inducible in the single animal showed a significantly elevated fraction of animals ≥3 (⁎: P=0.013) and N4 (⁎⁎: P=0.015) inducible episodes
in the deficient mice; animals ≥10 inducible episodes (19 testings) were only present in anxA7−/−. E: The probability of VT induction in the total animals (left
side) and the subgroups of inducible animals (right side) showed a significant elevated VT-susceptibility (⁎: Pb0.001) in anxA7−/−.
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Fig. 3. Fractional shortening (A) and the fura-2 transient (B) measured at increasing stimulation frequencies in murine ventricular cardiomyocytes of anxA7−/−
(n=18 from 4 hearts) and WT (n=16 from 6 hearts). C: Fibers were first maximally Ca2+-activated, then the sarcoplasmic reticulum (SR) was loaded under
definite conditions and finally caffeine was added to the solution. The ratio of caffeine-induced tension over the maximal Ca2+-activated tension is a parameter
characterizing the amount of Ca2+released out of the SR. D: Caffeine-induced Ca2+release was significantly reduced in anxA7−/−. This reduction was reversible
by PKA-dependent phosphorylation (E). F: The density of the β1-adrenoceptors was significantly down-regulated in anxA7−/−G: No alterations were observed
in the expression of the heteromeric G-proteins.⁎: Pb0.05.
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2.6. Determination of intracellular Ca2+transients
The intracellular Ca2+transient was measured using the
cell permeable Ca2+-indicator fura-2 acetoxymethylester
(fura-2 AM, Molecular Probes, Eugene). Isolated cardiomyo-
cytes were incubated at 37 °C for 30 min with fura-2 AM
(7 μM) and afterwards incubated without fura-2 AM for
10 min for de-esterification. Fura-2 loaded cells were sited in
the inverted microscope, perfused with a Tyrode's solution at
32 °C and stimulated electrically. Fluorescence was detected
by a photomultiplier system, recorded and digitally stored
(Scientific Instruments, Heidelberg, Germany). The dual
excitation of fura-2 at 380 nm (Ca2+-free indicator) and
340 nm (Ca2+-bound indicator) allows determination of the
intracellular Ca2+concentration. Emission was measured at
530 nm. The relativeCa2+signalwas calculatedfromthe ratio
340 nm over 380 nm. Data are presented as ratio of
fluorescence intensity at 340 nm and 380 nm excitation.
2.7. Measurement of the caffeine-induced sarcoplasmic
reticulum (SR) Ca2+-release
To prepare the skinned fibers, hearts were placed in ice-
cold solution of 50% glycerol and (in mmol/l) imidazole 20,
NaN310, ATP 5, MgCl25, EGTA 4, dithiotreitol 2 (pH 7.0).
Small sections of the left ventricle were dissected, yielding
muscle fibers of a 0.2 mm diameter and 2–3 mm in length.
The fibre strips were skinned by incubation in the above
solution with addition of saponin (0.05 mg/ml, 30 min)
. The fibers were transferred into a fresh solution
without detergent and stored at −20 °C. Caffeine was used to
open the ryanodine receptors of the SR and to induce SR Ca2+
release. Experiments were further performed as previously
2.8. β1- and β2-adrenoceptor binding studies
Membrane preparations of cardiac tissue were performed
as described before . β-adrenoceptors in cardiac tissue
homogenates were investigated using3H-CGP 12.177 [(−)-
dazol-2-one)] as the radio-labelled ligand (specific activity
50 Ci/M). Specific binding was determined as the difference
of binding in the absence and presence of 10 μM DL-
propranolol. β-adrenoceptor subtypes were determined by
competition experiments using the β1-selective antagonist
CGP 207.12A (0.3 μM) and the β2-selective antagonist
ICI 118.551 (0.05 μM). β2to β1ratio was calculated as
previously described .
After homogenisation of ventricular tissue, protein was
determined as outlined before . For western blot analysis,
4% stacking gel and a sodium dodecyl sulfate polyacrylamid
(SDS)-12% gel under constant current (70 mA [60 min]
and 120 mA [180–240 min]) and transferred onto a poly-
vinyldiene fluoride membrane (PVDF, Roche, Mannheim,
2.10. Statistical analysis
Statistical analysis included a two-tailed Student's t-test
or multivariate ANOVA with post-hoc subgroup testing
when appropriate (i.e. Tukey's test). Discrete variables were
analyzed by 2-sided Fisher's exact test. A P-value≤0.05
was regarded as statistically significant.
3.1. Surface ECG and EPI
We first carried out ECG recordings (Fig. 1B and Table 1).
