- Email: [email protected]
Characterization of the effects of Ryanodine, TTX, E-4031 and 4-AP on the sinoatrial and atrioventricular nodes
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Progress in Biophysics and Molecular Biology 96 (2008) 452–464 www.elsevier.com/locate/pbiomolbio
Review
Characterization of the effects of Ryanodine, TTX, E-4031 and 4-AP on the sinoatrial and atrioventricular nodes Mohammed R. Nikmarama,1, Jie Liuc,1, Mohamed Abdelrahmanc, Halina Dobrzynskib, Mark R. Boyettb, Ming Leib, a
Department of Physiology, Iran University of Medical Sciences, Tehran, Iran Cardiovascular Research Group, Division of Cardiovascular and Endocrine Sciences, University of Manchester, 46 Grafton Street, Manchester M13 9NT, UK c Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK
b
Available online 1 August 2007
Abstract Aims: To characterize the effects of inhibition of Ryanodine receptor (RyR), TTX-sensitive neuronal Na+ current (iNa), ‘‘rapidly activating’’ delayed rectifier K+ current (iKr) and ultrarapid delayed rectifier potassium current (IKur) on the pacemaker activity of the sinoatrial node (SAN) and the atrioventricular node (AVN) in the mouse. Methods: The structure of mouse AVN was studied by histology and immunolabelling of Cx43 and hyperpolarizationactivated, cyclic nucleotide-binding channels (HCN). The effects of Ryanodine, TTX, E-4031 and 4-AP on pacemaker activities recorded from mouse intact SAN and AVN preparations have been investigated. Results: Immuno-histological characterization delineated the structure of the AVN showing the similar molecular phenotype of the SAN. The effects of these inhibitors on the cycle length (CL) of the spontaneous pacemaker activity of the SAN and the AVN were characterized. Inhibition of RyR by 0.2 and 2 mM Ryanodine prolonged CL by 42712.3% and 64718.1% in SAN preparations by 163772.3% and 241791.2% in AVN preparations. Inhibition of TTX-sensitive iNa by 100 nM TTX prolonged CL by 2276.0% in SAN preparations and 53713.6% in the AVN preparations. Block of iKr by E-4031 prolonged CL by 68712.5% in SAN preparations and 2873.4% in AVN preparations. Inhibition of iKur by 50 mM 4-AP prolonged CL by 2073.4% in SAN preparations and 1873.0% in AVN preparations. Conclusion: Mouse SAN and AVN showed distinct different response to the inhibition of RyR, TTX-sensitive INa, IKr and iKur, which reflects the variation in contribution of these currents to the pacemaker function of the cardiac nodes in the mouse. Our data provide valuable information for developing virtual tissue models of mouse SAN and AVN. r 2007 Published by Elsevier Ltd. Keywords: Sinoatrial node; Atrioventricular node; Pacemaking; TTX-sensitive neuronal Na+ current (iNa); ‘‘Rapidly activating’’ delayed rectifier K+ current (iKr); Ultrarapid delayed rectifier potassium current (IKur)
Corresponding author. Tel.: +44 161 275 1194; fax: +44 161 275 1183. 1
E-mail address: [email protected] (M. Lei). Joint first authors.
0079-6107/$ - see front matter r 2007 Published by Elsevier Ltd. doi:10.1016/j.pbiomolbio.2007.07.003
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Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Electrical recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Statistics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Immuno-histological characterization of the AVN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Characterization of SAN and AVN spontaneous pacemaker activities . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Effect of Ryanodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Effect of TTX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Effect of E-4031 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Effect of 4-AP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Delineation of AV junction pacemaker structure by HCN4 and Cx43 . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Effect of Ryanodine, TTX, E-4031 and 4-AP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction There is an effort to build anatomically and biophysically detailed models of different regions of the heart. Such models require knowledge of anatomy, physiology, and molecular and cellular biology of the different regions. In mammalian heart, each heartbeat is normally initiated from the sinoatrial node (SAN), the primary pacemaker of the heart. The SAN contains special ‘‘pacemaker’’ cells generating the electrical signals that control the pace and rhythm of the heart. The signals travel from the SAN to the AVN. From the AVN, the signals are conducted along pathways and spread into the ventricles, causing them to contract and pump blood into the lungs and throughout the body. The function of the AVN is normally to act as a conduction pathway between the atria and ventricles under physiological condition, but it can also act as a major subsidiary pacemaker, initiating heartbeat by generating the junctional rhythm in the circumstance of failure of the SAN. Although all parts of cardiac conduction system have the capability of generating rhythmic heartbeat, i.e. the capacity of automaticity, such capacity varies regionally. Thus, the SAN has a highest automaticity; its discharge rate determines the heart rate that is normally within the physiological range (e.g. 60–120 beats/min in human). The automaticity of the AVN is lower than that of the SAN and it generates the junctional rhythm (sub-physiological range) having much slower rate than the sinus rhythm (e.g. o60 beats/min in human). The more down stream of the conduction system (i.e. Purkinje fibres) is, the lower automaticity has. The pacemaking mechanisms for cardiac conduction tissue, the SAN in particular, have been extensively investigated over the past two to three decades and have been largely defined (for reviews, see Irisawa et al., 1993; Boyett et al., 2000; Kleber and Rudy, 2004). The mechanism underlying the regional difference in automaticity within the conduction system, however, has been rarely explored. Recent studies suggest that the diversity in electrophysiology in different regions of the heart (e.g. SAN and AVN) is likely attributed to variable expression of ion channel gene products in different regions of the heart. For example, high-density real-time RT-PCR analysis suggested a specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart (Marionneau et al., 2005). Further detailed characterization of the
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functional roles of individual ion channel gene products in different parts of the conduction system will deepen our understanding of regional difference in automaticity of this system. In the present study, we have characterized the structure of the AVN studied by histology and immunolabelling of Cx43 and HCN channels. The effects of Ryanodine, TTX, E-4031 and 4-AP on pacemaker activity recorded from intact mouse SAN and AVN preparations have been investigated. From the results obtained, possible roles of RyR, TTX-sensitive neuronal Na+ current (iNa), ‘‘rapidly activating’’ delayed rectifier K+ current (iKr) and ultrarapid delayed rectifier potassium current (IKur) in mouse SAN and AVN have been assessed. Our data, therefore, provide valuable information for developing virtual tissue models of mouse SAN and AVN. 2. Materials and methods 2.1. Animals C57BL/6J mice either sex weighing 20–30 g (age, 10–12 weeks; from Charles River UK Ltd., Kent, UK) were used. Mice were killed by cervical dislocation and the heart quickly removed. All animal procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986. 2.2. Electrical recording The SAN and AVN preparations were isolated from the mouse heart as described previously (Lei et al., 2004). In brief, the right atrium was separated from the rest of the heart and opened by the longitudinal incision from the rest of the heart. SAN and AVN were separated by cutting through atrial tissue superior to the coronary sinus. The SAN preparation contains intercaval region and some surrounding atrial tissue. AVN preparation contains a region that includes the triangle of Koch. The nodal preparations (endocardial surface up) are fixed in a tissue bath and superfused with Tyrode solution with or without blocker at 32 1C at a rate of 4 ml/min through a heat exchanger. To record the extracellular signals, two modified bipolar electrodes (Yamamoto et al., 1998) were placed in the SAN and AVN regions separately. Thus, the extracellular potential and the firing activities (the rate and cycle length (CL, calculate by measuring R–R interval)) of the SAN and the AVN preparations were recorded simultaneously. 2.3. Solutions For electrical recording, the composition of the solution was as follows (in mM): 93 NaCl, 20 NaHCO3, 1 Na2HPO4, 5 KCl, 2 CaC12, 1 MgSO4, 20 sodium acetate, and 10 glucose. The solution was equilibrated with 95% Oz–5% CO2 to give a pH of 7.4. 2.4. Drugs E-4031 (1-[2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulphonylaminobenzoyl)-piperidine, Eisai Pharmaceuticals, Tokyo, Japan) and TTX (Tetrodotoxin, Sigma-Aldrich, Poole, UK) were dissolved in deionized water to make 10 mM stock solutions, and stored at 4 1C. Ryanodine (Sigma-Aldrich, Poole, UK). A stock solution of 1 M 4-aminopyridine (4-AP) (Sigma-Aldrich, Poole, UK) was prepared in deionized water, titrated with HCl to pH 7.4 and stored at 4 1C. 2.5. Histology Sections were stained with Masson’s trichrome to show histology; with this technique, connective tissue is stained blue, cardiac myocytes are stained red and nuclei are stained dark blue. Images of tissue sections were obtained using a Leica DC camera mounted on a Leica DMIRB inverted microscope in conjunction with Leica TWAIN software.
