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Supplementary Information
Supplementary Methods
Genotyping and Primers. HCN4 animals were genotyped by PCR. The floxed (L2) and the
knockout (L1) allele were detected by using a three primer strategy. Two primers flanking the loxP
sites were placed in intron 3 and intron 4 (forward: 5´-CACCCAAAAGGAGGACAGTGAAG T3´, reverse: 5´-CAGGGAGGACTGGCCATAACTAT-3´), one primer was placed in exon 4
(forward: 5´-CTGCCCTCATCCAGTCGCTAGAC3´). The LacZ transgene was detected using the
primer pair 5´-ACCAGAAGCGGTGCCGAA AA-3´ and 5´-CCCGTAGGTAGTCACGCAAC-3´.
The CAGG-CreTM transgene was identified using primers localized in the CAGG promoter (5´CTCT AGAGCCTCTGCTAACC-3´) and in the Cre coding region (5´-CGCCGCATA
ACCAGTGAAAC-3´).
X-Gal staining. LacZ staining was done as described (Kuhbandner et al, 2000).
In-situ hybridization. Isolated tissue was fixed in 4% paraformaldehyde, embedded in paraffin
wax and cut into 10 m sections. In-situ hybridization was done as described (Moosmang et al,
2001).
Quantitative RT-PCR. The following TaqMan gene expression assays (Applied Biosystems,
Darmstadt, Germany) were used: Cav1.2, Mm00437917_m1; Cav1.3, Mm00551384_m1; Cav3.1,
Mn00486549_m1;
HCN1,
Mm00468832_m1;
HCN2,
Mm00468538_m1;
HCN3,
Mm00468543_m1; NCX1, Mm00441524_m1; PLB, Mm00452263_m1; RyR2, Mm00465877_m1;
Kir2.1, Mm00434619_m1; Kir3.1, Mm00434618_m1; mERG, Mm00465370_m1; KvLQT1,
Mm00434641_m1; MiRP1, Mm00506492_m1.
Immunofluorescence on isolated SAN cells. SAN cells were isolated by enzymatic digestion,
plated onto polylysine coated slides (Menzel, Germany) using a cytospin centrifuge and fixed with
2% paraformaldehyde for 15 min at 4°C. Endogenous peroxidase activity was inhibited by
incubation in 0.3 % H2O2 / PBS for 10 min. Cells were incubated with primary antibody (rabbit
anti-HCN1, 1:100 dilution or rabbit anti-HCN2, 1:100 dilution, Alomone Labs, Israel) over night at
4°C. After washing, cells were incubated with a HRP-conjugated secondary antibody (goat antirabbit IgG, 1:1000 dilutions, Jackson ImmunoResearch). The signal was amplified by the TSA-
2
Cyanine 3 system according to the manufacturer’s protocol (NEN Life Science Products).
Immunolabelled samples were viewed with a Zeiss LSM 510 laser scanning confocal microscope
equipped with a helium-neon-laser.
Isolation of sinoatrial node cells. Mice were deeply anesthetized with 100 mg/kg ketamine
(Ketavet, Pharmacia & Upjohn GmbH, Erlangen, Germany) and 10 mg/kg xylazine (Rompun ,
Bayer AG, Leverkusen, Germany), injected i.p. 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 SAN region was excised, minced and placed into
modified Tyrode solution containing: 140 mM NaCl, 5.4 mM KCl, 0.2 mM CaCl 2, 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 (both Roche, Mannheim, 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 L-glutamic 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 calcium
containing solutions.
Electrophysiological recordings and analyses. SAN cells for electrophysiological recordings were
chosen according to their typical shape and small, thin, transverse tubuli-lacking appearance (see
Supplementary Fig. S2) in contrast to ventricular (usually not present in our preparation) or atrial
cells. The presence of spontaneous contractions was not relied on as a solely verifying criterion for
SAN cells since isolated atrial cells also tend to rhythmically contract in calcium containing
solutions at 37°C. Moreover, knockout SAN cells were often silent under basal conditions.
If from SAN cells was recorded with the whole cell patch clamp recording technique at a
temperature of 22  2°C. Patch pipettes were pulled from borosilicate glass and had a resistance of
2-5 M when filled with intracellular solution, containing: 10 mM NaCl, 30 mM KCl, 90 mM
potassium aspartate, 1 mM MgSO4, 5 mM EGTA, 10 mM HEPES, pH adjusted to 7.4 with KOH.