At equal baseline heart rates, anxA7−/−exhibited significantly
shorter P-wave and QRS duration. Evaluation of standard
intracardiac electrophysiological parameters on atrial, AV-
nodal and ventricular level showed no significant differences
among the groups (Table 1).
3.2. Atrial fibrillation
After atrial burst stimulation, anxA7−/−exhibited a signifi-
cant elevated susceptibility to induction of AF (Fig. 1C, D),
regarding inducible episodes/animal (4.4±1.7 versus 2.7±
2.0 in WT; Pb0.02). A not yet significant number of the
anxA7−/−animals showed long lasting AF episodes ≥1 min
(42% in anxA7−/−versus 14% in WT; P=0.131; Fig. 1E).
Sustained AF episodes lasting ≥30 min were exclusively
observed in 4 of 19 anxA7−/−(Fig. 1E), whereas the longest
AF episode in one WT lasted just 480 s before spontaneous
termination. In the mutant animals with sustained AF, ven-
tricular stimulation procedure was performed despite persist-
ing AF after 30 min. Analogously to longer duration, the
number of multiple inducible animals (≥4 episodes/10 test-
ings at different stimulation-CLs) was significantly higher in
Protein expression of SERCA2a, phospholamban (PLB), the SERCA2a/
PLB-ratio as well as of NCX measured in crude membrane preparations
(densitometric units/mg protein)
(densitometric units/mg protein)
(densitometric units/mg protein)
(densitometric units/mg protein)
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anxA7−/−, too, and indicates elevated arrhythmogeneity on
atrial level in these mice (Fig. 1E).
We defined the probability of AF induction as inducible
episodes divided by total testing maneuvers applied in the
groups. This surrogate parameter was significantly elevated
in the deficient mice (Fig. 1F). Deficient mice were sig-
nificantly more susceptible to AF induction in the total group
of all animals (anxA7−/−: 79 inducible episodes/190 testings;
WT: 38 episodes/140 testings) and in the group of inducible
animals only (anxA7−/−: 79 episodes/170 testings; WT: 38
elevated electrical instability on atrial level in the anxA7−/−
3.3. Ventricular tachyarrhythmia (VT)
WTwerelessprone to VTinduction and only short lasting,
polymorphic VTs were inducible by burst and programmed
extra-stimulation protocols (Fig. 2A). The susceptibility
towards longer lasting VTs was elevated in the anxA7−/−
mice under both the programmed and burst stimulation
protocols (Fig. 2B). Moreover, these VT episodes lasted
significantly longer in anxA7−/−(0.88±1.3 s versus 0.25±
0.07 s; Pb0.0001). All inducible VTs were self-terminating.
Analysis of the number of total inducible VT episodes per
inducible animal revealed a significant higher incidence
in anxA7−/−animals (total: 8.1±5.3 versus 2.4±1.5 in WT;
Fig. 4. At the electron microscopic level, no abnormalities of the cardiomyocyte ultrastructure could be observed in anxA−/−(A, B, C) as compared to WT (D),
while a distinct alteration is found in the intercellular spaces of myocardium from anxA−/−. At lower magnification, a homogeneous electron dense material (⁎) is
visible in the enlarged intercellular space between a capillary (CA) and two cardiomyocytes (CM) (A). At higher magnification it is recognizable that the spaces
between the cardiomyocytes also are filled by such a homogeneous material (⁎) (B). In contrast to WT (D), which reveals a normal structured basement
membrane (arrows) covering the adjacent cardiomyocytes, such basement membranes cannot be identified in intermyocardial space (⁎: material; arrows: missing
basement membrane) of anxA−/−myocardium (C). Bar: A=4 μm; B=400 nm, C=200 nm, D=400 nm.
