Cardiac pacemaker function of HCN4 channels in mice is confined to embryonic development and requires cyclic AMP
2008; Springer Nature; Volume: 27; Issue: 4 Linguagem: Inglês
10.1038/emboj.2008.3
ISSN1460-2075
AutoresDagmar Harzheim, K Pfeiffer, Larissa Fabritz, Elisabeth Kremmer, Thorsten Buch, Ari Waisman, Paulus Kirchhof, U. Benjamin Kaupp, Reinhard Seifert,
Tópico(s)Cardiomyopathy and Myosin Studies
ResumoArticle24 January 2008free access Cardiac pacemaker function of HCN4 channels in mice is confined to embryonic development and requires cyclic AMP Dagmar Harzheim Dagmar Harzheim Forschungszentrum Jülich, Institut für Neurowissenschaften und Biophysik, Abteilung Zelluläre Biophysik, Jülich, Germany Present address: Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT UK Search for more papers by this author K Holger Pfeiffer K Holger Pfeiffer Forschungszentrum Jülich, Institut für Neurowissenschaften und Biophysik, Abteilung Zelluläre Biophysik, Jülich, Germany Search for more papers by this author Larissa Fabritz Larissa Fabritz Medizinische Klinik und Poliklinik C, Universitätsklinikum Münster und IZKF Münster, Münster, Germany Search for more papers by this author Elisabeth Kremmer Elisabeth Kremmer GSF, Institut für Molekulare Immunologie, München, Germany Search for more papers by this author Thorsten Buch Thorsten Buch Institut für Experimentelle Immunologie, Universität Zürich, Zürich, Switzerland Search for more papers by this author Ari Waisman Ari Waisman I Medizinische und Poliklinik, Johannes Gutenberg-Universität Mainz, Mainz, Germany Search for more papers by this author Paulus Kirchhof Paulus Kirchhof Medizinische Klinik und Poliklinik C, Universitätsklinikum Münster und IZKF Münster, Münster, Germany Search for more papers by this author U Benjamin Kaupp Corresponding Author U Benjamin Kaupp Forschungszentrum Jülich, Institut für Neurowissenschaften und Biophysik, Abteilung Zelluläre Biophysik, Jülich, Germany Present address: Stiftung Caesar, Ludwig-Erhard-Allee 2, 53175 Bonn Search for more papers by this author Reinhard Seifert Corresponding Author Reinhard Seifert Forschungszentrum Jülich, Institut für Neurowissenschaften und Biophysik, Abteilung Zelluläre Biophysik, Jülich, Germany Search for more papers by this author Dagmar Harzheim Dagmar Harzheim Forschungszentrum Jülich, Institut für Neurowissenschaften und Biophysik, Abteilung Zelluläre Biophysik, Jülich, Germany Present address: Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT UK Search for more papers by this author K Holger Pfeiffer K Holger Pfeiffer Forschungszentrum Jülich, Institut für Neurowissenschaften und Biophysik, Abteilung Zelluläre Biophysik, Jülich, Germany Search for more papers by this author Larissa Fabritz Larissa Fabritz Medizinische Klinik und Poliklinik C, Universitätsklinikum Münster und IZKF Münster, Münster, Germany Search for more papers by this author Elisabeth Kremmer Elisabeth Kremmer GSF, Institut für Molekulare Immunologie, München, Germany Search for more papers by this author Thorsten Buch Thorsten Buch Institut für Experimentelle Immunologie, Universität Zürich, Zürich, Switzerland Search for more papers by this author Ari Waisman Ari Waisman I Medizinische und Poliklinik, Johannes Gutenberg-Universität Mainz, Mainz, Germany Search for more papers by this author Paulus Kirchhof Paulus Kirchhof Medizinische Klinik und Poliklinik C, Universitätsklinikum Münster und IZKF Münster, Münster, Germany Search for more papers by this author U Benjamin Kaupp Corresponding Author U Benjamin Kaupp Forschungszentrum Jülich, Institut für Neurowissenschaften und Biophysik, Abteilung Zelluläre Biophysik, Jülich, Germany Present address: Stiftung