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2.6. Immunohistochemistry Immunohistochemistry was carried out using established methods as described previously (Lei et al., 2004). Briefly, sections were fixed in 10% formalin (Sigma) for 30 min and washed with 0.01 M phosphate buffer solution (PBS) three times at 10 min intervals. Sections were then permeabilized by incubating them in PBS containing 0.1% Triton X-100 for 30 min, after which they were washed with PBS and then blocked in 10% normal serum in PBS for 1 h at room temperature. Sections were incubated with the primary antibody (diluted in 1% bovine serum albumin, BSA) at 4 1C overnight, after which they were washed three times with PBS over 30 min. Sections were incubated with secondary antibodies for 1–2 h at room temperature. After washing three times in PBS, coverslips were mounted on the microscope slides and the coverslips were sealed with nail polish. Slides were stored in the dark at 4 1C. Immunolabelled tissue was viewed with a Leica TCS SP or Zeiss LSM 510 laser scanning confocal microscope equipped with argon and helium–neon lasers, which allowed excitation at 488 nm wavelength for the detection of FITC/Alex 488. All images presented are single optical sections. Images were saved and later processed using Corel Photo-Paint and Corel Draw software (Corel, Ottawa, Canada). 2.7. Antibodies Rabbit anti-HCN4 IgG raised against residues 119–155 of human HCN4 (1:100 dilution; Alomone Labs) and anti-Cx43 (connexin43) IgG raised against residues 363–382 of human Cx43 (1:1000 dilution; Sigma, Poole, UK). To detect the primary IgGs, goat anti-rabbit IgG conjugated to Alexa Fluro 488 (Molecular Probes, Eugene, USA), or donkey anti-rabbit IgG conjugated to FITC (Chemicon) were used. The specificity of anti-Cx43 (Dobrzynski et al., 2003) and anti-HCN4 (Dobrzynski et al., 2003) IgGs has been checked previously. No labelling above background was obtained when the primary or secondary antibodies were omitted (data not shown). 2.8. Statistics Where applicable, a Student t-test is applied. Data are presented as mean7S.E.M. (n ¼ number of preparations). Po0.05 is considered as significant. 3. Results 3.1. Immuno-histological characterization of the AVN We recently characterized the structure of mouse SAN by histology and immunolabelling of ANP, Cx43 and hyperpolarization-activated cyclic nucleotide-gated cation channels (HCNs) (Liu et al., 2006). We adapted the SAN approach for the AVN in the present study. Fig. 1A shows an example of histological features of the different regions along the conduction axis of AVN (posterior to anterior along the AV junction). Tissue sections cut perpendicular to the AV junction. Cardiac myocytes were stained purple, connective tissue was stained blue, cell nuclei were stained dark blue. The serial of tissue sections from the same preparation studied by histology were then immunolabelled with the two molecular markers, Cx43 and HCN4, used for delineation of SAN (Liu et al., 2006). As shown in Fig. 1B, HCN4 was used as a positive marker of pacemaker cells. In the serial tissue sections, HCN4 highlighted the AVN region that presumably has capacity to produce jnuctional rhythm. From the 0 mm section, HCN4-positive cells appeared as a small cluster of cells located at the ventricular septum above the tricuspid valve. The location and size of this AVN region was almost identical as those determined in their sister sections by histology. In the 0.4 mm section, the entire nodal region measured about 0.8 mm 0.4 mm, which included the compact AVN (showing as a bright knot) and the transitional region facing the atrial septum. The region of the compact node (boxed region in 0.4 mm section). Cells at the compact node were small and interwoven. To confirm the delineation by HCN4, a negative marker, Cx43, was applied to the same set of tissue sections. Fig. 1C shows the immunolabelling of Cx43. The region devoid of green fluorescence appeared larger than the HCN4-positive region in the sister
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Fig. 1. (Continued)
sections. There was connective tissue around the nodal region as shown in histological staining, the quantity of the connective tissue varied from the posterior to anterior region of the AVN. This connective tissue showed negative immunoreactivity for Cx43, thus the Cx43-negative region around AVN was composed of nodal region and the surrounding connective tissue. However, positive immunoreactivity of Cx43 was found specifically in the His bundle (boxed region in 0.8 mm). The positive immunoreactivity appeared as positive clusters and the size of which was smaller than those of the atrial or ventricular. The small Cx43 clusters were not found in the PNE, compact AVN, or bundle branches, but solely in the His bundle. Same results were obtained from four preparations. 3.2. Characterization of SAN and AVN spontaneous pacemaker activities The spontaneous firing rate of the SAN and the AVN were compared by extracellular potential recording. Fig. 2A shows a typical mouse SAN–AVN preparation for electrical extracellular potential recording. Fig. 1. Delineation of AVN. Serial sections cut perpendicular to the AV junction were stained by either histology, or anti-HCN4 or antiCx43. (A) Masson’s trichrome stained sections at different levels (0–1.2 mm) through the AVN. Broad arrows indicate the connective tissue separating AVN from ventricle, triangle arrows indicate connective tissue at the transitional region merging with atrium. PNE, posterior nodal extension; CN, compact node; CF, centre fibrous body; HB, His bundle; LBB, left bundle branch; RBB, right bundle branch; TV, tricuspid valve. (B) Anti-HCN4 immunostaining. Regions that were highlighted by green fluorescence labelled HCN4 antibody (within the red dotted lines) were the AVN region. White dashed lines indicate the tissue border. Boxed region in the compact node region (0.4 mm section) was magnified in the bottom as CN (compact node). (C) Anti-Cx43 immunostaining. Serial sections were stained with anti-Cx43 antibody. Regions devoid of green fluorescence labelled Cx43 antibody were indicated as the red dotted lines. Boxed region in the 0.8 mm section was magnified in at the bottom (HB). Scale ¼ 200 mm for all panels.
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Fig. 2. Recording of spontaneous electrical activities from SAN and AVN. (A) The opened right side of mouse heart was illustrated in with the SAN region and AVN region circled in red. (B) Two nodes are separated by cutting through the black dashed line and two electrodes are positioned in two nodal regions for extracellular potential recording. SVC, superior vena cava; SEP, interatrial septum; IVC, inferior vena cava; RA, right atrial appendage; CT, crista terminalis. (C) An example of spontaneous pacemaker activity simultaneously recorded from the SAN and the AVN preparations.
The non-separated tissue preparation showed single beating rhythm of rate of 300 bpm, presumably originating from the SAN. Once the preparation is separated into two parts (as indicated in Fig. 2C), two nodes showed different rhythms. The firing rate of AVN is much slower that of SAN. Fig. 2C shows examples of spontaneous activities recorded from SAN and AVN preparations simultaneously. After the two pieces of tissue stabilized for 430 min (to allow them to recover from the injury during dissection), the spontaneous firing rate of the SAN and AVN preparations measured as cycle length (CL) are 246.8735.9 ms (SAN, n ¼ 8) and 495.2761.8 ms (AVN, n ¼ 8), respectively. 3.3. Effect of Ryanodine First, Ryanodine, a selective inhibitor of RyR, was applied for characterization of the role of sarcoplasmic reticulum (SR) function in pacemaking of the SAN and the AVN. Fig. 3 shows the effect of Ryanodine. Ryanodine had been applied for 20–30 min to obtain a steady-state response. In SAN preparations, 0.2 and 2 mM Ryanodine prolonged CL by 47715.7% and 70721.8% from 247735.9 ms in the control condition to 356757.0 and 444782.7 ms in the presence of 0.2 and 2 mM Ryanodine, respectively (n ¼ 8) (Po0.05). In AVN preparations, the same doses of Ryanodine prolonged CL by 163772.3% and 241791.2% from 495761.8 ms in the control condition to 11727245.2 and 15727423.8 ms, respectively (n ¼ 8, Po0.01). Thus, Ryanodine slows down pacemaker activity of both SAN and AVN, but its effect was greater in the AVN than in the SAN. Ryanodine does not cease the spontaneous beating of either the SAN or the AVN preparations after even more than 1 h perfusion of the drug at 10 mM concentration. The effect of 2 mM Ryanodine on the SAN and the AVN was also examined in two additional species, rat and rabbit. Ryanodine caused an increase of CL of SAN rhythm by 40712.8% in rat preparations and by 3076.8% in rabbit preparations. The same dose Ryanodine caused an increase of CL of AVN rhythm by 186757.6% in rat preparations (n ¼ 9) and by
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Fig. 3. The effect of Ryanodine on electrical activities of SAN and the AVN. (A) CL of the SAN under control conditions and after application of 0.2 and 2 mM Ryanodine. (B) CL of the AVN under control conditions and after the application of 0.2 and 2 mM Ryanodine. (C) The percentage change in CL of SAN and AVN after application of 2 mM Ryanodine vs. control. Mean7S.E.M.s are shown. *Significantly different (Po0.05) from control. Mean value of CL for each preparation was averaged from 50 cycles.
65710.8% in rabbit preparations (n ¼ 6). Thus, the effects on the SAN and the AVN are consistent with that of the mouse. 3.4. Effect of TTX Second, the effect of TTX on the SAN and AVN was compared. The majority of Na+ channels in the heart are composed of the tetrodotoxin (TTX)-resistant (Kd, 2–6 mM) Nav1.5 isoform; however, recently it has been shown that TTX-sensitive (Kd, 1–10 nM) neuronal Na+ channel isoforms (Nav1.1, Nav1.3 and Nav1.6) are also present and functionally important in the myocytes of the ventricles and the SAN. In this study, 100 nM TTX, a dose that causes complete block of TTX-sensitive neuronal Na+ channel isoforms in single SAN cells (Lei et al., 2004), was applied to the SAN and the AVN preparations. TTX had been applied for 20–30 min to obtain a steady-state response. In SAN preparations, TTX prolonged CL by 2276.0% from 212713.5 ms in the control condition to 263729.2 ms in the presence of TTX (Fig. 4) (n ¼ 7, Po0.05). In AVN preparations, while the same dose of TTX prolonged CL by 53713.6% from 578768.2 ms in the control condition to 856771.8 ms in the presence of TTX (Fig. 4) (n ¼ 5, Po0.005). 3.5. Effect of E-4031 Third, the effect of E-4031 on the SAN and the AVN was then examined. A ‘‘rapidly activating’’ delayed rectifier K+ current (IKr) has been previously characterized in mouse SAN pacemaker cells (Cho et al., 2003; Clark et al., 2004). In the present study, we compared the blocking effect of 1 mM E-4031, a selective block of IKr, on pacemaker activity of the SAN and the AVN. In SAN preparations, 1 mM E-4031 increased CL by 68712.5% from 215711.9 ms in the control condition to 360733.8 ms in the presence of E-4031 (Fig. 5) (n ¼ 6, Po0.005). In AVN preparations, the same dose of E-4031 increases in CL by 2873.4% from 452720 ms in control condition to 580735.8 ms in the presence of E-4031 (Fig. 5) (n ¼ 6, Po0.005). Thus, the block effect of E-4031 is greater in the SAN than in the AVN. 3.6. Effect of 4-AP Finally, the effect of 4-AP on the SAN and the AVN was studied. Ultrarapid delayed rectifier potassium current (IKur) was first identified in atrial myocytes (Feng et al., 1997). Its functional role has not been
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Fig. 4. The effect of 100 nM TTX on electrical activities of SAN and the AVN. (A) CL of the SAN under control conditions and after application of TTX. (B) CL of the AVN under control conditions and after the application of TTX. (C) The percentage change in CL of SAN and AVN after application of TTX vs. control. Mean7S.E.M.s are shown. *Significantly different (Po0.05) from control. Mean value of CL for each preparation was averaged from 50 cycles.
