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1 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, 3 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 4 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. 5 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. 6 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: 7 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. 8 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. 9 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). 10 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). 11 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. 12 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. 13 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. 14 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. 15 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. 16 Supplementary references Akagi K, Sandig V, Vooijs M, Van der Valk M, Giovannini M, Strauss M, Berns A (1997) Cremediated somatic site-specific recombination in mice. 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