Caesar, Ludwig-Erhard-Allee 2, 53175 Bonn Search for more papers by this author Reinhard Seifert Corresponding Author Reinhard Seifert Forschungszentrum Jülich, Institut für Neurowissenschaften und Biophysik, Abteilung Zelluläre Biophysik, Jülich, Germany Search for more papers by this author Author Information Dagmar Harzheim1,6, K Holger Pfeiffer1, Larissa Fabritz2, Elisabeth Kremmer3, Thorsten Buch4, Ari Waisman5, Paulus Kirchhof2, U Benjamin Kaupp 1,7 and Reinhard Seifert 1 1Forschungszentrum Jülich, Institut für Neurowissenschaften und Biophysik, Abteilung Zelluläre Biophysik, Jülich, Germany 2Medizinische Klinik und Poliklinik C, Universitätsklinikum Münster und IZKF Münster, Münster, Germany 3GSF, Institut für Molekulare Immunologie, München, Germany 4Institut für Experimentelle Immunologie, Universität Zürich, Zürich, Switzerland 5I Medizinische und Poliklinik, Johannes Gutenberg-Universität Mainz, Mainz, Germany 6Present address: Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT UK 7Present address: Stiftung Caesar, Ludwig-Erhard-Allee 2, 53175 Bonn *Corresponding authors: Institut fuer Neurowissenschaften, Forschungszentrum Juelich, INB-1, Jülich 52425, Germany. Tel.: +49 2461 6140 41; Fax: +49 2461 6142 16; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2008)27:692-703https://doi.org/10.1038/emboj.2008.3 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Important targets for cAMP signalling in the heart are hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels that underlie the depolarizing 'pacemaker' current, If. We studied the role of If in mice, in which binding of cAMP to HCN4 channels was abolished by a single amino-acid exchange (R669Q). Homozygous HCN4R669Q/R669Q mice die during embryonic development. Prior to E12, homozygous and heterozygous embryos display reduced heart rates and show no or attenuated responses to catecholaminergic stimulation. Adult heterozygous mice display normal heart rates at rest and during exercise. However, following β-adrenergic stimulation, hearts exhibit pauses and sino-atrial node block. Our results demonstrate that in the embryo, HCN4 is a true cardiac pacemaker and elevation of HCN4 channel activity by cAMP is essential for viability. In adult mice, an important function of HCN4 channels is to prevent sinus pauses during and after stress while their role as a pacemaker of the murine heart is put into question. Most importantly, our results indicate that HCN4 channels can fulfil their physiological function only when cAMP is bound. Introduction Spontaneous activity of the mammalian heart is generated in the sino-atrial node (SAN). How pacemaker activity is generated in SAN cells and how pacemaking is regulated by the autonomous nervous system is still a matter of debate. A number of different ion channels and signalling events have been implicated in pacemaker activity (for review see, Couette et al, 2006). Key players controlling the diastolic depolarization are the hyperpolarization-activated current If and the L-type calcium current ICa,L (DiFrancesco, 1993; Verheijck et al, 1999; Biel et al, 2002; Zhang et al, 2002; Mangoni et al, 2003; Barbuti et al, 2007). The If current is mediated by hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels. These channels have a number of particular features that enable them to open at voltages near the maximal diastolic potential (MDP). First, channels are activated on hyperpolarization of the membrane potential. Second, channel activity is dependent on the intracellular cAMP concentration ([cAMP]i) (DiFrancesco and Tortora, 1991). When cAMP binds to the cyclic nucleotide-binding domain (CNBD) of HCN channels, the activation curve shifts