Fig. 5. The effect of 1 mM E-4031 on electrical activities of SAN and the AVN. (A) CL of the SAN under control conditions and after application of E-4031. (B) CL of the AVN under control conditions and after the application of E-4031. (C) The percentage change in CL of SAN and AVN after application of E-4031 vs. control. Mean7S.E.M.s are shown. *Significantly different (Po0.05) from control. Mean value of CL for each preparation was averaged from 50 cycles.
previously investigated in the SAN and the AVN. The effect 50 mM 4-AP, a concentration that blocks IKur, on pacemaker activity of SAN and AVN was then examined. In SAN preparations, 50 mM 4-AP prolonged CL by 2073.4% from 272725.0 ms in the control condition to 328730.9 ms in the presence of 4-AP (Fig. 6) (n ¼ 5, Po0.005). In AVN preparations, while the same dose of 4-AP prolonged CL by 1873.0% from 480722.9 ms in the control condition to 566730.8 ms in the presence of 1873.0% (Fig. 6) (n ¼ 5, Po0.005). Therefore, there is no significant difference of the effect of 4-AP on the SAN and the AVN.
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Fig. 6. The effect of 50 mM 4-AP on electrical activities of SAN and the AVN. (A) CL of the SAN under control conditions and after application of 4-AP. (B) CL of the AVN under control conditions and after the application of 4-AP. (C) The percentage change in CL of SAN and AVN after application of 4-AP vs. control. Mean7S.E.M.s are shown. *Significantly different (Po0.05) from control. Mean value of CL for each preparation was averaged from 50 cycles.
4. Discussion The main findings of this study are: (i) immuno-histological characterization of A–V junction revealed the structure of the AV junction pacemaker of the mouse; (ii) mouse SAN and AVN show different response to the inhibition of RyR, TTX-sensitive iNa, iKr and IKur, thus, the effects of Ryanodine and TTX are greater on the AVN, while, in contrast, the effect of E-4031 is greater on the SAN, while 4-AP has a similar effect on both nodes. 4.1. Delineation of AV junction pacemaker structure by HCN4 and Cx43 The study delineated the mouse AV junction pacemaker structure with histology and immunolabelling. Based on immunolabelling of HCN4 and Cx43, different cell types were identified in and around the AVN. Comparison of Fig. 1A–C shows that myocytes that expressed Cx43 did not express HCN4, whereas the myocytes that did not express Cx43 express HCN4. The Cx43-positive/HCN4-negative tissue is assumed to be atrial muscle. The Cx43-negative/HCN4-positive tissue is assumed to be AVN tissue with pacemaker generation potential. Cx43-negative/HCN4-positive cells were found throughout the posterior nodal extension (PNE), AVN, His bundle and bundle branches without significant difference in expression level. Previous functional studies showed the site of initial pacemaker in the AVN was found at the compact AVN, the NH region or the PNE region (Meijler and Jalife, 1999; Dobrzynski et al., 2003; Efimov et al., 2004; Meijler and Janse, 1988). The molecular basis for particular region to act as the leading pacemaker site in A–V junctional region is still in controversy. Moreover, when the atrial fibres were peeled off, pacemaking depolarizations were presented in all regions of AVN with the steepest ones located at the middle or lower nodal regions (Tse, 1973). It was suggested that, in the intact preparation, atrial myocardium might shrink pacemaker activity in the upper node by electronic interaction with the nodal cells. In this study, intermingling of nodal cells (HCN4-positive/Cx43-negative cells) and atrial myocytes (HCN4-negative/Cx43-positive cells) were also found in the upper nodal region where the AVN meet atrium. The interdigitation of AVN and atrium provides the structural substrate for this kind of electrical interaction at this region. 4.2. Effect of Ryanodine, TTX, E-4031 and 4-AP Application of Ryanodine (0.2–2 mM), TTX (100 nM), E-4031 (1 mM) and 4-AP (50 mM) revealed the regional difference in responding to the inhibition of SR function, TTX-sensitive iNa, iKr and IKur in mouse
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conduction system. Firstly, the effect of Ryanodine on the SAN was characterized. Recent studies have suggested that the SAN pacemaker activity is also influenced by transient changes in cytosolic Ca2+ (Vinogradova et al., 2005), the disruption of SR function causes a slowing of spontaneous pacemaker activity in isolated SAN cells (Huser et al., 2000; Bogdanov et al., 2001; Lancaster et al., 2004; Vinogradova et al., 2005) and intact SAN preparations. As shown in Fig. 3, 0.2 and 2 mM of Ryanodine prolonged CL by 46% and 70% in SAN preparations, whereas the same doses of Ryanodine caused a much greater prolongation of CL in AVN preparations (by 163% and 241%, respectively). A high dose of Ryanodine (10 mM) did not cause cessation of spontaneous activity of both SAN and AVN preparations. Thus, it appears that SR calcium release is a significant modulator, but not an essential player to pacemaking of cardiac nodes, its effect is also greater in the AVN than in the SAN. Previous studies on single SAN cells (guinea-pig) (Bassani et al., 1997) and intact SAN preparations of the SAN (rabbit) (Honjo et al., 2003), and isolated right atria (dog, rat) (Lipsius, 1987; Bassani et al., 1999) have shown that Ryanodine caused a modest slowing of SAN pacemaker activity. High concentrations (30 mM) did not cause cessation of spontaneous activity (rabbit) (Honjo et al., 2003) nor did cyclopiazonic acid (guinea-pig) (Rigg and Terrar, 1996), thapsigargin (rat) (Bassani et al., 1997) by disruption of SR function by these manoeuvres. Thus, our result is principally in consistent with the previous studies on single cell and isolated SAN preparations (Bassani et al., 1997; Honjo et al., 2003; Rigg and Terrar, 1996). The different response of the SAN and the AVN to Ryanodine (greater in the AVN) is likely due to the difference in the intracellular calcium regulation between these regions. For example, the level of the transcripts for type 2 Ryanodine receptor, calcium pump (SERCA) is much higher in the AVN than that in the SAN in the mouse (Marionneau et al., 2005). The higher density of RyR2 and SERCA may reflect a greater role of SR function in the AVN than in the AVN. Secondly, the effect of 100 nM TTX (a dose that causes complete block of TTX-sensitive neuronal Na+ channel isoforms in single SAN cells) was studied. 100 nM TTX prolonged CL in SAN preparations by 22%, while the same dose of TTX caused a much greater prolongation in CL in AVN preparations (53%), which suggests a greater role of TTX-sensitive INa in the AVN than in the SAN. Recent studies by ourselves and others have suggested complex yet specific regional distributions of Na+ channel isoforms in the cardiac conduction system particularly through the SAN and AV junction (Lei et al., 2004; Maier et al., 2003). Thus, there is a co-expression of multiple neuronal and cardiac Na+ channel isoforms in the heart (Maier et al., 2002). Both ourselves (Lei et al., 2004) and Maier et al. (2003) have confirmed that Nav1.1 is uniformly expressed across the SAN; our own recent work also demonstrated a complex distribution of Nav1.5, which was not expressed in its intercaval, central, region but which did occur in peripheral SAN, on the endocardial face of the crista terminalis, thereby explaining electrophysiological findings of relatively fast conduction in peripheral SAN (Lei et al., 2004). In the AVN (rat), both Nav1.1 and Nav1.5 were distributed in a similar manner in the AV junction regions (Yoo et al., 2006). The distinct different response of SAN and AVN to inhibition of TTX-sensitive INa is likely due to difference in expression of neuronal type Na+ channels (presumably Nav1.1 channels) in these two regions, quantitative analysis indicated that the expression of Nav1.1 transcript is much higher in the AVN than in the SAN in mouse heart (8–10:1) (Marionneau et al., 2005). Thirdly, the effects of inhibition of two repolarizating currents, IKr and iKur, by E-4031 and 4-AP were examined on pacemaker activity of mouse SAN and AVN. The data show that, in contrast to the effect of Ryanodine and TTX, the effect of E-4031 on pacemaker activity is greater in the SAN than in the AVN, while 4-AP has similar effect on both nodes. 1 mM E-4031 significantly decrease in firing rate in SAN preparations (68%) and in AVN preparations (28%). In isolated SAN cells, Clark et al. (2004) found that in isolated mouse SAN cells, outward currents evoked by depolarizing steps (greater than 40 mV) were strongly inhibited by 1 mM E-4031. 1 mM E-4031 slowed the spontaneous pacing rate of Langendorff-perfused, isolated adult mouse hearts by an average of 36.5% Cho et al. (2003) also reported that E4031-sensitive, rapidly activating delayed rectifier K+ current (IKr) was activated by depolarization with the amplitude of 38.3 pA at 0 mV. However, the chromanol 293B-sensitive, slowly activating delayed rectifier K+ current (IKs) was not present (Cho et al., 2003). Therefore, these studies suggest that IKr is the major, perhaps the only channel, contributes to the delayed rectifier K+ current in mouse SAN pacemaker cells. The previous study on profiles of ion channel transcripts of mouse hearts indicated that the levels of the transcripts (mERG) for IKr is similar in both regions (Marionneau et al., 2005). Therefore, it is unlikely that the functional discrepancy of IKr in two nodes is related
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to the expression of the channels. Whether such discrepancy is the result of different basal rate between the SAN and the AVN requires a further investigation. We could speculate that the effect of blocking of IKr in greater in higher basal rate in the case of the SAN. Clearly, such rate-dependent effect needs to be examined in isolated single cells. The effect of 50 mM 4-AP suggests a role of iKur in mouse SAN and AVN. A decade ago, Yue et al. (1996) first reported iKur from dog atrial myocytes. It showed very rapid activation and deactivation. The current was insensitive to E-4031, dendrotoxin and chloride substitution, but was inhibited by barium, with an Kd50 of 1.65 mM. Single-channel activity was strongly inhibited by 50 mM 4-AP or 10 mM TEA. Both 4-AP and TEA decreased open time, suggesting open-channel block. Selective inhibition of IKur with 50 mM 4-AP prolonged canine atrial action potentials, indicating that IKur contributes to canine atrial repolarization (Yue et al., 1996). This current, however, has not been characterized in cardiac pacemaker cells. Profiling ion channel distribution in mouse heart, Kv1.5, the transcript for IKur channel, has been identified in both SAN and AVN regions. Our data showed that 50 mM 4-AP slowed firing rate of the SAN preparations and the AVN preparations equally (20% and 18%) (Fig. 6). Thus, our data indicate a possible presence and functional role of iKur in mouse SAN and AVN. The previous study on profiles of ion channel transcripts of mouse hearts indicated that the levels of the transcripts (mERG and Kv1.5) for IKr and IKur are similar in two nodes (Marionneau et al., 2005). This may explain the similar effect of 4-AP on two nodes. In conclusion, mouse SAN and AVN showed the different response to the inhibition of RyR, TTX-sensitive INa, IKr and iKur, which reflects the variation in contribution of these currents to the pacemaker function of the SAN and the AVN in the mouse.