Fig. 5. A: Activation mapping of 15 left and 15 right epicardial ventricular areas of anxA−/−and WT-hearts was performed. The figure shows representative
examples of conduction properties of these epicardial mappings. In contrast to the homogeneous conduction under spontaneous conditions in WT, irregular
breakthrough areas can be seen in anxA−/−, resulting in more heterogeneous transduction in spontaneous sinus rhythm beats (spont). This effect is even more
pronounced under epicardial fixed rate stimulation (stim) and extra-stimulation (premature). Distinct blockages of conduction in the mutant mice are present
during these stimulation protocols (upper trace, stim, premature). In contrast, WT show homogeneous centrifugal conduction from the stimulation site (white
ovals), and no relevant blocking. B: The anisotropic ratio was calculated as described before  for all mapped areas (longitudinal conduction velocity (CV)
divided by transversal CV with regard to fibre orientation that was determined as described before ). This analysis showed significantly enhanced global
anisotropy provoked by premature stimulation (⁎: P=0.029).These results indicate preservation of directed conduction under more basal conditions, but loss of
physiological and directed global impulse propagation under early extrabeat stimulation performed close to refractoriness, simulating early premature ventricular
beats. C: The inhomogeneity index (median of phase delay divided by P5-P95 interval of phase delay calculated for every single electrode compared to all
adjacent electrodes of the array) showed highly significantly elevated heterogeneity of conduction in anxA−/−under fixed rate (⁎: P=0.00001) and premature
stimulation conditions (⁎⁎: P=0.0004). D: Mean number of adjacent electrodes of the whole array with significant conduction delay defined as CV b0.1 mm/ms
was significantly elevated in the deficient mice under fixed rate (⁎: P=0.00001) and premature stimulation (⁎⁎: P=0.0007), but not sinus rhythm.
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burst-stimulation: 4.6±2.9 versus 2.0±0.7; programmed
stimulation: 4.9±2.6 versus 2.3±1.5; Pb0.05). Significantly
more anxA7−/−animals were susceptible to VT induction by
programmed extra-stimulation as compared to WT (Fig. 2C).
AnxA7−/−also showed a higher number of inducible VT
episodes per individual animal (19 testings; Fig. 2D). The
probabilityofVTinductionwas calculatedasdescribed above
for AF and was significantly elevated in the deficient mice
independent from the stimulation protocol (Fig. 2E). Thus
ventricular vulnerability is markedly pronounced in the
annexin A7-deficient mice.
an analysis of possible Ca2+-dependent mechanisms underly-
ing the incidence of arrhythmias in anxA7−/−mice was per-
formed. Intracellular Ca2+transients and cell shortening were
measured in ventricular cardiomyocytes at increasing stimula-
tion frequencies [0.5–5 Hz (=30–300 bpm)]. Under basal
conditions, i.e. stimulation frequency of 0.5 Hz, fractional cell
shortening was 2.32±0.40% in WT (16 measurements from
6 mice) and 2.97±0.40% in anxA7−/−mice (18 measurements
from 4 mice; Fig. 3A). By raising the stimulation frequency to
5 Hz, fractional cell shortening increased in WT (5 Hz: 4.28±
0.52%), but declined in anxA7−/−mice (5 Hz: 1.68±0.30%).
The diastolic cell length was not changed.
Analyses of the intracellular Ca2+transients at 0.5 Hz
determined fura-2 amplitude that were similar in anxA7−/−
and WT animals (0.16±0.02 versus 0.20±0.01). At higher
stimulation frequencies up to 5 Hz, the fura-2 transient
was not significantly altered in anxA7−/−, but increased in
WT (Fig. 3B). Also in this setting, the diastolic fura-2
ratio was unchanged (0.5 versus 5 Hz; WT: 1.35±0.06
versus 1.44±0.07; anxA7−/−: 1.13±0.05 versus 1.18±
0.06%). AnxA7−/−thus shows a systolic, but not dias-
tolic dysregulation of the cardiomyocytal Ca2+homeo-
To investigate Ca2+release from the SR during systole,
the caffeine-induced tension development was measured in
saponin-skinned fibers (Fig. 3C). Under basal conditions,
(i.e. absence of PKA-dependent stimulation of the SR Ca2+-
release), the caffeine-induced tension was significantly
reduced (anxA7−/−: 86.1±6.4%, WT: 101.9±1.2% of maxi-
mal tension; Fig. 3D). Incubation of the saponin-skinned
fibers with the catalytic subunit of PKA significantly
increased the caffeine-induced tension only in anxA7−/−
(Fig. 3E). In anxA7−/−, protein expression of SERCA2a was
significantly decreased, whereas that of phospholamban was
not altered, leading to a significantly decreased SERCA2a/
phospholamban ratio (Table 2). This change results in a
reduced reuptake of Ca2+into the SR. We detected a com-
pensating upregulation of the protein level of the sodium-
calcium antiporter (NCX; Table 2). We furthermore investi-
gated the distribution of the β1- and β2-adrenoceptors in
cardiac membrane preparations. β1-adrenoceptors were sig-
nificantly down-regulated in anxA7−/−(Fig. 3F), without
alterations of the β2-adrenoceptors or the expression of the
Gi- and Gs-proteins (Fig. 3G,H). These results indicate rele-
vant changes of Ca2+-handling in the hearts of anxA7−/−that
are analogous to human heart failure.