towards more positive voltages, thereby enhancing channel activity. In addition, channels with cAMP bound activate faster and deactivate slower. Thus, an increase of [cAMP]i, for example, during β-adrenergic stimulation, enhances If, whereas a decrease of [cAMP]i, for example, during muscarinic stimulation, attenuates If (DiFrancesco et al, 1986; DiFrancesco and Tromba, 1988). It has been proposed that cAMP modulation of If is the primary pathway by which the autonomous nervous system controls the heart rate (Bucchi et al, 2003). Consistent with this model, mutations in the HCN4 channel gene, the major isoform in the SAN, are associated with sinus bradycardia and arrhythmia (Schulze-Bahr et al, 2003; Milanesi et al, 2006). The other key player in cardiac pacemaking is the voltage-dependent Ca2+ channel Cav1.3 that carries ICa,L. The ICa,L is activated during the early phase of the diastolic depolarization, and inhibition of ICa,L induces bradycardia in vivo (Lande et al, 2001). Moreover, Cav1.3 knockout mice are bradycardic and show sino-atrial arrhythmia (Platzer et al, 2000; Zhang et al, 2002; Mangoni et al, 2003). These observations have led to the proposal that in SAN cells Cav1.3 channels generate a significant electrical signal contributing to the diastolic depolarization (Couette et al, 2006). Within the framework of this model, the increase of ICa,L during cAMP stimulation enhances the pacing component. In addition to If and ICa,L, at least three other ionic pathways contribute to the regulation of the heart rate. One pathway is ICa,T, which is almost exclusively mediated by Cav3.1 channels (Mangoni et al, 2006). Cav3.1 knockout mice exhibit pronounced bradycardia and a significant slowing of atrioventricular (AV) conduction (Mangoni et al, 2006). Another current implied in pacemaker generation is a sustained inward current, Ist, which was identified in spontaneously beating SAN cells (Guo et al, 1995). Its proposed role in cardiac pacemaking is largely based on numerical simulations of pacemaker activity (Shinagawa et al, 2000; Zhang et al, 2002). The impact of Ist on pacing the heart is difficult to estimate due to its unidentified molecular nature and the lack of specific pharmacological tools to target this current. Finally, rhythmic Ca2+ oscillations have been proposed to be involved in cardiac pacemaking. This proposal challenges the major role of If in primary pacemaker activity of the SAN and its role in the autonomous regulation of the heartbeat (Bogdanov et al, 2001; Kodama et al, 2002; Lipsius and Bers, 2003). Instead of If, electrogenic Na+/Ca2+ exchange has been proposed to provide the major depolarizing pacemaking current. According to this model, SAN cells generate rhythmic, submembrane Ca2+ oscillations via spontaneous opening of ryanodine receptors in the sarcoplasmatic reticulum (SR) (Bogdanov et al, 2001). Thereby, the intracellular Ca2+ concentration is locally increased and the Na+/Ca2+ exchanger in the plasma membrane is activated. An inward current is generated that depolarizes the cell. The frequency of spontaneous Ca2+ release events is tightly controlled by the efficiency of Ca2+ uptake into the SR by the Ca2+ ATPase SERCA. The pumping rate of SERCA is highly sensitive to changes in [cAMP]i. An increase of [cAMP]i stimulates SERCA activity (for review see, Colyer, 1998; Kamp and Hell, 2000), leading to a higher SR Ca2+ uptake and spontaneous Ca2+ events occur more frequently. The heart beats faster. Here, we investigate the role of HCN4 in cardiac pacemaking of mice using a knock-in mouse model in which cAMP binding to the HCN4 channel has been abolished. Our results are both intriguing and unexpected. They