During the recordings, cells were continuously superfused with extracellular (bath) solution,
containing: 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM HEPES, 10 mM
Glucose, 2 mM BaCl2, 0.3 mM CdCl2, pH adjusted to 7.4 with NaOH. HCN currents from HEK293
cells, transfected with either m(murine)HCN1, mHCN2 or mHCN4, were recorded under the same
conditions. The effects of isoproterenol and cAMP was measured by superfusing cells with bath
solution containing 1 µM isoproterenol or 100 µM 8-Br-cAMP (both Sigma, Taufkirchen,
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Germany). Integrity of the cell and identity of If was tested by applying 2 mM cesium followed by a
washout with bath solution which restored the original current amplitude. Data were acquired using
an Axopatch 200B amplifier and pClamp7 software or a MultiClamp 700A/B amplifier and
pClamp9/10 software (all Axon Instruments/Molecular Devices, Union City, USA). Analysis was
done offline with Origin 6.1. (MicroCal Software, Northampton, USA) or Clampfit9 (Molecular
Devices, Union City, USA). Membrane and action potentials from SAN cells were recorded at 36 
1°C using the perforated patch technique with 200 µg/ml Amphotericin B added to the intracellular
solution which contained: 10 mM NaCl, 130 mM potassium aspartate, 0.04 mM CaCl2, 2 mM MgATP, 6.6 mM creatine-phosphate, 0.1 mM Na-GTP, 10 mM HEPES, pH 7.3. Bath solution was the
same as described above for current recordings except for the omittance of BaCl2 and CdCl2.
Standard If and AP parameters were calculated as described (Stieber et al, 2005; Stieber et al, 2006).
The current amplitude was defined as the amplitude at the end of the activation pulse minus the
amplitude of the initial lag, current density was obtained by dividing the amplitude through the
capacity of the cell. Time constants of activation (act) were obtained by fitting the current traces of
the –130 to –90 mV steps after the initial lag with the sum of two exponential functions: y0 + A1e(-x/1)
+ A2e(-x/2),
where 1 and 2 are the fast and slow time constants of activation, respectively; 1 is
consequently referred to as act since the slow component (A2) generally accounts for <20% of the
current amplitude. To obtain voltage dependent steady-state activation curves, tail currents
measured immediately after the final step to –140 mV were normalized by the maximal current
(Imax) and plotted as a function of the preceding membrane potential. The curves were fitted with the
Boltzmann function: (I-Imin)/(Imax-Imin) = (A1-A2)/(1+e(V-V1/2)/k)) + A2, where Imin is an offset caused
by a nonzero holding current and is not included in the current amplitude, V is the test potential,
V1/2 is the membrane potential for half-maximal activation, and k is the slope factor.
Telemetric ECG recordings in mice. Mice were housed in single cages in a 12 hour dark-lightcycle environment with free access to food and water. Radiotelemetric ECG transmitters (DSI, St.
Paul, USA) were implanted into the peritoneal cavity under general anesthesia with isoflurane/O2.
The ECG leads were sutured subcutaneous onto the upper right chest muscle and the upper left
abdominal wall muscle. This resulted approximately in the Lead II position of the ECG electrodes.
The animals were allowed to recover for at least 3 weeks before any experiments or training
sessions. Data were acquired using the DSI acquisition system. For long-term ECG recordings, data
was sampled for 20 s every 10 minutes. For pharmacological experiments, solutions of
isoproterenol, carbachol (both Sigma, Taufkirchen, Germany) and cilobradine (Boehringer,
Biberach, Germany) were prepared in sterile 0.9% NaCl (Braun AG, Melsungen, Germany) on the
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day of the experiment, 2-Chloro-N6-cyclopentyladenosine (CCPA) was dissolved using ultrasound
in 0.9% NaCl containing 0.1% DMSO. Control solution was 0.9% NaCl. For these pharmacological
experiments, ECG signals were sampled every minute for 20 seconds. After a one hour pre-run, the
mice were injected i.p. with the drug. The ECGs were recorded for 3 to 24 hours thereafter. The
animals were allowed to recover for at least 48 hours between experiments. For forced physical
activity ("exercise"), animals were trained to run on a custom made, electrically driven treadmill at
a speed of approximately 0.4 m/s and 20% ascending slope for 10 to 20 minutes.
As appropriate, before and after the injection of the drug/exercise, ECG parameters were analyzed.
The definitions of the ECG intervals are: RR, duration from R to R; PQ, beginning of P to peak of
Q; QT, peak of Q to end of T, where end of T is the return of any T deviation to the isoelectric line;
QTc, heart rate-corrected QT interval using the equation
QT
RR / 100
(Mitchell et al, 1998); TP, end
of T to beginning of next ECG complex's P. The heart rate in beats per minute was calculated from
the RR duration (in ms) as: 60000/RR. The ED50 values for the isoproterenol effect were obtained
by fitting the semi-logarithmically plotted data (heart rate vs. drug concentration) with a sigmoidal
fit/logistic model: y=A2+(A1-A2)/(1+(x/x0)p), where x0 corresponds to ED50.