Acknowledgements This work was supported by Iran University of Medical Sciences, the Wellcome Trust and the British Heart Foundation.
References Bassani, R.A., Bassani, J.W., Lipsius, S.L., Bers, D.M., 1997. Diastolic SR Ca efflux in atrial pacemaker cells and Ca-overloaded myocytes. Am. J. Physiol. 273, H886–H892. Bassani, J.W., Godoy, C.M., Bassani, R.A., 1999. Effect of ryanodine on sinus node recovery time determined in vitro. Braz. J. Med. Biol. Res. 32, 1039–1043. Bogdanov, K.Y., Vinogradova, T.M., Lakatta, E.G., 2001. Sinoatrial nodal cell ryanodine receptor and Na+-Ca2+ exchanger: molecular partners in pacemaker regulation. Circ. Res. 88, 1254–1258. Boyett, M.R., Honjo, H., Kodama, I., 2000. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc. Res. 47, 658–687. Cho, H.-S., Takano, M., Noma, A., 2003. The electrophysiological properties of spontaneously beating pacemaker cells isolated from mouse sinoatrial node. J. Physiol. (Lond.) 550, 169–180. Clark, R.B., Mangoni, M.E., Lueger, A., Couette, B., Nargeot, J., Giles, W.R., 2004. A rapidly activating delayed rectifier K+ current regulates pacemaker activity in adult mouse sinoatrial node cells. Am. J. Physiol. 286, H1757–H1766. Dobrzynski, H., Nikolski, V.P., Sambelashvili, A.T., Greener, I.D., Yamamoto, M., Boyett, M.R., Efimov, I.R., 2003. Site of origin and molecular substrate of atrioventricular junctional rhythm in the rabbit heart. Circ. Res. 93, 1102–1110. Efimov, I.R., Nikolski, V.P., Rothenberg, F., Greener, I.D., Li, J., Dobrzynski, H., Boyett, M., 2004. Structure–function relationship in the AV junction. Anat. Rec. A Discov. Mol. Cell Evol. Biol. 280 (2), 952–965. Feng, J., Wible, B., Li, G.R., Wang, Z., Nattel, S., 1997. Antisense oligodeoxynucleotides directed against Kv1.5 mRNA specifically inhibit ultrarapid delayed rectifier K+ current in cultured adult human atrial myocytes. Circ. Res. 80, 572–579. Honjo, H., Inada, S., Lancaster, M.K., Yamamoto, M., Niwa, R., Jones, S.A., Shibata, N., Mitsui, K., Horiuchi, T., Kamiya, K., Kodama, I., Boyett, M.R., 2003. Sarcoplasmic reticulum Ca2+ release is not a dominating factor in sinoatrial node pacemaker activity. Circ. Res. 92, e41–e44. Huser, J., Blatter, L.A., Lipsius, S.L., 2000. Intracellular Ca2+ release contributes to automaticity in cat atrial pacemaker cells. J. Physiol. (Lond.) 524 (Pt. 2), 415–422. Irisawa, H., Brown, H.F., Giles, W., 1993. Cardiac pacemaking in the sinoatrial node. Physiol. Rev. 73, 197–227. Kleber, A.G., Rudy, Y., 2004. Basic mechanisms of cardiac impulse propagation and associated arrhythmias. Physiol. Rev. 84, 431–488. Lancaster, M.K., Jones, S.A., Harrison, S.M., Boyett, M.R., 2004. Intracellular Ca2+ and pacemaking within the rabbit sinoatrial node: heterogeneity of role and control. J. Physiol. (Lond.) 556, 481–494.
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Lei, M., Jones, S.A., Liu, J., Lancaster, M.K., Fung, S.S.-M., Dobrzynski, H., Camelliti, P., Maier, S.K.G., Noble, D., Boyett, M.R., 2004. Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking. J. Physiol. (Lond.) 559, 835–848. Lipsius, S.L., 1987. Triggered rhythms in atrial muscle. J. Electrocardiol. 20, 33–37. Liu, J., Dobrzynski, H., Abdelrahman, M., Boyett, M., Lei, M., 2006. Organization of the mouse sinoatrial node: structure and expression of HCNs. Cardiovasc. Res. 73, 729. Maier, S.K., Westenbroek, R.E., Schenkman, K.A., Feigl, E.O., Scheuer, T., Catterall, W.A., 2002. An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart. Proc. Natl. Acad. Sci. USA 99, 4073–4078. Maier, S.K., Westenbroek, R.E., Yamanushi, T.T., Dobrzynski, H., Boyett, M.R., Catterall, W.A., Scheuer, T., 2003. An unexpected requirement for brain-type sodium channels for control of heart rate in the mouse sinoatrial node. Proc. Natl. Acad. Sci. USA 100, 3507–3512. Marionneau, C., Couette, B., Liu, J., Li, H., Mangoni, M.E., Nargeot, J., Lei, M., Escande, D., Demolombe, S., 2005. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J. Physiol. (Lond.) 562, 223–234. Meijler, F.L., Jalife, J., 1999. Electrophysiology of the atrioventricular node during atrial fibrillation. I. Ventricular rhythm. Ned. Tijdschr. Geneeskd. 143, 1245–1252. Meijler, F.L., Janse, M.J., 1988. Morphology and electrophysiology of the mammalian atrioventricular node. Physiol. Rev. 68 (2), 608–647. Rigg, L., Terrar, D.A., 1996. Possible role of calcium release from the sarcoplasmic reticulum in pacemaking in guinea-pig sino-atrial node. Exp. Physiol. 81, 877–880. Tse, W.W., 1973. Evidence of presence of automatic fibers in the canine atrioventricular node. Am. J. Physiol. 225 (3), 716–723. Vinogradova, T., Maltsev, V., Bogdanov, K., Lyashkov, A.E., Lakatta, E., 2005. Rhythmic Ca2+ oscillations drive sinoatrial nodal cell pacemaker function to make the heart tick. Ann. N. Y. Acad. Sci. 1047, 138–156. Yamamoto, M., Honjo, H., Niwa, R., Kodama, I., 1998. Low-frequency extracellular potentials recorded from the sinoatrial node. Cardiovasc. Res. 39, 360–372. Yoo, S., Dobrzynski, H., Fedorov, V.V., Xu, S.-Z., Yamanushi, T.T., Jones, S.A., Yamamoto, M., Nikolski, V.P., Efimov, I.R., Boyett, M.R., 2006. Localization of Na+ channel isoforms at the atrioventricular junction and atrioventricular node in the rat. Circulation 114, 1360–1371. Yue, L., Feng, J., Li, G.R., Nattel, S., 1996. Characterization of an ultrarapid delayed rectifier potassium channel involved in canine atrial repolarization. J. Physiol. (Lond.) 496, 647–662.