demonstrate that (1) in the embryo, HCN4 is a powerful pacemaker but only when cAMP is bound and (2) in adult mice, HCN4 does not seem to contribute to cardiac pacemaking, but rather ensures stable heart rhythm during and after stress. Results Generation of HCN4R669Q knock-in mice To define the physiological function of If and its modulation by cAMP more precisely, we generated knock-in mice that harbour a single amino-acid exchange (R669Q) in the CNBD of the HCN4 channel. This arginine residue is crucial for the binding of cAMP because it interacts with the negatively charged phosphate group (McKay et al, 1982; Bubis et al, 1988; Zagotta et al, 2003). To ascertain that the mutation abolishes regulation by cAMP, we analysed the wild-type HCN4 and the mutant HCN4R669Q channel in Flp-In-293 cells. In the absence of cAMP, the voltage-dependent activation of HCN4R669Q mutant and wild-type channels was similar (Figure 1A). Voltages of half-maximal activation V1/2 were −94.3±3.7 mV (n=8) for wild-type and −99.5±3.4 mV (n=7) for HCN4R669Q channels. Cyclic AMP shifted the activation curve of the wild-type HCN4 channel by +24 mV (V1/2=−70.6±2.7 mV (n=3)), whereas no shift was observed for the HCN4R669Q mutant (V1/2=−96.3±7.1 mV (n=5)). We introduced the mutation into the HCN4 gene locus of mice via homologous recombination in embryonic stem cells (Figure 1B). Integration of the targeting vector, germ-line transmission, and deletion of the neomycin resistance gene were confirmed by Southern blot and PCR analysis (Figure 1C and D, and data not shown). Heterozygous HCN4+/R669Q mice were viable, bred normally, and were indistinguishable from wild-type littermates. However, no homozygous HCN4R669Q/R669Q pups were born from heterozygous matings (HCN4+/+: 57/155; HCN4+/R669Q: 98/155). Analysis of timed matings revealed that HCN4R669Q/R669Q embryos developed normally until E11, but were dead from E12 on (Figure 1E), indicating that homozygous embryos die between E11 and E12. Figure 1.Embryonic death of HCN4R669Q/R669Q mice. (A) Voltage-dependent activation of heterologously expressed HCN4 (circles) and HCN4R669Q channels (squares). Voltages of half-maximal activation (±s.d.) in the absence (open symbols) and presence (filled symbols) of cAMP (100 μM) were −94.3±3.7 (n=8) and −70.6±2.7 mV (n=3) for HCN4, and −99.5±3.4 (n=7) and −96.3±7.1 mV (n=5) for HCN4R669Q, respectively. (B) Targeting strategy. Only exons IV–VIII of the HCN4 locus are shown (white boxes). The targeting vector carried the mutation in exon VII (red arrow) and the neomycin resistance gene (neo), flanked by two loxP elements (red arrowheads). neo was deleted by Cre-mediated recombination. Homologous recombination and deletion of neo were detected through Southern blot using the XbaI restriction sites (X) and the 3′ probe (black box), and through PCR (primers 3 and 4, grey arrows). XbaI fragments that are recognized by the 3′ probe are depicted in blue. (C) Southern blot and PCR analysis of genomic DNA from HCN4+/+ (lane 1), HCN4+/neoR669Q (lane 2), and HCN4+/R669Q (lane 3) mice. Upper panel: Southern hybridization of XbaI-digested DNA with the 32P-radiolabelled 3′ probe. Middle panel: amplification of the wild-type allele (primers 1 and 2, 586 bp). Lower panel: amplification of the recombinant allele (primers 3 and 4, 1804 or 610 bp). (D) Genotyping of embryos from one litter. Upper panel: PCR amplifying the wild-type allele (primers 1 and 2). Middle panel: PCR amplifying the recombinant allele (primers 3 and 4). Lower panel: genotypes of analysed litter. (E) Number of embryos found alive (E9.5–E12.5; red: HCN4+/+; green: HCN4+/R669Q; blue: HCN4R669Q/R669Q). Download figure Download PowerPoint The