CVR-2013-230R2
The cardiac sodium-calcium exchanger NCX1 is a key player in initiation and maintenance of a
stable heart rhythm
Stefan Herrmann, Peter Lipp, Kathrina Wiesen, Juliane Stieber, Huong Nguyen, Elisabeth Kaiser,
Andreas Ludwig
Supplementary Methods
Echocardiography
Animals were investigated 8-9 weeks after tam injection by using a Vevo 770 (Visualsonics, Canada).
Anesthesia was induced by 3 % isoflurane and continued with 1.5 % isoflurane. Body temperature was
controlled by a rectal probe. A 15-45 MHz scanhead was used to perform B- and M-mode imaging in
long and short axis views. Pulse-waved Doppler was employed to image the trans-mitral flow pattern.
ECG recording in anesthetized mice
Mice were anesthetized with isoflurane. The adequate depth of anesthesia was determined by a
negative toe-pinch reflex. Body temperature was maintained at 37°C using an infrared light source
controlled by a rectal probe. ECG signals from needle electrodes were collected and amplified using a
Power Lab 8/30 and Bio Amp ML136 system (AD Instruments). Data were analyzed using Chart 5
Pro (AD Instruments). ECGs were recorded twice a week for over two months. QT intervals were
corrected for heart rate using the formula QTC =
QT
.
RR/100
ECG recording in conscious mice.
Mice were housed in single cages in a 12 hour dark-light-cycle environment. Radiotelemetric ECG
transmitters (DSI, USA) were implanted into the peritoneal cavity under isoflurane anesthesia. The
adequate depth of anesthesia was determined by a negative toe-pinch reflex. ECG leads were sutured
subcutaneously onto the upper right chest muscle and the upper left abdominal wall muscle
(approximately Lead II). The animals were allowed to recover for at least 3 weeks. For long-term ECG
recordings, data were sampled for 20 s every 10 minutes. Isoproterenol, atropine, propranolol (Sigma)
2-chloro-N-cyclopentyladenosine (Tocris Bioscience, United Kingdom) were prepared on the day of
the experiment and ECG signals were sampled every minute for 20 seconds. After a one hour control
period, the mice were injected i.p. and ECGs were recorded for 3 to 24 hours thereafter. The end of the
drug response interval was defined as the time point where no significant difference to the mean
control value could be detected anymore. The animals were allowed to recover for at least 48 hours
between experiments.
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CVR-2013-230R2
Heart tissue isolation
Mice were sacrified by cervical dislocation. For protein and RNA isolation hearts were quickly
removed and placed in ice-cold phosphate-buffered saline, pH 7.4, containing 4 mM EDTA. The right
atrium including the superior vena cava was isolated under a dissecting microscope (Zeiss SV6) and
cleared of connective tissue and fat. The sinus node region limited by crista terminalis, atrial septum
and orifice of the superior vena cava, was carefully dissected. To avoid contamination with the
supraventricular conduction system, tissue specimens of the remaining right atrium were taken in
sufficient distance to the extracted sinus node area and to the atrial septum. Specimens of ventricular
tissue were obtained from the free wall of the right and left ventricle from a region midway between
heart valves and apex. All tissue samples were immediately flash-frozen in liquid nitrogen. For
immunohistochemistry hearts were removed, frozen and embedded in GSV1 Tissue Embedding
Medium (Slee Technik, Germany).
Isolation of sinoatrial node cells
Mice were sacrificed by cervical dislocation. The hearts were quickly removed and placed in
prewarmed Tyrode solution containing: 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5
mM HEPES and 5.5 mM glucose, pH 7.4. The SN region was excised, minced and placed into
modified Tyrode solution containing: 140 mM NaCl, 5.4 mM KCl, 0.2 mM CaCl2, 0.5 mM MgCl2, 1.2
mM KH2PO4, 50 mM taurine, 5 mM HEPES and 5.5 mM glucose, pH 6.9. Enzymatic digestion of the
tissue was carried out for 30 minutes at 35°C with 1.75 mg/ml collagenase B, 0.4 mg/ml elastase
(Roche, Germany) and 1 mg/ml BSA added to the modified Tyrode solution. After digestion, the
modified Tyrode solution was replaced by Storage Solution containing: 25 mM KCl, 80 mM Lglutamic acid, 20 mM taurine, 10 mM KH2PO4, 3 mM MgCl2, 10 mM glucose, 10 mM HEPES and
0.5 mM EGTA. The pH was adjusted to 7.4 with KOH. Cells were kept at least 3 hours at 4°C in
Storage Solution before they were slowly readapted to Ca2+ containing solutions.