ceptor contribution usually associated to heart failure, a
detailed echocardiographical examination showed no sig-
nificant differences in cardiac contractile function among the
groups (analyses included: left ventricular end-systolic
volume, left ventricular end-diastolic volume, ejection-vol-
ume, diastolic left ventricular mass and left ventricular
ejection fraction; data not shown). There were no signi-
ficant alterations in the heart-to body weight ratio between
anxA7−/−and WT (data not shown), indicating an absence of
cardiac hypertrophy or dilatation as possible arrhythmogenic
factors. Histological examination showed neither enhanced
fibrosis nor changes in the number or morphology of
cardiomyocytes (data not shown).
ventricular level that may be associated with the induction of
arrhythmia, atrial and ventricular tissues were examined by
electron microscopy (Fig. 4). In contrast to the WT-group
(Fig. 4D), the whole intercellular space of all 5 anxA7−/−
myocardia that were analyzed was filled by a homogeneous
electron dense material of unknown origin (Fig. 4) that in-
terfered with the basement membranes (Fig. 4C) normally
covering the cardiomyocytes and capillaries in WT (Fig. 4D).
Moreover, collagen fibres in these intercellular spaces were
not detectable (Fig. 4A and B).
3.7. Epicardial mapping
Under conditions of spontaneous sinus beating, epicardial
ventricular activation mapping revealed a more heteroge-
neous conduction in anxA7−/−as compared to WT (Fig. 5A).
At comparatively fast conduction velocities, color-coded re-
construction of isochrone lines showed more heterogeneous
wave breakthrough in the ventricular myocardium of
anxA7−/−. This heterogeneity of conduction successively
increased under simulation and extrastimulus administration,
resulting in significant conduction blocking in the anxA7−/−
mice (Fig. 5A, upper row). This effect was not seen in WT-
animals. The anisotropic ratio was higher in anxA7−/−
animals after premature beat stimulation (Fig. 5B). The cal-
culation of the inhomogeneity index revealed a significantly
enhanced heterogeneity of global conduction (Fig. 5C), and
the mean number of adjacent electrodes with significant
in the deficient animals (Fig. 5D).
266J.W. Schrickel et al. / Cardiovascular Research 76 (2007) 257–268
by guest on February 18, 2013
Atrial mapping revealed analogous effects as present in
ventricular myocardium with significantly elevated inhomo-
geneity indices in the mutant mice under fixed rate
stimulation (3.9±2.0 versus 2.9±0.8; P=0.048) and prema-
ture stimulation (5.8±2 versus 3.5±1.7, P=0.013), but not
basal conditions (4.1±2.2 versus 3.2±1.3; P=0.153).
These results demonstrate that disturbances of the con-
ductive properties in the anxA7−/−mice go along with a
significantly elevated susceptibility to arrhythmia on the
atrial and ventricular levels, particularly under conditions of
stimulation and premature beats. These conduction delays
are less distinct in sinus rhythm and seem thus in part be
compensated under basal conditions.
Depletion of anxA7 is deleterious to the electrical stability
of the murine heart in vivo and leads to an enhanced sus-
ceptibility towards induction of long-lasting atrial and ven-
tricular tachyarrhythmias. We have performed a variety of
analytical approaches to determine possible molecular and
structural alterations underlying this overt pathology. We
found a distinct heterogeneity of epicardial conduction pro-
perties and relevant blockages under conditions of epicardial
stimulation. This is likely due to a severe widening of the
intercellular spacing caused by accumulation of a non-
collagenous substrate and associated disturbances in direct
AnxA7 is a relevant protein in the investigated murine
ventricular tissue (0.04% of total heart protein) and known
to play a role in Ca2+-binding and Ca2+-dependent signalling
processes. It therefore is proposed to dominantly influence the
Ca2+buffering system. In analogy to human heart failure,
anxA7−/−cardiomyocytes show negative force-frequency
relation and downregulation of β1-adrenoceptors, but these
contractility under basal in vivo conditions. Abnormal Ca2+
homeostasis is known to contribute to human arrhythmia, i.e.