amino-acid exchange R669Q does not alter protein expression of HCN4 We were concerned about the similar lethal phenotype of HCN4−/− (Stieber et al, 2003) and HCN4R669Q/R669Q mice. To investigate whether the pattern or level of expression of the channel mutant is altered, we analysed the cellular HCN4 distribution during embryonic development prior to E11.5. Different HCN4-specific antibodies labelled the same region in the heart of HCN4+/+ and HCN4R669Q/R669Q embryos (Figure 2A). No obvious difference existed in the cellular distribution of the HCN4 channel in wild type compared to the HCN4R669Q channel in homozygous embryos (Figure 2A, middle panel). A monoclonal, HCN4-specific antibody (specificity of HCN4 antibodies shown in Figure 2C) labelled two bands in western blots of membrane proteins from Flp-In-293 cells that expressed the HCN4 or the HCN4R669Q channel (Figure 2B). Treatment with PNGase F revealed that the upper band represents a glycosylated form of the HCN4 channel. In western blots of proteins isolated from wild-type, heterozygous, and homozygous embryonic hearts, the antibody recognized the same protein bands with equal intensities, demonstrating that the mutation does not affect the level of HCN4 expression (Figure 2D). Figure 2.The HCN4R669Q channel alters the embryonic heartbeat. (A) Upper panels: cryo-sections from HCN4+/+ (left) and HCN4R669Q/R669Q embryos (right) stained with an HCN4-specific antibody (PPc73K; bar: 100 μm). Middle panels: higher magnification of the labelled region (bar: 20 μm). Lower panels: Two independent HCN4-specific antibodies label identical structures in the heart of HCN4R669Q/R669Q embryos (left: PPc73K; middle: SHG-1E5; right: overlay; bar: 20 μm). (B) Western blot analysis of membrane proteins from Flp-In-293 cells, expressing HCN4 (left) or HCN4R669Q (right), labelled with the HCN4-β antibody. NT: non-transfected cells, +: treatment with PNGase F. (C) Left, upper panel: lysates from HEK293-mHCN1 cells. The following antibodies have been used to show the isoform specificity: 1, HCN4-β; 2, HCN4-specific PG2-7H9; 3, HCN1-specific RTQ-7C3. Middle, upper panel: lysates from HEK293-mHCN2 cells. Antibodies used: 4, HCN4-β; 5, HCN4-specific PG2-7H9; 6, HCN2-α. Right, upper panel: lysates from HEK293-mHCN4 cells. Antibodies used: 7, HCN4-β; 8, HCN4-specific PG2-7H9. All lower panels: loading control with anti-actin antibody. (D) Upper panel, lanes 1 and 2: membrane proteins from Flp-In-293 cells (1, non-transfected; 2, transfected with mHCN4) labelled with the HCN4-specific antibody PG2-7H9 (exposure time: 1 min). Lanes 3–5: total proteins from embryonic hearts (3, HCN4+/+; 4, HCN4+/R669Q; 5, HCN4R669Q/R669Q; 10 hearts per genotype) labelled with PG2-7H9 (exposure time: 10 min). Lower panel: loading control with anti-actinin antibody. (E) Basal embryonic heart rate at E9.5, E10.5, and E11 (HCN4+/+, red; HCN4+/R669Q, green; HCN4R669Q/R669Q, blue). The number of embryos analysed is indicated. Student's t-test: *P<0.05 compared to HCN4+/+ at the same developmental stage; ϕP<0.05 compared to the same genotype at E9.5. (F) Effect of isoproterenol (2 μM) on the embryonic heart rate (HCN4+/+, red; HCN4+/R669Q, green; HCN4R669Q/R669Q, blue). Data have been normalized to the rates during superfusion with BM. (G) Increase in heart rate (at 9 min) during perfusion with isoproterenol. Data have been normalized to the corresponding heart rate during superfusion with BM (grey, basal heart rate; yellow, heart rate at 9 min). Statistical analysis: student's t-test; *P<0.05 compared to basal heart rate; ϕP<0.05 compared to heart rate of the other genotypes at 9 min. (H) See F for NKH477 (100 μM). (I) See G for NKH477. Data