Calcium imaging
Isolated sinoatrial node cells plated on coverslips were mounted in custom made chambers and loaded
with fura2 (0.75 µM, 30 min with additional 10 min de-esterification time, room temperature). The
cells were placed on the stage of a video imaging system based on an inverted microscope (TE 2000U, NIKON, Japan) comprising a fast switching excitation light source (Polychrome V; TILL
Photonics, Germany) and a CCD camera for detection (Pike F145B, Allied Vision Technologies
GmbH, Germany). Imaging was performed through a 20x oil immersion objective (20x/NA 0.75, Plan
Fluor, NIKON Japan) to maximize the number of cells in the field of view. Fura2 was excited by
switching the excitation wavelength between 350 nm and 380 nm and the resulting emission was
integrated for 12 ms each on the camera in 8x8 binning mode (resulting image size was 172x128
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CVR-2013-230R2
pixel). Three different protocols (each lasting 24 s) were employed: (i) spontaneous activity was
assessed by monitoring myocytes that were not field stimulated; (ii) steady-state behavior was
investigated by electrical field stimulation of the field of view at 1 Hz for at least 3 minutes (7 V, 5 ms
square pulses with alternating polarity); (iii) SR-Ca2+ content and NCX activity were analysed with
brief caffeine pulses (10 mM) through a gravity driven fast local perfusion system controlled by
solenoid valves. After recording, the data was transferred into a data management system (OME, Open
Microscopy Environment).1 Analyses of single cellular Ca2+ transients was performed in several steps.
Raw fura2 fluorescence over time data was obtained in ImageJ (W. Rasband, NIH, USA) running
custom made macros and transferred into Igor Software (Wavemetrics, USA) for further analysis.
Custom written code in Igor helped to automatically calculate background corrected fura2 ratios.
Because of the complex Ca2+ signals recorded, analysis of all parameters was performed manually and
blinded. The amplitudes of Ca2+ transients were defined as the difference between the diastolic and
peak fura2 ratio while the decay was characterized by fitting a monoexponential decay to the recovery
phase. The amplitudes and decay (during caffeine application) of caffeine-evoked Ca2+ transients were
characterized accordingly. The frequency of spontaneous Ca2+ signals was obtained after counting
Ca2+ transients during the 25 s recording period. Time to peak values were measured by analyzing the
time between end diastole and the peak of the Ca2+ signal. All experiments were performed at 37°C.
Immunofluorescence
Heart cryosections (10 µm) were fixed in 4% paraformaldehyde in PBS, pH 7.4 for 30 minutes. After
two washing steps sections were permeabilized in 0.1% Triton X100 for 30 minutes. Endogenous
peroxidase activity was quenched by incubation for 15 minutes in a solution of 1.5 ml MeOH, 30 ml
H2O2, 118.5 ml PBS. Subsequently sections were incubated in blocking solution (5% normal goat
serum) for 1 hour. NCX1 rabbit polyclonal antibody (1:250, Swant) or HCN4 antibody (1:200,
Alomone) was used overnight at 4°C. After washing, slides were incubated with a Cy3 labeled
secondary antibody (1:200, goat anti rabbit, Dianova). SN cells were isolated by enzymatic digestion,
plated onto polylysine coated slides (Menzel, Germany) using a cytospin centrifuge and fixed with 4%
paraformaldehyde for 10 min. After a blocking step with 5% normal goat serum in TritonX100 cells
were co-incubated with HCN4 (Alomone, 1:100) and NCX1 monoclonal antibody (Swant, 1:150) over
night at 4°C. After washing, cells were incubated with a secondary antibody (goat anti rabbit Cy3
conjugated, 1:400, Dianova and goat anti mouse FITC conjugated, 1:100, SantaCruz) for 1 hour at
room temperature. Images were acquired using a Zeiss LSM 5 Pascal confocal microscope. Control
stainings without the primary antibody gave no signal.
RT-PCR
SAN tissue was pulverized under liquid nitrogen. Total RNA was isolated using RNeasy Fibrous
Tissue Micro Kit (Quiagen) and cDNA was amplified using Superscript II (Invitrogen) or a OneStep
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CVR-2013-230R2
RT-PCR Kit (Qiagen). Primer pairs for amplification of NCX1 and GAPDH were intron-spanning to
preclude amplification of genomic DNA. Two to three sinoatrial nodes were pooled for RNA
isolation.
Quantitative RT-PCR
One-tube RT-PCR was performed using a Quantitect Probe RT-PCR Kit (Qiagen). Expression of
genes was determined by TaqMan assays on an ABI Prism 7900. Gene specific primers and probes
were purchased from Applied Biosystems. For each RT-PCR, the threshold cycle (Ct) defined as the
cycle at which the fluorescence exceeds 10 times the standard deviation of the mean baseline emission
for cycles 3 to 10 was determined. The Ct value of each gene was normalized to GAPDH according to
the following formula: Ct = Ct(examined gene) – Ct(GAPDH). Values were averaged and then used
for the 2- Ct x 100 calculation.