Progress in Biophysics and Molecular Biology 96 (2008) 452–464 www.elsevier.com/locate/pbiomolbio
Review
Characterization of the effects of Ryanodine, TTX, E-4031 and 4-AP on the sinoatrial and atrioventricular nodes Mohammed R. Nikmarama,1, Jie Liuc,1, Mohamed Abdelrahmanc, Halina Dobrzynskib, Mark R. Boyettb, Ming Leib, a
Department of Physiology, Iran University of Medical Sciences, Tehran, Iran Cardiovascular Research Group, Division of Cardiovascular and Endocrine Sciences, University of Manchester, 46 Grafton Street, Manchester M13 9NT, UK c Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK
b
Available online 1 August 2007
Abstract Aims: To characterize the effects of inhibition of Ryanodine receptor (RyR), TTX-sensitive neuronal Na+ current (iNa), ‘‘rapidly activating’’ delayed rectifier K+ current (iKr) and ultrarapid delayed rectifier potassium current (IKur) on the pacemaker activity of the sinoatrial node (SAN) and the atrioventricular node (AVN) in the mouse. Methods: The structure of mouse AVN was studied by histology and immunolabelling of Cx43 and hyperpolarizationactivated, cyclic nucleotide-binding channels (HCN). The effects of Ryanodine, TTX, E-4031 and 4-AP on pacemaker activities recorded from mouse intact SAN and AVN preparations have been investigated. Results: Immuno-histological characterization delineated the structure of the AVN showing the similar molecular phenotype of the SAN. The effects of these inhibitors on the cycle length (CL) of the spontaneous pacemaker activity of the SAN and the AVN were characterized. Inhibition of RyR by 0.2 and 2 mM Ryanodine prolonged CL by 42712.3% and 64718.1% in SAN preparations by 163772.3% and 241791.2% in AVN preparations. Inhibition of TTX-sensitive iNa by 100 nM TTX prolonged CL by 2276.0% in SAN preparations and 53713.6% in the AVN preparations. Block of iKr by E-4031 prolonged CL by 68712.5% in SAN preparations and 2873.4% in AVN preparations. Inhibition of iKur by 50 mM 4-AP prolonged CL by 2073.4% in SAN preparations and 1873.0% in AVN preparations. Conclusion: Mouse SAN and AVN showed distinct different response to the inhibition of RyR, TTX-sensitive INa, IKr and iKur, which reflects the variation in contribution of these currents to the pacemaker function of the cardiac nodes in the mouse. Our data provide valuable information for developing virtual tissue models of mouse SAN and AVN. r 2007 Published by Elsevier Ltd. Keywords: Sinoatrial node; Atrioventricular node; Pacemaking; TTX-sensitive neuronal Na+ current (iNa); ‘‘Rapidly activating’’ delayed rectifier K+ current (iKr); Ultrarapid delayed rectifier potassium current (IKur)
Corresponding author. Tel.: +44 161 275 1194; fax: +44 161 275 1183. 1
E-mail address: [email protected] (M. Lei). Joint first authors.
0079-6107/$ - see front matter r 2007 Published by Elsevier Ltd. doi:10.1016/j.pbiomolbio.2007.07.003
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Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Electrical recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Statistics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Immuno-histological characterization of the AVN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Characterization of SAN and AVN spontaneous pacemaker activities . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Effect of Ryanodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Effect of TTX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Effect of E-4031 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Effect of 4-AP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Delineation of AV junction pacemaker structure by HCN4 and Cx43 . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Effect of Ryanodine, TTX, E-4031 and 4-AP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction There is an effort to build anatomically and biophysically detailed models of different regions of the heart. Such models require knowledge of anatomy, physiology, and molecular and cellular biology of the different regions. In mammalian heart, each heartbeat is normally initiated from the sinoatrial node (SAN), the primary pacemaker of the heart. The SAN contains special ‘‘pacemaker’’ cells generating the electrical signals that control the pace and rhythm of the heart. The signals travel from the SAN to the AVN. From the AVN, the signals are conducted along pathways and spread into the ventricles, causing them to contract and pump blood into the lungs and throughout the body. The function of the AVN is normally to act as a conduction pathway between the atria and ventricles under physiological condition, but it can also act as a major subsidiary pacemaker, initiating heartbeat by generating the junctional rhythm in the circumstance of failure of the SAN. Although all parts of cardiac conduction system have the capability of generating rhythmic heartbeat, i.e. the capacity of automaticity, such capacity varies regionally. Thus, the SAN has a highest automaticity; its discharge rate determines the heart rate that is normally within the physiological range (e.g. 60–120 beats/min in human). The automaticity of the AVN is lower than that of the SAN and it generates the junctional rhythm (sub-physiological range) having much slower rate than the sinus rhythm (e.g. o60 beats/min in human). The more down stream of the conduction system (i.e. Purkinje fibres) is, the lower automaticity has. The pacemaking mechanisms for cardiac conduction tissue, the SAN in particular, have been extensively investigated over the past two to three decades and have been largely defined (for reviews, see Irisawa et al., 1993; Boyett et al., 2000; Kleber and Rudy, 2004). The mechanism underlying the regional difference in automaticity within the conduction system, however, has been rarely explored. Recent studies suggest that the diversity in electrophysiology in different regions of the heart (e.g. SAN and AVN) is likely attributed to variable expression of ion channel gene products in different regions of the heart. For example, high-density real-time RT-PCR analysis suggested a specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart (Marionneau et al., 2005). Further detailed characterization of the
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functional roles of individual ion channel gene products in different parts of the conduction system will deepen our understanding of regional difference in automaticity of this system. In the present study, we have characterized the structure of the AVN studied by histology and immunolabelling of Cx43 and HCN channels. The effects of Ryanodine, TTX, E-4031 and 4-AP on pacemaker activity recorded from intact mouse SAN and AVN preparations have been investigated. From the results obtained, possible roles of RyR, TTX-sensitive neuronal Na+ current (iNa), ‘‘rapidly activating’’ delayed rectifier K+ current (iKr) and ultrarapid delayed rectifier potassium current (IKur) in mouse SAN and AVN have been assessed. Our data, therefore, provide valuable information for developing virtual tissue models of mouse SAN and AVN. 2. Materials and methods 2.1. Animals C57BL/6J mice either sex weighing 20–30 g (age, 10–12 weeks; from Charles River UK Ltd., Kent, UK) were used. Mice were killed by cervical dislocation and the heart quickly removed. All animal procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986. 2.2. Electrical recording The SAN and AVN preparations were isolated from the mouse heart as described previously (Lei et al., 2004). In brief, the right atrium was separated from the rest of the heart and opened by the longitudinal incision from the rest of the heart. SAN and AVN were separated by cutting through atrial tissue superior to the coronary sinus. The SAN preparation contains intercaval region and some surrounding atrial tissue. AVN preparation contains a region that includes the triangle of Koch. The nodal preparations (endocardial surface up) are fixed in a tissue bath and superfused with Tyrode solution with or without blocker at 32 1C at a rate of 4 ml/min through a heat exchanger. To record the extracellular signals, two modified bipolar electrodes (Yamamoto et al., 1998) were placed in the SAN and AVN regions separately. Thus, the extracellular potential and the firing activities (the rate and cycle length (CL, calculate by measuring R–R interval)) of the SAN and the AVN preparations were recorded simultaneously. 2.3. Solutions For electrical recording, the composition of the solution was as follows (in mM): 93 NaCl, 20 NaHCO3, 1 Na2HPO4, 5 KCl, 2 CaC12, 1 MgSO4, 20 sodium acetate, and 10 glucose. The solution was equilibrated with 95% Oz–5% CO2 to give a pH of 7.4. 2.4. Drugs E-4031 (1-[2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulphonylaminobenzoyl)-piperidine, Eisai Pharmaceuticals, Tokyo, Japan) and TTX (Tetrodotoxin, Sigma-Aldrich, Poole, UK) were dissolved in deionized water to make 10 mM stock solutions, and stored at 4 1C. Ryanodine (Sigma-Aldrich, Poole, UK). A stock solution of 1 M 4-aminopyridine (4-AP) (Sigma-Aldrich, Poole, UK) was prepared in deionized water, titrated with HCl to pH 7.4 and stored at 4 1C. 2.5. Histology Sections were stained with Masson’s trichrome to show histology; with this technique, connective tissue is stained blue, cardiac myocytes are stained red and nuclei are stained dark blue. Images of tissue sections were obtained using a Leica DC camera mounted on a Leica DMIRB inverted microscope in conjunction with Leica TWAIN software.