represent mean±s.e.m. Download figure Download PowerPoint HCN4R669Q/R669Q embryonic mice display reduced basal heart rates We analysed the spontaneous beat frequency of hearts isolated from HCN4+/+, HCN4+/R669Q, and HCN4R669Q/R669Q embryos prior to E11.5. Under basal conditions, hearts from heterozygous and homozygous embryos beat regularly without obvious arrhythmias; however, the heart rate was significantly slower compared to hearts from wild-type embryos (Figure 2E and Table I). Furthermore, in wild-type embryos, the heart rate increased from E9 to E11 (Figure 2E and Table I), thereby enhancing supply of nutrients and oxygen necessary for proper development. This increase was not observed in HCN4+/R669Q embryos, and in HCN4R669Q/R669Q embryos, the heart rate was even further reduced from day 9.5 to 11.5 (Figure 2E). Notably, heart rates from HCN4R669Q/R669Q and HCN4−/− embryos at E9.5 (Stieber et al, 2003) are virtually identical, indicating that the Arg669Gln exchange completely eliminates the pacing function of the HCN4 channel. Table 1. Basal embryonic heart rate Basal heart beat (b.p.m.) E9.5 E10.5 E.11 HCN4+/+ 164.5±5.9 174.6±7.8 194.5±8.7 HCN4+/R669Q 129.7±9.0 127.9±7.9 141±10.5 HCN4R669Q/R669Q 99.3±7.3 75.6±8.2 72.2±6.5 Adrenergic stimulation does not accelerate the heart rate of HCN4R669Q/R669Q embryonic mice Isoproterenol superfusion increased the rate of isolated hearts from HCN4+/+ and HCN4+/R669Q embryos (37.4±4.5% (n=8) and 19.8±2.8% (n=6), respectively), whereas no increase was observed in hearts from HCN4R669Q/R669Q embryos (2.8±2.5% (n=5); Figure 2F and G). Similarly, superfusion with NKH477, a drug that specifically activates adenylyl cyclases, increased the heart rate from HCN4+/+ and HCN4+/R669Q embryos (22.6±1.7% (n=14) and 14.2±2.6% (n=13), respectively), but not from HCN4R669Q/R669Q embryos (1.9±1.9% (n=5); Figure 2H and I). These results demonstrate that HCN4 is the principal target for cAMP during the embryonic stages analysed in this study. An acceleration of heartbeat frequency during β-adrenergic stimulation is only possible when cAMP binds to HCN4. The If of HCN4R669Q/R669Q cardiomyocytes activates slower and deactivates faster To characterize genotypic differences of cardiomyocytes, we recorded If currents and action potentials from cultured cells of HCN4+/+, HCN4+/R669Q, and HCN4R669Q/R669Q embryonic hearts. Isolated beating cells were studied with the patch-clamp technique in the whole-cell configuration. The If current was activated by hyperpolarizing voltage steps from a holding voltage of −55 mV to test voltages up to −135 mV. Subsequently, a voltage step to −95 mV was applied to probe the activation state of HCN channels. We observed robust If currents in all three genotypes. Figure 3A and B shows currents from cardiomyocytes of wild-type and homozygous embryos, respectively. The If current of wild-type cardiomyocytes activated faster and more completely than that of HCN4R669Q/R669Q cells (Figure 3A and B). Therefore, we analysed the kinetics of If current activation and deactivation in more detail. Figure 3C shows current responses of wild-type and homozygous cells to steps from the holding voltage (−55 mV) to the test voltage (−95 mV) and back to the holding voltage. We fitted simple exponential models to the respective current traces ignoring the initial delay at the onset of activation (see Figure 3C). While for the homozygous genotype a single exponential with a time constant of 1757±132 ms (n=16) was sufficient to describe activation, two time constants of 264±35 ms (relative amplitude 0.35) and 1771±474 ms (relative amplitude 0.65) (n=11) were necessary to describe wild-type currents. Similarly, deactivation