by intracellular Ca2+overload under conditions of ischemia
heterogeneity of cardiac electrical properties . Despite the
identified dysregulation of intracellular Ca2+uptake, no sig-
nificant diastolic Ca2+overload was observed. This can be
explained with the measured increase of NCX-activity. It is
possible, however, that the NCX-dependent prevention of
arrhythmogenic diastolic Ca2+overload is limited at higher
heart rates up to 800 bpm (13.3 Hz) or short coupled extra-
beats, which were administered during atrial and ventricular
stimulation manoeuvres. Thus, altered Ca2+homeostasis may
partly contribute to elevated electric vulnerability, but most
likely it is not the predominant arrhythmogenic factor in this
Electron microscopy revealed an impaired integrity of
the basement membranes and a widening of the intercellular
space accompanied by alterations of the structure of the
basement membrane of cardiomyocytes. These alterations
strongly point towards a disturbed intercellular communica-
tion. Although connexin function and distribution were
not studied, such deposits are likely to influence cell-to-cell
communication resulting in impaired gap-junctional cou-
pling. In analogy to previous studies with connexin43 dis-
orders resulting in strong arrhythmogenic effects , we
could detect significant arrhythmia-predisposing heteroge-
neous conduction properties within the ventricular myo-
cardium, most probably related to these ultrastructural
alterations. This heterogeneous conduction was even more
pronounced under conditions of stress, such as extrastimulus
pacing, resulting in significantly more blockage and con-
duction slowing in the myocardium of anxA7−/−mice. Such
distinct proarrhythmic conduction disturbances represent a
relevant substrate for the development of arrhythmias .
Alterations of the intercellular spacing in the heart have been
shown to strongly influence cardiac excitability and myocar-
dial conduction properties [26,27]. Changes of the extracel-
lular matrix and altered collagen distribution also interfere
with local conduction in proarrhythmic atrial remodelling
models . Proper composition of the extracellular matrix
therefore is a major factor in the conservation of a regular
conductive function of the myocardium. Thus, we consider
these changes as the predominant proarrhythmic substrate
factor responsible for increased susceptibility to long-lasting
cardiac arrhythmias. It still remains unclear whether the
susceptibility and duration as compared to diseases with
collagenous aggregations. Furthermore, accompanying
arrhythmogenic co-factors such as alterations of ion channel
expression and function in cardiomyocytes cannot be ruled
out by our experiments and warrant future investigations.
The finding of shorter P and QRS durations is somehow
surprising in a mouse strain with elevated arrhythmia sus-
ceptibility and conduction blocks under ventricular stimula-
tion, but these findings are not contradictory for elevated
stress. Firstly, the surface ECG under basal conditions may
not always be a sensitive surrogate for abnormalities in
myocardial conductivity. Secondly, no alterations of conduc-
tion velocities or conductive blocks were present in the
epicardial recordings under basal, i.e. sinus rhythm condi-
alterations of cardiomyocytal ion-channel activation or en-
hanced adrenergic drive that also might account for the
shortening of P and QRS interval in vivo.
Analogously, myocardial ultrastructural alterations asso-
ciated with enhanced electrical instability are known to occur
in the absence of relevant surface ECG changes . We
showed a dramatic decrease of conduction velocity, enhance-
ment of heterogeneity of conduction and significantly more
blockages under provocation manoeuvres, i.e. fixed rate and
in particular extrastimulus pacing. These findings suggest an
elevated probability for the co-existence of adjacent fast and
slow conducting areas under such conditions. This predis-
poses the electrical wavefront to hitting refractory tissue,
267 J.W. Schrickel et al. / Cardiovascular Research 76 (2007) 257–268
by guest on February 18, 2013
with an elevated possibility for the development of micro- Download full-text
reentries, increasing susceptibility and probability of perpet-
uation of arrhythmias [17,18]. Our electrophysiological
findings point towards this pathomechanism as predominant
arrhythmogenic factor that might be further pronounced in
association with the additional changes found in the in the
hearts of anxA7−/−mice, i.e. the disturbed Ca2+handling.
The nature of the accumulated extracellular substrate likely
accounting in a relevant part for this electrophysiological
phenomenon remains yet unclear and has to be further
We like to thank M. Rick for biometric measurements.
Thanks to H.J.M. van Rijen and C. de Bakker for their
technical assistance regarding the methodological approach
for the epicardial mapping system. Thanks to H. Begerau for
programming and designing the software for the analysis of
the epicardial activation maps.
This work was supported by an institutional grant from
the University of Bonn (to J.W.S.: BONFOR O-109.0008),
grants from the Deutsche Forschungsgemeinschaft (to C.G.
and R.M), grants from the Bundesministerium für Bildung
und Forschung BMBF (to T.L.; AF-Network), grants from
the Center for Molecular Medicine, Cologne (to A.A.N.),
and grants of the Pinguin Foundation (to K.B., R.H.G.S.)
and Köln Fortune (to K.B.).
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