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2.6. Immunohistochemistry Immunohistochemistry was carried out using established methods as described previously (Lei et al., 2004). Briefly, sections were fixed in 10% formalin (Sigma) for 30 min and washed with 0.01 M phosphate buffer solution (PBS) three times at 10 min intervals. Sections were then permeabilized by incubating them in PBS containing 0.1% Triton X-100 for 30 min, after which they were washed with PBS and then blocked in 10% normal serum in PBS for 1 h at room temperature. Sections were incubated with the primary antibody (diluted in 1% bovine serum albumin, BSA) at 4 1C overnight, after which they were washed three times with PBS over 30 min. Sections were incubated with secondary antibodies for 1–2 h at room temperature. After washing three times in PBS, coverslips were mounted on the microscope slides and the coverslips were sealed with nail polish. Slides were stored in the dark at 4 1C. Immunolabelled tissue was viewed with a Leica TCS SP or Zeiss LSM 510 laser scanning confocal microscope equipped with argon and helium–neon lasers, which allowed excitation at 488 nm wavelength for the detection of FITC/Alex 488. All images presented are single optical sections. Images were saved and later processed using Corel Photo-Paint and Corel Draw software (Corel, Ottawa, Canada). 2.7. Antibodies Rabbit anti-HCN4 IgG raised against residues 119–155 of human HCN4 (1:100 dilution; Alomone Labs) and anti-Cx43 (connexin43) IgG raised against residues 363–382 of human Cx43 (1:1000 dilution; Sigma, Poole, UK). To detect the primary IgGs, goat anti-rabbit IgG conjugated to Alexa Fluro 488 (Molecular Probes, Eugene, USA), or donkey anti-rabbit IgG conjugated to FITC (Chemicon) were used. The specificity of anti-Cx43 (Dobrzynski et al., 2003) and anti-HCN4 (Dobrzynski et al., 2003) IgGs has been checked previously. No labelling above background was obtained when the primary or secondary antibodies were omitted (data not shown). 2.8. Statistics Where applicable, a Student t-test is applied. Data are presented as mean7S.E.M. (n ¼ number of preparations). Po0.05 is considered as significant. 3. Results 3.1. Immuno-histological characterization of the AVN We recently characterized the structure of mouse SAN by histology and immunolabelling of ANP, Cx43 and hyperpolarization-activated cyclic nucleotide-gated cation channels (HCNs) (Liu et al., 2006). We adapted the SAN approach for the AVN in the present study. Fig. 1A shows an example of histological features of the different regions along the conduction axis of AVN (posterior to anterior along the AV junction). Tissue sections cut perpendicular to the AV junction. Cardiac myocytes were stained purple, connective tissue was stained blue, cell nuclei were stained dark blue. The serial of tissue sections from the same preparation studied by histology were then immunolabelled with the two molecular markers, Cx43 and HCN4, used for delineation of SAN (Liu et al., 2006). As shown in Fig. 1B, HCN4 was used as a positive marker of pacemaker cells. In the serial tissue sections, HCN4 highlighted the AVN region that presumably has capacity to produce jnuctional rhythm. From the 0 mm section, HCN4-positive cells appeared as a small cluster of cells located at the ventricular septum above the tricuspid valve. The location and size of this AVN region was almost identical as those determined in their sister sections by histology. In the 0.4 mm section, the entire nodal region measured about 0.8 mm 0.4 mm, which included the compact AVN (showing as a bright knot) and the transitional region facing the atrial septum. The region of the compact node (boxed region in 0.4 mm section). Cells at the compact node were small and interwoven. To confirm the delineation by HCN4, a negative marker, Cx43, was applied to the same set of tissue sections. Fig. 1C shows the immunolabelling of Cx43. The region devoid of green fluorescence appeared larger than the HCN4-positive region in the sister
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Fig. 1. (Continued)
sections. There was connective tissue around the nodal region as shown in histological staining, the quantity of the connective tissue varied from the posterior to anterior region of the AVN. This connective tissue showed negative immunoreactivity for Cx43, thus the Cx43-negative region around AVN was composed of nodal region and the surrounding connective tissue. However, positive immunoreactivity of Cx43 was found specifically in the His bundle (boxed region in 0.8 mm). The positive immunoreactivity appeared as positive clusters and the size of which was smaller than those of the atrial or ventricular. The small Cx43 clusters were not found in the PNE, compact AVN, or bundle branches, but solely in the His bundle. Same results were obtained from four preparations. 3.2. Characterization of SAN and AVN spontaneous pacemaker activities The spontaneous firing rate of the SAN and the AVN were compared by extracellular potential recording. Fig. 2A shows a typical mouse SAN–AVN preparation for electrical extracellular potential recording. Fig. 1. Delineation of AVN. Serial sections cut perpendicular to the AV junction were stained by either histology, or anti-HCN4 or antiCx43. (A) Masson’s trichrome stained sections at different levels (0–1.2 mm) through the AVN. Broad arrows indicate the connective tissue separating AVN from ventricle, triangle arrows indicate connective tissue at the transitional region merging with atrium. PNE, posterior nodal extension; CN, compact node; CF, centre fibrous body; HB, His bundle; LBB, left bundle branch; RBB, right bundle branch; TV, tricuspid valve. (B) Anti-HCN4 immunostaining. Regions that were highlighted by green fluorescence labelled HCN4 antibody (within the red dotted lines) were the AVN region. White dashed lines indicate the tissue border. Boxed region in the compact node region (0.4 mm section) was magnified in the bottom as CN (compact node). (C) Anti-Cx43 immunostaining. Serial sections were stained with anti-Cx43 antibody. Regions devoid of green fluorescence labelled Cx43 antibody were indicated as the red dotted lines. Boxed region in the 0.8 mm section was magnified in at the bottom (HB). Scale ¼ 200 mm for all panels.
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Fig. 2. Recording of spontaneous electrical activities from SAN and AVN. (A) The opened right side of mouse heart was illustrated in with the SAN region and AVN region circled in red. (B) Two nodes are separated by cutting through the black dashed line and two electrodes are positioned in two nodal regions for extracellular potential recording. SVC, superior vena cava; SEP, interatrial septum; IVC, inferior vena cava; RA, right atrial appendage; CT, crista terminalis. (C) An example of spontaneous pacemaker activity simultaneously recorded from the SAN and the AVN preparations.
The non-separated tissue preparation showed single beating rhythm of rate of 300 bpm, presumably originating from the SAN. Once the preparation is separated into two parts (as indicated in Fig. 2C), two nodes showed different rhythms. The firing rate of AVN is much slower that of SAN. Fig. 2C shows examples of spontaneous activities recorded from SAN and AVN preparations simultaneously. After the two pieces of tissue stabilized for 430 min (to allow them to recover from the injury during dissection), the spontaneous firing rate of the SAN and AVN preparations measured as cycle length (CL) are 246.8735.9 ms (SAN, n ¼ 8) and 495.2761.8 ms (AVN, n ¼ 8), respectively. 3.3. Effect of Ryanodine First, Ryanodine, a selective inhibitor of RyR, was applied for characterization of the role of sarcoplasmic reticulum (SR) function in pacemaking of the SAN and the AVN. Fig. 3 shows the effect of Ryanodine. Ryanodine had been applied for 20–30 min to obtain a steady-state response. In SAN preparations, 0.2 and 2 mM Ryanodine prolonged CL by 47715.7% and 70721.8% from 247735.9 ms in the control condition to 356757.0 and 444782.7 ms in the presence of 0.2 and 2 mM Ryanodine, respectively (n ¼ 8) (Po0.05). In AVN preparations, the same doses of Ryanodine prolonged CL by 163772.3% and 241791.2% from 495761.8 ms in the control condition to 11727245.2 and 15727423.8 ms, respectively (n ¼ 8, Po0.01). Thus, Ryanodine slows down pacemaker activity of both SAN and AVN, but its effect was greater in the AVN than in the SAN. Ryanodine does not cease the spontaneous beating of either the SAN or the AVN preparations after even more than 1 h perfusion of the drug at 10 mM concentration. The effect of 2 mM Ryanodine on the SAN and the AVN was also examined in two additional species, rat and rabbit. Ryanodine caused an increase of CL of SAN rhythm by 40712.8% in rat preparations and by 3076.8% in rabbit preparations. The same dose Ryanodine caused an increase of CL of AVN rhythm by 186757.6% in rat preparations (n ¼ 9) and by
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Fig. 3. The effect of Ryanodine on electrical activities of SAN and the AVN. (A) CL of the SAN under control conditions and after application of 0.2 and 2 mM Ryanodine. (B) CL of the AVN under control conditions and after the application of 0.2 and 2 mM Ryanodine. (C) The percentage change in CL of SAN and AVN after application of 2 mM Ryanodine vs. control. Mean7S.E.M.s are shown. *Significantly different (Po0.05) from control. Mean value of CL for each preparation was averaged from 50 cycles.
65710.8% in rabbit preparations (n ¼ 6). Thus, the effects on the SAN and the AVN are consistent with that of the mouse. 3.4. Effect of TTX Second, the effect of TTX on the SAN and AVN was compared. The majority of Na+ channels in the heart are composed of the tetrodotoxin (TTX)-resistant (Kd, 2–6 mM) Nav1.5 isoform; however, recently it has been shown that TTX-sensitive (Kd, 1–10 nM) neuronal Na+ channel isoforms (Nav1.1, Nav1.3 and Nav1.6) are also present and functionally important in the myocytes of the ventricles and the SAN. In this study, 100 nM TTX, a dose that causes complete block of TTX-sensitive neuronal Na+ channel isoforms in single SAN cells (Lei et al., 2004), was applied to the SAN and the AVN preparations. TTX had been applied for 20–30 min to obtain a steady-state response. In SAN preparations, TTX prolonged CL by 2276.0% from 212713.5 ms in the control condition to 263729.2 ms in the presence of TTX (Fig. 4) (n ¼ 7, Po0.05). In AVN preparations, while the same dose of TTX prolonged CL by 53713.6% from 578768.2 ms in the control condition to 856771.8 ms in the presence of TTX (Fig. 4) (n ¼ 5, Po0.005). 3.5. Effect of E-4031 Third, the effect of E-4031 on the SAN and the AVN was then examined. A ‘‘rapidly activating’’ delayed rectifier K+ current (IKr) has been previously characterized in mouse SAN pacemaker cells (Cho et al., 2003; Clark et al., 2004). In the present study, we compared the blocking effect of 1 mM E-4031, a selective block of IKr, on pacemaker activity of the SAN and the AVN. In SAN preparations, 1 mM E-4031 increased CL by 68712.5% from 215711.9 ms in the control condition to 360733.8 ms in the presence of E-4031 (Fig. 5) (n ¼ 6, Po0.005). In AVN preparations, the same dose of E-4031 increases in CL by 2873.4% from 452720 ms in control condition to 580735.8 ms in the presence of E-4031 (Fig. 5) (n ¼ 6, Po0.005). Thus, the block effect of E-4031 is greater in the SAN than in the AVN. 3.6. Effect of 4-AP Finally, the effect of 4-AP on the SAN and the AVN was studied. Ultrarapid delayed rectifier potassium current (IKur) was first identified in atrial myocytes (Feng et al., 1997). Its functional role has not been
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Fig. 4. The effect of 100 nM TTX on electrical activities of SAN and the AVN. (A) CL of the SAN under control conditions and after application of TTX. (B) CL of the AVN under control conditions and after the application of TTX. (C) The percentage change in CL of SAN and AVN after application of TTX vs. control. Mean7S.E.M.s are shown. *Significantly different (Po0.05) from control. Mean value of CL for each preparation was averaged from 50 cycles.