of homozygous currents was well described by a single time constant of 172±23 ms (n=9), while for wild-type currents two time constants of 200±38 and 1690±314 ms (n=8) were necessary for a satisfactory fit. The additional time constant for wild-type currents was fast in the case of activation and slow in the case of deactivation, demonstrating that in wild type, currents activated faster and deactivated slower compared to currents of homozygous cells. Table II summarizes the results of the kinetic analysis. We have included in Table II also the kinetic analysis of the activation of heterologously expressed HCN4 and HCN4R669Q channels. The activation properties of wild-type cardiomyocytes are strikingly similar to those of heterologously expressed HCN4 currents in the presence of cAMP (see Table II). Figure 3.If currents and action potentials recorded from isolated embryonic cardiomyocytes. (A) Upper panel: voltage protocol for the recording of If. Lower panel: If current in an HCN4+/+ cell. Tail currents at −95 mV were used to analyse the voltage dependence of activation. The red line in the current progression indicates the zero current level. (B) If current in an HCN4R669Q/R669Q cell. The red line in the current progression indicates the zero current level. (C) Left part: activation and deactivation of HCN4+/+ channels. Right part: activation and deactivation of HCN4R669Q/R669Q channels. The time course of activation and deactivation of If was fitted with two (HCN4+/+) or a single (HCN4R669Q/R669Q) exponential term (red traces). (D) Voltage-dependent activation of If from HCN4+/+ (filled circles), HCN4+/R669Q (filled triangles), and HCN4R669Q/R669Q (open circles) cells. The solid line was calculated with the Boltzmann equation with the following parameters (mean±s.d.): HCN4+/+, V1/2=−82.5±3.2 mV and s=5.8±2.4 mV (n=12); HCN4+/R669Q, V1/2=−89.3±6.6 mV and s=6.6±1.1 mV (n=12); HCN4R669Q/R669Q, V1/2=−95.7±4.5 mV and s=6.5±2.6 mV (n=9). (E) Summary of beat frequencies (in b.p.m.) of isolated cells recorded in current-clamp mode. Upper panel: frequencies recorded from HCN4+/+ cells. Lower panel: frequencies recorded from HCN4R669Q/R669Q cells. (F) Action potentials of an HCN4+/+ (upper panel) or an HCN4R669Q/R669Q (middle and lower panels) cell. In the experiment shown in the lower panel, a depolarizing current of 20 pA was injected. (G) Voltages V1/2 of half-maximal activation of HCN4+/+ (red), HCN4+/R669Q (green), and HCN4R669Q/R669Q (blue) under control conditions, with 500 μM cAMP (HCN4+/+ and HCN4R669Q/R669Q) or 100 μM cAMP (HCN4+/R669Q) in the pipette solution, or with 10 μM carbachol in the bath, or with 10 μM MDL12330 in the pipette solution. (H) Current densities at −135 mV (determined as If, −135 mV per cell capacitance) of cells from HCN4+/+ (red), HCN4+/R669Q (green), and HCN4R669Q/R669Q (blue) embryonic hearts at defined developmental stages. Recordings were made between 24 and 36 h after preparation. (I) Voltages V1/2 of half-maximal activation of If from HCN4+/+ (red) and HCN4R669Q/R669Q (blue) cells under control conditions (data from G) and from cells pre-incubated with wortmannin (10 μM) for at least 30 min. Data represent mean±s.e.m. except when explicitly stated; *P<0.05 as indicated; ⊗P<0.05 compared to the same genotype under control conditions. Download figure Download PowerPoint Table 2. Kinetics of If activation and deactivation τfast (ms) Relative amplitude A1/(A1+A2) τslow (ms) Relative amplitude A2/(A1+A2) n Activation HCN4a — — 1873±132 1.0 19 HCN4 (100 μM cAMP)a 290±32 0.55±0.09 1522±426 0.45±0.09 7 HCN4R669Q a — — 1749±185 1.0 9 HCN4+/+ b 264±35 0.35±0.03 1771±474 0.65±0.03 11 HCNR669Q/R669Q b — — 1757±132 1.0 16 