Western blot
Two to three sinoatrial nodes were pooled for protein isolation. Snap-frozen SN tissues were
homogenized in extraction buffer (50 mM Tris/HCl, pH 7.6, 150 mM NaCl, 1 % Triton X-100, 1 %
Na-deoxycholat, 0.1 % SDS, 1 mM EDTA) containing protease (complete mini, Roche) and
phosphatase inhibitors (Inhibitor Cocktail 3, Sigma). The homogenates were centrifuged at 13000 rpm
for 5 min at 4°C and supernatants were stored at -80°C. Protein samples (10-30 µg protein) were
heated at 70°C for 10 minutes with a 6xSDS probe buffer (containing 10% SDS), fractionated on
SDS-PAGE with Tris-HCl/SDS running buffer and transferred to PVDF membrane (Millipore). A
7.5% SDS-PAGE gel was used to detect SERCA2, NCX1 and PMCA. A 15% SDS-PAGE gel was
used for the detection of PLN and phosphor-Ser-16-PLN. After blocking nonspecific binding by 5%
nonfat powdered dry milk in TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20),
membranes were incubated either with anti-SERCA2 (monoclonal mouse, 1:1000, Abcam), antiNCX1 (monoclonal mouse, 1:1000, Swant), anti-PMCA (monoclonal mouse, 1:1000, Thermo
Scientific), anti-phospho-Ser16 (polyclonal rabbit, 1:1000, Millipore) or anti-PLN (monoclonal
mouse, 1:1000, Pierce) antibodies. After washing membrane was incubated with horseradish
peroxidase-conjugated secondary antibodies (Dianova). Blots were then stripped (ReBlot plus,
Millipore) and probed with an anti-tubulin antibody (polyclonal rabbit, Santa Cruz Biotechnology).
Bound antibodies were visualized by the ECL system (NEN). Levels of SERCA2, PMCA, total PLN,
PLN-phospho-Ser-16 and Tubulin were quantified using an imaging system (ChemiDoc XRS,
BioRad) and quantification software (Quantity One, Biorad). Ratios of SERCA2/Tubulin,
PMCA/Tubulin, PLN/Tubulin and PLN-phospho-Ser-16/Tubulin were calculated.
Patch-clamp recordings
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CVR-2013-230R2
8-12 hours after isolation of SN cells, voltage activated Ica was recorded using an Axopatch 200B
amplifier and pClamp10-software (Axon Instruments, USA). Analysis was done offline with Clampfit
10-software (Axon Instruments, USA). Patch pipettes were pulled from borosilicate glass, heat
polished and had a resistance of 3-6 Mwhen filled with intracellular solution. Current recordings
were performed in the whole cell configuration at 23  1°C. The extracellular (bath) solution
contained (in mM): 130 Tetraethylammonium(TEA-)chlorid, 10 4-Aminopyridine, 1 MgCl2, 2 CaCl2,
25 HEPES, 10 Glucose, pH adjusted to 7.4 with TEA-OH. The intracellular (pipette) solution
contained (in mM): 10 NaCl, 120 CsCl, 1 MgSO4, 5 EGTA, 10 HEPES, pH adjusted to 7.4 with CsOH. The membrane potential was held at –80 mV. To elicit Ica, a 50 ms prepuls of -40 mV was
applied, followed by Ica-activating step pulses from -60 mV to +50 mV for 500 ms. The Ica-amplitude
was determined as the difference between the peak current and minimal current at the end of the pulse.
The current density was calculated as the amplitude divided by the cell capacitance.
References:
1
Allan C, Burel JM, Moore J, Blackburn C, Linkert M, Loynton S et al.. Omero: Flexible,
model-driven data management for experimental biology. Nature methods. 2012;9:245-253.
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CVR-2013-230R2
Supplementary Figures
Supplementary Figure 1. Temporally controlled deletion of NCX1 in SN and AV nodal tissue.
RT-PCR analysis of cardiac tissues from heterozygous floxed cpNCX KO (NCX1 flox/+, HCN4
CreERT2/+) animals after (+) tamoxifen treatment. Amplicons correspond to wildtype (WT) and
recombined knockout (KO) alleles. Cre transcript expression is present in tissue isolated from
sinoatrial node (SN) and atrioventricular node (AVN), but absent from left (LA) and right atrial (RA)
tissue.