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Supplementary Tables
Cre-lines analyzed for efficiency of recombination in the primary cardiac conduction system
Line
X-Gal/HCN4
Promoter
Expression
Inducible
endogenous unknown
ubiquitous
tamoxifen
incomplete
endogenous ROSA26
ubiquitous
tamoxifen
incomplete
muscle creatine kinase
heart, skeletal
muscle
no
incomplete
-myosin heavy chain
heart
Ru486
incomplete
-myosin heavy chain
heart
tamoxifen
incomplete
chimeric CMV-IE and
chicken -actin
ubiquitous
tamoxifen
complete
deletion
GTEV-Cre
(Vallier et al,
2001)
ROSA-Cre
(Vooijs et al,
2001)
MCK-Cre
(Bruning et al,
1998)
MHC-Cre
(Minamino et
al, 2001)
MerCreMer
(Sohal et al,
2001)
CAGG-Cre
(Hayashi &
McMahon,
2002)
Table S1: Cre transgenic lines were crossed to the ACZL reporter strain (Akagi et al, 1997).
Sections through the sinoatrial node of resulting double transgenic descendants were analyzed by
X-Gal staining. Cre lines demonstrating appropriate staining were crossed to floxed HCN4 mice
and the deletion of HCN4 examined by immunohistochemistry and western blot. The column “XGal/HCN4-deletion” indicates the extent of X-Gal staining and deletion of HCN4 protein in the
SAN.
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Control (L2)
Basal
P
PQ
13.0  1.1
37.0  3.6
8.6  0.7
44.3  10.2 38.9  8.7
13.4  1.2
38.0 6.1
7.8  0.6
42.2  9.7
37.2  8.9
33.1  1.0
6.8  1.2
39.8  0.8
44.8  3.7
11.0  0.5
34.6  2.8
7.2  1.2
39.7  7.2
42.4  7.1
43.4  3.6
Exercise 9.0  0.5
QRS
KO
QT
QTc
P
PQ
QRS
QT
QTc
Iso
10.7  1.6
36.3  2.9
7.9  1.0
40.8  4.2
44.2  4.3
12.0  1.5
39.9  2.7
8.3  0.8
40.1  3.3
Carb
13.7  0.9
38.5  2.3
8.2  0.4
64.8  4.7* 33.6  8.4
13.4  1.8
35.6  3.1
8.3  0.8
122.0  44* 39.8  18
CCPA
12.4  2.2
39.5  2.4
8.4  0.9
63.4  17*
12.4  2.2
36.6  6.5
8.7  1.2
107.6  21* 31.1  4.0
26.9  7.7
Table S2: ECG parameters of knockout and control (L2) mice taken from the experiments for Fig. 4d of the main paper.
All values are given in ms as mean  SD. The definition of the ECG parameters (P, PQ, QRS, QT) is shown below. Exercise, forced running on a
treadmill; Iso, isoproterenol; Carb, carbachol; CCPA, 2-Chloro-N6-cyclopentyladenosine
* QT values are significantly different between control and KO, but heart rate corrected QTc values are NOT significantly different.
Definition
of
ECG
parameters:
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Supplementary Figures
Fig. S1. The CAGGCre-ERTM transgenic line displays high recombination efficiency in the
sinoatrial node. (A-C) Hearts were isolated from tamoxifen-induced ACZL -Galtg/0; CAGGCreERTM double transgenic animals and whole-mount stained with X-Gal (left). In addition, SAN
sections from double transgenic animals were stained with anti--Gal (middle) and HCN4antibodies (right). (Left) Whole mount X-Gal stained right atrium shows robust ubiquitous
recombination (blue). The red bar indicates the location of sections shown to the right. (Middle)
Cre-mediated recombination analyzed by an anti--Gal antibody (brown). (Right) Adjacent sections
were labeled with an HCN4-antibody (blue) (D) Higher magnification of the SAN region from the
sections in (B) demonstrates complete overlapping expression of -Gal (Cre-recombinase activity)
and HCN4. SVC, superior vena cava; SA, sinoatrial node artery; RA, right atrium.
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Fig. S2. Expression of HCN1 and HCN2 in the primary cardiac conduction system. (A) In-situ
hybridization. Adjacent sections through the SAN region of wild-type animals labeled with an
HCN1- (left) and HCN2- (right) specific riboprobe. The dark-field micrographs (upper panels)
display transcripts of HCN1 and HCN2 located in the sinoatrial node. Lower panels show the same
sections viewed under bright-field illumination. RA, right atrium; SVC, superior vena cava; SAN,
sinoatrial node; DF, dark-field; BF, bright-field. (B) Immunolabeling of HCN1 (left) and HCN2
(right) in isolated sinoatrial node cells. Left panel, cyanine 3 immunofluorescence; middle panel,
SAN cell under transmitted-light; right panel, overlay of left and middle panel; scale bars, 50 m.