Deactivation HCN4+/+ b 200±38 0.43±0.04 1690±314 0.57±0.04 8 HCNR669Q/R669Q b 172±23 1.0 — — 9 a Recorded in HEK293 cells. b Genotype of embryonic cardiomyocytes. The equation I(t)=A0+A1exp(−t/τfast)+A2exp(−t/τslow) was used to describe the activation and deactivation of hyperpolarization activated currents (A1 and A2, amplitudes of the fast and slow components, respectively; τfast and τslow, fast and slow time constants, respectively). The voltage-dependent activation of If in cardiomyocytes differs among genotypes Next, we analysed tail currents as in Figure 3A and B to determine the voltage dependence of activation of If for the different genotypes. The voltage of half-maximal activation (V1/2) in the three genotypes differed significantly: V1/2 was −82.5±3.2 mV (n=12) for wild-type, −89.3±6.6 mV (n=12) for heterozygous, and −95.7±4.5 mV (n=9) for homozygous cardiomyocytes (Figure 3D). This result is reminiscent of the differences observed with heterologously expressed HCN4 and HCN4R669Q channels in the presence of cAMP (compare Figure 2A). However, for the experiments presented here, no exogenous cAMP was added. We interpret this result to indicate that even in the unstimulated state, the If in wild-type and heterozygous cardiomyocytes is significantly upregulated by cAMP. Isolated HCN4R669Q/R669Q cardiomyocytes beat with lower frequencies We recorded action potentials from isolated cells of wild-type and homozygous hearts to investigate whether the differences in If are reflected in the spontaneous rates of cardiomyocytes. Action potentials were highly variable from cell to cell, indicating that cardiomyocytes at this developmental stage have already undergone substantial differentiation. Spontaneous rates of HCN4+/+ cells ranged from 60 to 337 b.p.m. (Figure 3E, upper panel), the action potential duration (APD) varied from 30 to 360 ms, and the MDP varied from −62 to −87 mV. Rates from HCN4R669Q/R669Q cells varied from 15 to 225 b.p.m. (Figure 3E, lower panel), ADPs from 5 to 800 ms, and MDPs from −61.5 to −87 mV. On average, rates were significantly lower in HCN4R669Q/R669Q cells (99±11 b.p.m. (n=32)) than in HCN4+/+ cells (156±11 b.p.m. (n=37), P<0.05). We reasoned that cells with the highest spontaneous rates eventually pace the embryonic heart. In wild-type embryos, we identified pacemaker-like cardiomyocytes (9 of 32 cells) with particularly high average rates of 211±20 b.p.m. (n=9), MDPs of −75.7±0.7 mV, APDs<100 ms, and robust If currents when voltage clamped. In the presence of cAMP (100 μM in the pipette solution), this cell population displayed beat frequencies of 338±41 b.p.m., indicating a pronounced stimulatory effect of cAMP. Action potentials of a typical wild-type cell from this population are shown in Figure 3F (upper panel). The cell is spontaneously active at about 240 b.p.m. We then searched for HCN4R669Q/R669Q cells that produced the same action potential shape (APD<100 ms) characteristic of this particular class of cells. In total, 8 of 32 cells were found with action potential shapes similar to the ones we were looking for. These cells were characterized by a relatively slow rate of 92±9 b.p.m. (n=8) and MDPs of −82.6±0.7 mV. Figure 3F (middle panel) shows a typical example. The cell was active with 75 b.p.m. and had an MDP of −83 mV. When we injected a depolarizing current of 20 pA into this cell (Figure 3F, lower panel) to compensate for the poor activation of the HCN4R669Q/R669Q channel, action potential rates increased drastically to values otherwise not observed in HCN4R669Q/R669Q cells. Under these conditions, rate, action potential shape, and MDP were very simi
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