600
Heart rate (bpm)
500
C tr, night
C tr, day
400
KO, night
KO, day
300
200
1
3
5
7
weeks after tam
9
Supplementary Figure 2. Telemetric ECG recordings in freely moving animals. Average weekly
heart rate of controls (n= 9, open symbols) and cpNCX1KO (n= 9, closed symbols) after tamoxifen
induction plotted against time. Diamonds indicate averaged heart rates during daytime recorded from
07:00 am to 3:00 pm. Squares indicate averaged heart rate during nighttime recorded from 8:00 pm to
5:00 am. One week after tamoxifen treatment mean heart rates during night and day were significantly
(p< 0.05) reduced in cpNCXKO as compared to control, afterwards knockout heart rates were highly
significantly reduced (p < 0.001).
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CVR-2013-230R2
A
60
**
SDRR (ms)
50
40
***
C tr
KO
30
20
**
*
10
0
-1
1
2
3
>4
weeks after tam
tam
B
C
C tr
KO
***
50
**
50
*
*
***
***
40
*
30
PR interval (ms)
PR interval (ms)
60
C tr
KO
***
40
30
20
10
0
2
tam
6
10
14
weeks after tam
Supplementary Figure 3. Deletion of NCX1 results in increased RR interval variability and AV
nodal conduction time. A, Standard deviation of RR intervals (SDRR) of controls and cpNCXKO are
plotted against time. B, PR-intervals of controls and cpNCXKO mice plotted against time. C, Averaged
PR interval of controls and knockouts one month after tam treatment. Arrows indicate the five
tamoxifen treatment days. N=12 controls and knockouts, respectively.
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CVR-2013-230R2
Supplementary Figure 4. Deletion of NCX1 had no effect on the expression of other genes
implicated in cellular calcium handling, pacemaking and rate modulation. Real-time RT–PCR
analysis of SAN tissue. The relative mRNA expression level of the indicated genes differed not
significantly between control (n=8, open bars) and cpNCXKO (n=8, solid bars). Cav1.2 and Cav1.3, LType calcium channel 1C and 1D; Cav3.1, T-Type calcium channel 1G; RyR2 and RyR3, ryanodine
receptor 2 and 3; HCN4, hyperpolarization-activated, cyclic nucleotide-gated cation channel 4;
Kchip2, KV channel-interacting protein 2 and KV4.2, potassium voltage-gated channel subfamily D
member 2 contribute to the cardiac transient outward potassium current (Ito); ERG, Ether-à-go-goRelated Gene is the alpha subunit of the 'rapid' delayed rectifier current (IKr); KiR2.1, inward-rectifier
potassium ion channel; KiR3.1, potassium inwardly-rectifying channel is the alpha subunit of the G
protein-gated potassium channel (IKACh); Sk2, potassium calcium-activated potassium channel,
member 2; KVLQT1, alpha subunit of the slow delayed rectifying potassium current (IKs); β1R, betaadrenergic receptor type 1; M2R, muscarinic receptor type 2.
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CVR-2013-230R2
Supplementary Figure 5. Deletion of NCX1 did not result in significant altered voltagedependent calcium currents. Current-voltage relationship of the peak calcium current amplitude (A)
and current density (B) determined in isolated sinoatrial node cells from control (n=9 cells) and
knockout (n =11 cells). Cell capacitance did not differ significantly between genotypes (control: 42.6
±9.6 pF, n= 9; knockout: 40.6 ± 12.0 pF, n=11, p>0.05).
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CVR-2013-230R2
Supplementary Table 1
Echocardiography Analysis
Control (n=4)
cpNCX1KO (n=8)
Measurement
Interventricular septum, diastole
mm
LV internal diameter, diastole
mm
LV posterior wall, diastole
mm
LV anterior wall, diastole
mm
Interventricular septum, systole
mm
LV internal diameter, systole
mm
LV posterior wall, systole
mm
LV anterior wall, systole
mm
Mitral valve E wave velocity
mm/s
Mitral valve A wave velocity
mm/s
Isovolumic relaxation time
ms
0,67  0,03
4,39  0,05
0,70  0,03
4,75  0,09*
0,57  0,02
0,65  0,02
0,61  0,02
0,60  0,03
0,82  0,05
3,41  0,24
0,85  0,02
3,62  0,07
0,88  0,09
0,84  0,04
0,85  0,03
0,81  0,03
 144,38
957,30 
397,12  35,36
1155,38  82,22
459,43  66,91
20,63  1,88
23,46  0,95

Calculation
LV volume, diastole
ul
LV volume, systole
ul
Ejection fraction
%
Fractional shortening
%
LV mass
mg
LV anterior wall mass
mg
Mitral valve E/A ratio

 2,68
87,45 
48,30  7,84
 4,53*
105,10 
55,25  2,41
45,20  7,49
22,66  4,56
47,12  1,80
23,73  1,10
99,25  1,03
97,65  0,94
121,07  2,76**
109,83  2,68*
2,40 
 0,15
2,86 
 0,38
All values are mean  SEM
*P<0.05, **P<0.01 as compared with control.
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