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Fig. S3. Deletion of HCN4 had no effect on the expression of various genes implicated in cardiac
pacemaking and on the phosphorylation level of phospholamban. (A) Real-time RT–PCR analysis
of SAN tissue. Shown are CT values  SEM, i.e. the threshold cycles (CT) for each gene
normalized to -actin (CT = CT (examined gene)- CT (-actin)). The relative mRNA expression
levels differed not significantly between control (n=5, solid bars) and knock-out (n=5, open bars; p<
0,05). Please note that smaller CT values represent higher levels of mRNA. The following genes
involved in cardiac depolarization were analyzed: Cav1.2 and Cav1.3, L-Type calcium channel 1C
and 1D; Cav3.1, T-Type calcium channel 1G ; RyR2, ryanodine receptor 2; PLB, phospholamban;
NCX1, Na+/Ca2+ exchanger 1. Examined genes involved in cardiac repolarization: Kir2.1 and
Kir3.1; Inward-rectifier K+-channel 2.1 and 3.1; mERG, murine homologue of HERG; KvLQT1.
(B) Immunoblot analysis of phosphorylated (at serine 16) and total PLB in SAN tissue. The blot
was incubated with an anti-phospho-PLB antibody and revealed no difference in protein kinase A
dependent phosphorylation. Total PLB was used as loading control (anti-PLB antibody, Dianova).
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A
Ctr
KO
B
Fig. S4. Basic morphological characteristics of isolated knockout SAN cells were not different from
wild-type. SAN cells from control and wild-type mice had comparable sizes and shapes (A) and
capacities (B).
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Fig. S5. Comparison of If current densities and absolute current amplitudes of control and knockout
cells (same cells as in Fig. 2B). If was elicited by –100 mV. The approximately 75% reduction of
the current density in knockout cells equally applies to the absolute current amplitude.
Fig. S6. Activation kinetics of control and knockout If as compared to murine HCN1, 2 and 4
expressed in HEK293 cells. The activation kinetic of the knockout If is significantly different from
control If. This analysis suggests that the residual If in knockout cells is most probably carried by
HCN1 and HCN2 channels.
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A
B
Fig S7. Long-term ECG recordings of 8 control (L1/L2, - Tam) mice and the same 8 mice after
deletion of HCN4 (L1/L2, + Tam). (A) Heart rate analysis before and 4 weeks after Tam injection
reveals that a normal circadian rhythm concerning heart rates is not altered by the deletion of
HCN4. Similarly, the circadian rhythm of activity is unaltered (not shown) (B) Dependence of sinus
pause frequency on the activity of HCN4 knockout mice. Significantly more sinus pauses occurred
when the mice were less active or sleeping and had a low heart rate, e.g. during the day/light phase.
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A
B
(a) maximum heart rate
(b) area under the curve (AUC)
Fig. S8. Dose-response curves of isoproterenol. (A) Definition of "maximum heart rate" and "area
under the curve" as used in this study. Shown is the example of a mouse injected with 0.5 mg/kg
and 1 mg/kg isoproterenol at t=1 h on different days. To establish dose-response curves, the
maximum heart rate (green line) or the area under the curve (AUC, gray area) was used as
indicated. (B) The dose-response curves constructed using the maximum heart rate (a) or area under
the curve (b) did not differ significantly between knockout and L2 (control) mice. This
demonstrates the undisturbed ability of HCN4 knockout animals to maintain fast heart rates.
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Fig. S9. No sinus pauses during stimulated heart rates. Example traces of ECGs, measured during
maximum stimulation after injection of 0.5 mg/kg isoproterenol. The enlargements demonstrate
correct ECG complexes without arrhythmic episodes in both genotypes.
Fig. S10. Severe sinoatrial node dysfunction after stimulation. Example traces of ECGs, measured
1.5 h after the injection of 0.5 mg/kg isoproterenol (same traces as in Fig. 4B (phase II) of the main
paper) when the heart rates have just returned to the basal level. This phase is challenging also for
L2 (control) animals as demonstrated by the slightly dysrhythmic episode in this example. The
enlargements demonstrate correct ECG complexes in both control and knockouts. However, the
sinus pauses are 1.5 - 2fold longer (up to 900 ms) and much more numerous than before the
stimulation.
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Fig. S11: Enlargement of the example ECG trace displayed in Fig. 4 of the main paper. I.p.
injection of 0.5 mg/kg of the sinus node inhibitor cilobradine prolonged the sinus pauses in HCN4
knockout mice, but ECG complexes in between the pauses showed no abnormalities.
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