Fig. 5. The effect of 1 mM E-4031 on electrical activities of SAN and the AVN. (A) CL of the SAN under control conditions and after application of E-4031. (B) CL of the AVN under control conditions and after the application of E-4031. (C) The percentage change in CL of SAN and AVN after application of E-4031 vs. control. Mean7S.E.M.s are shown. *Significantly different (Po0.05) from control. Mean value of CL for each preparation was averaged from 50 cycles.
previously investigated in the SAN and the AVN. The effect 50 mM 4-AP, a concentration that blocks IKur, on pacemaker activity of SAN and AVN was then examined. In SAN preparations, 50 mM 4-AP prolonged CL by 2073.4% from 272725.0 ms in the control condition to 328730.9 ms in the presence of 4-AP (Fig. 6) (n ¼ 5, Po0.005). In AVN preparations, while the same dose of 4-AP prolonged CL by 1873.0% from 480722.9 ms in the control condition to 566730.8 ms in the presence of 1873.0% (Fig. 6) (n ¼ 5, Po0.005). Therefore, there is no significant difference of the effect of 4-AP on the SAN and the AVN.
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Fig. 6. The effect of 50 mM 4-AP on electrical activities of SAN and the AVN. (A) CL of the SAN under control conditions and after application of 4-AP. (B) CL of the AVN under control conditions and after the application of 4-AP. (C) The percentage change in CL of SAN and AVN after application of 4-AP vs. control. Mean7S.E.M.s are shown. *Significantly different (Po0.05) from control. Mean value of CL for each preparation was averaged from 50 cycles.
4. Discussion The main findings of this study are: (i) immuno-histological characterization of A–V junction revealed the structure of the AV junction pacemaker of the mouse; (ii) mouse SAN and AVN show different response to the inhibition of RyR, TTX-sensitive iNa, iKr and IKur, thus, the effects of Ryanodine and TTX are greater on the AVN, while, in contrast, the effect of E-4031 is greater on the SAN, while 4-AP has a similar effect on both nodes. 4.1. Delineation of AV junction pacemaker structure by HCN4 and Cx43 The study delineated the mouse AV junction pacemaker structure with histology and immunolabelling. Based on immunolabelling of HCN4 and Cx43, different cell types were identified in and around the AVN. Comparison of Fig. 1A–C shows that myocytes that expressed Cx43 did not express HCN4, whereas the myocytes that did not express Cx43 express HCN4. The Cx43-positive/HCN4-negative tissue is assumed to be atrial muscle. The Cx43-negative/HCN4-positive tissue is assumed to be AVN tissue with pacemaker generation potential. Cx43-negative/HCN4-positive cells were found throughout the posterior nodal extension (PNE), AVN, His bundle and bundle branches without significant difference in expression level. Previous functional studies showed the site of initial pacemaker in the AVN was found at the compact AVN, the NH region or the PNE region (Meijler and Jalife, 1999; Dobrzynski et al., 2003; Efimov et al., 2004; Meijler and Janse, 1988). The molecular basis for particular region to act as the leading pacemaker site in A–V junctional region is still in controversy. Moreover, when the atrial fibres were peeled off, pacemaking depolarizations were presented in all regions of AVN with the steepest ones located at the middle or lower nodal regions (Tse, 1973). It was suggested that, in the intact preparation, atrial myocardium might shrink pacemaker activity in the upper node by electronic interaction with the nodal cells. In this study, intermingling of nodal cells (HCN4-positive/Cx43-negative cells) and atrial myocytes (HCN4-negative/Cx43-positive cells) were also found in the upper nodal region where the AVN meet atrium. The interdigitation of AVN and atrium provides the structural substrate for this kind of electrical interaction at this region. 4.2. Effect of Ryanodine, TTX, E-4031 and 4-AP Application of Ryanodine (0.2–2 mM), TTX (100 nM), E-4031 (1 mM) and 4-AP (50 mM) revealed the regional difference in responding to the inhibition of SR function, TTX-sensitive iNa, iKr and IKur in mouse
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conduction system. Firstly, the effect of Ryanodine on the SAN was characterized. Recent studies have suggested that the SAN pacemaker activity is also influenced by transient changes in cytosolic Ca2+ (Vinogradova et al., 2005), the disruption of SR function causes a slowing of spontaneous pacemaker activity in isolated SAN cells (Huser et al., 2000; Bogdanov et al., 2001; Lancaster et al., 2004; Vinogradova et al., 2005) and intact SAN preparations. As shown in Fig. 3, 0.2 and 2 mM of Ryanodine prolonged CL by 46% and 70% in SAN preparations, whereas the same doses of Ryanodine caused a much greater prolongation of CL in AVN preparations (by 163% and 241%, respectively). A high dose of Ryanodine (10 mM) did not cause cessation of spontaneous activity of both SAN and AVN preparations. Thus, it appears that SR calcium release is a significant modulator, but not an essential player to pacemaking of cardiac nodes, its effect is also greater in the AVN than in the SAN. Previous studies on single SAN cells (guinea-pig) (Bassani et al., 1997) and intact SAN preparations of the SAN (rabbit) (Honjo et al., 2003), and isolated right atria (dog, rat) (Lipsius, 1987; Bassani et al., 1999) have shown that Ryanodine caused a modest slowing of SAN pacemaker activity. High concentrations (30 mM) did not cause cessation of spontaneous activity (rabbit) (Honjo et al., 2003) nor did cyclopiazonic acid (guinea-pig) (Rigg and Terrar, 1996), thapsigargin (rat) (Bassani et al., 1997) by disruption of SR function by these manoeuvres. Thus, our result is principally in consistent with the previous studies on single cell and isolated SAN preparations (Bassani et al., 1997; Honjo et al., 2003; Rigg and Terrar, 1996). The different response of the SAN and the AVN to Ryanodine (greater in the AVN) is likely due to the difference in the intracellular calcium regulation between these regions. For example, the level of the transcripts for type 2 Ryanodine receptor, calcium pump (SERCA) is much higher in the AVN than that in the SAN in the mouse (Marionneau et al., 2005). The higher density of RyR2 and SERCA may reflect a greater role of SR function in the AVN than in the AVN. Secondly, the effect of 100 nM TTX (a dose that causes complete block of TTX-sensitive neuronal Na+ channel isoforms in single SAN cells) was studied. 100 nM TTX prolonged CL in SAN preparations by 22%, while the same dose of TTX caused a much greater prolongation in CL in AVN preparations (53%), which suggests a greater role of TTX-sensitive INa in the AVN than in the SAN. Recent studies by ourselves and others have suggested complex yet specific regional distributions of Na+ channel isoforms in the cardiac conduction system particularly through the SAN and AV junction (Lei et al., 2004; Maier et al., 2003). Thus, there is a co-expression of multiple neuronal and cardiac Na+ channel isoforms in the heart (Maier et al., 2002). Both ourselves (Lei et al., 2004) and Maier et al. (2003) have confirmed that Nav1.1 is uniformly expressed across the SAN; our own recent work also demonstrated a complex distribution of Nav1.5, which was not expressed in its intercaval, central, region but which did occur in peripheral SAN, on the endocardial face of the crista terminalis, thereby explaining electrophysiological findings of relatively fast conduction in peripheral SAN (Lei et al., 2004). In the AVN (rat), both Nav1.1 and Nav1.5 were distributed in a similar manner in the AV junction regions (Yoo et al., 2006). The distinct different response of SAN and AVN to inhibition of TTX-sensitive INa is likely due to difference in expression of neuronal type Na+ channels (presumably Nav1.1 channels) in these two regions, quantitative analysis indicated that the expression of Nav1.1 transcript is much higher in the AVN than in the SAN in mouse heart (8–10:1) (Marionneau et al., 2005). Thirdly, the effects of inhibition of two repolarizating currents, IKr and iKur, by E-4031 and 4-AP were examined on pacemaker activity of mouse SAN and AVN. The data show that, in contrast to the effect of Ryanodine and TTX, the effect of E-4031 on pacemaker activity is greater in the SAN than in the AVN, while 4-AP has similar effect on both nodes. 1 mM E-4031 significantly decrease in firing rate in SAN preparations (68%) and in AVN preparations (28%). In isolated SAN cells, Clark et al. (2004) found that in isolated mouse SAN cells, outward currents evoked by depolarizing steps (greater than 40 mV) were strongly inhibited by 1 mM E-4031. 1 mM E-4031 slowed the spontaneous pacing rate of Langendorff-perfused, isolated adult mouse hearts by an average of 36.5% Cho et al. (2003) also reported that E4031-sensitive, rapidly activating delayed rectifier K+ current (IKr) was activated by depolarization with the amplitude of 38.3 pA at 0 mV. However, the chromanol 293B-sensitive, slowly activating delayed rectifier K+ current (IKs) was not present (Cho et al., 2003). Therefore, these studies suggest that IKr is the major, perhaps the only channel, contributes to the delayed rectifier K+ current in mouse SAN pacemaker cells. The previous study on profiles of ion channel transcripts of mouse hearts indicated that the levels of the transcripts (mERG) for IKr is similar in both regions (Marionneau et al., 2005). Therefore, it is unlikely that the functional discrepancy of IKr in two nodes is related
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to the expression of the channels. Whether such discrepancy is the result of different basal rate between the SAN and the AVN requires a further investigation. We could speculate that the effect of blocking of IKr in greater in higher basal rate in the case of the SAN. Clearly, such rate-dependent effect needs to be examined in isolated single cells. The effect of 50 mM 4-AP suggests a role of iKur in mouse SAN and AVN. A decade ago, Yue et al. (1996) first reported iKur from dog atrial myocytes. It showed very rapid activation and deactivation. The current was insensitive to E-4031, dendrotoxin and chloride substitution, but was inhibited by barium, with an Kd50 of 1.65 mM. Single-channel activity was strongly inhibited by 50 mM 4-AP or 10 mM TEA. Both 4-AP and TEA decreased open time, suggesting open-channel block. Selective inhibition of IKur with 50 mM 4-AP prolonged canine atrial action potentials, indicating that IKur contributes to canine atrial repolarization (Yue et al., 1996). This current, however, has not been characterized in cardiac pacemaker cells. Profiling ion channel distribution in mouse heart, Kv1.5, the transcript for IKur channel, has been identified in both SAN and AVN regions. Our data showed that 50 mM 4-AP slowed firing rate of the SAN preparations and the AVN preparations equally (20% and 18%) (Fig. 6). Thus, our data indicate a possible presence and functional role of iKur in mouse SAN and AVN. The previous study on profiles of ion channel transcripts of mouse hearts indicated that the levels of the transcripts (mERG and Kv1.5) for IKr and IKur are similar in two nodes (Marionneau et al., 2005). This may explain the similar effect of 4-AP on two nodes. In conclusion, mouse SAN and AVN showed the different response to the inhibition of RyR, TTX-sensitive INa, IKr and iKur, which reflects the variation in contribution of these currents to the pacemaker function of the SAN and the AVN in the mouse.
Acknowledgements This work was supported by Iran University of Medical Sciences, the Wellcome Trust and the British Heart Foundation.
References Bassani, R.A., Bassani, J.W., Lipsius, S.L., Bers, D.M., 1997. Diastolic SR Ca efflux in atrial pacemaker cells and Ca-overloaded myocytes. Am. J. Physiol. 273, H886–H892. Bassani, J.W., Godoy, C.M., Bassani, R.A., 1999. Effect of ryanodine on sinus node recovery time determined in vitro. Braz. J. Med. Biol. Res. 32, 1039–1043. Bogdanov, K.Y., Vinogradova, T.M., Lakatta, E.G., 2001. Sinoatrial nodal cell ryanodine receptor and Na+-Ca2+ exchanger: molecular partners in pacemaker regulation. Circ. Res. 88, 1254–1258. Boyett, M.R., Honjo, H., Kodama, I., 2000. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc. Res. 47, 658–687. Cho, H.-S., Takano, M., Noma, A., 2003. The electrophysiological properties of spontaneously beating pacemaker cells isolated from mouse sinoatrial node. J. Physiol. (Lond.) 550, 169–180. Clark, R.B., Mangoni, M.E., Lueger, A., Couette, B., Nargeot, J., Giles, W.R., 2004. A rapidly activating delayed rectifier K+ current regulates pacemaker activity in adult mouse sinoatrial node cells. Am. J. Physiol. 286, H1757–H1766. Dobrzynski, H., Nikolski, V.P., Sambelashvili, A.T., Greener, I.D., Yamamoto, M., Boyett, M.R., Efimov, I.R., 2003. Site of origin and molecular substrate of atrioventricular junctional rhythm in the rabbit heart. Circ. Res. 93, 1102–1110. Efimov, I.R., Nikolski, V.P., Rothenberg, F., Greener, I.D., Li, J., Dobrzynski, H., Boyett, M., 2004. Structure–function relationship in the AV junction. Anat. Rec. A Discov. Mol. Cell Evol. Biol. 280 (2), 952–965. Feng, J., Wible, B., Li, G.R., Wang, Z., Nattel, S., 1997. Antisense oligodeoxynucleotides directed against Kv1.5 mRNA specifically inhibit ultrarapid delayed rectifier K+ current in cultured adult human atrial myocytes. Circ. Res. 80, 572–579. Honjo, H., Inada, S., Lancaster, M.K., Yamamoto, M., Niwa, R., Jones, S.A., Shibata, N., Mitsui, K., Horiuchi, T., Kamiya, K., Kodama, I., Boyett, M.R., 2003. Sarcoplasmic reticulum Ca2+ release is not a dominating factor in sinoatrial node pacemaker activity. Circ. Res. 92, e41–e44. Huser, J., Blatter, L.A., Lipsius, S.L., 2000. Intracellular Ca2+ release contributes to automaticity in cat atrial pacemaker cells. J. Physiol. (Lond.) 524 (Pt. 2), 415–422. Irisawa, H., Brown, H.F., Giles, W., 1993. Cardiac pacemaking in the sinoatrial node. Physiol. Rev. 73, 197–227. Kleber, A.G., Rudy, Y., 2004. Basic mechanisms of cardiac impulse propagation and associated arrhythmias. Physiol. Rev. 84, 431–488. Lancaster, M.K., Jones, S.A., Harrison, S.M., Boyett, M.R., 2004. Intracellular Ca2+ and pacemaking within the rabbit sinoatrial node: heterogeneity of role and control. J. Physiol. (Lond.) 556, 481–494.
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Lei, M., Jones, S.A., Liu, J., Lancaster, M.K., Fung, S.S.-M., Dobrzynski, H., Camelliti, P., Maier, S.K.G., Noble, D., Boyett, M.R., 2004. Requirement of neuronal- and cardiac-type sodium channels for murine sinoatrial node pacemaking. J. Physiol. (Lond.) 559, 835–848. Lipsius, S.L., 1987. Triggered rhythms in atrial muscle. J. Electrocardiol. 20, 33–37. Liu, J., Dobrzynski, H., Abdelrahman, M., Boyett, M., Lei, M., 2006. Organization of the mouse sinoatrial node: structure and expression of HCNs. Cardiovasc. Res. 73, 729. Maier, S.K., Westenbroek, R.E., Schenkman, K.A., Feigl, E.O., Scheuer, T., Catterall, W.A., 2002. An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart. Proc. Natl. Acad. Sci. USA 99, 4073–4078. Maier, S.K., Westenbroek, R.E., Yamanushi, T.T., Dobrzynski, H., Boyett, M.R., Catterall, W.A., Scheuer, T., 2003. An unexpected requirement for brain-type sodium channels for control of heart rate in the mouse sinoatrial node. Proc. Natl. Acad. Sci. USA 100, 3507–3512. Marionneau, C., Couette, B., Liu, J., Li, H., Mangoni, M.E., Nargeot, J., Lei, M., Escande, D., Demolombe, S., 2005. Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart. J. Physiol. (Lond.) 562, 223–234. Meijler, F.L., Jalife, J., 1999. Electrophysiology of the atrioventricular node during atrial fibrillation. I. Ventricular rhythm. Ned. Tijdschr. Geneeskd. 143, 1245–1252. Meijler, F.L., Janse, M.J., 1988. Morphology and electrophysiology of the mammalian atrioventricular node. Physiol. Rev. 68 (2), 608–647. Rigg, L., Terrar, D.A., 1996. Possible role of calcium release from the sarcoplasmic reticulum in pacemaking in guinea-pig sino-atrial node. Exp. Physiol. 81, 877–880. Tse, W.W., 1973. Evidence of presence of automatic fibers in the canine atrioventricular node. Am. J. Physiol. 225 (3), 716–723. Vinogradova, T., Maltsev, V., Bogdanov, K., Lyashkov, A.E., Lakatta, E., 2005. Rhythmic Ca2+ oscillations drive sinoatrial nodal cell pacemaker function to make the heart tick. Ann. N. Y. Acad. Sci. 1047, 138–156. Yamamoto, M., Honjo, H., Niwa, R., Kodama, I., 1998. Low-frequency extracellular potentials recorded from the sinoatrial node. Cardiovasc. Res. 39, 360–372. Yoo, S., Dobrzynski, H., Fedorov, V.V., Xu, S.-Z., Yamanushi, T.T., Jones, S.A., Yamamoto, M., Nikolski, V.P., Efimov, I.R., Boyett, M.R., 2006. Localization of Na+ channel isoforms at the atrioventricular junction and atrioventricular node in the rat. Circulation 114, 1360–1371. Yue, L., Feng, J., Li, G.R., Nattel, S., 1996. Characterization of an ultrarapid delayed rectifier potassium channel involved in canine atrial repolarization. J. Physiol. (Lond.) 496, 647–662.