Published online before print December 20, 2007, doi: 10.1161/CIRCRESAHA.107.161778
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© 2008 American Heart Association, Inc.
Cellular Biology |
Functional Roles of a Ca2+-Activated K+ Channel in Atrioventricular Nodes
From the Division of Cardiovascular Medicine (Q.Z., V.T., L.L., N.L., A.S., M.K.L., N.C.), Department of Internal Medicine, University of California, Davis, Calif; Department of Veterans Affairs (N.C.), Northern California Health Care System, Mather, Calif; Vollum Institute (C.T.B., J.P.A.), Oregon Health and Science University, Portland; and Department of Physiology (Q.Z.), University of Zhengzhou, Henan, People’s Republic of China.
Correspondence to Nipavan Chiamvimonvat, Division of Cardiovascular Medicine, University of California, One Shields Ave, GBSF 6315, Davis, CA 95616. E-mail nchiamvimonvat{at}ucdavis.edu
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Abstract |
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Since the first description of the anatomical atrioventricular nodes (AVNs), a large number of studies have provided insights into the heterogeneity of the structure as well as a repertoire of ion channel proteins that govern this complex conduction pathway between the atria and ventricles. These studies have revealed the intricate organization of multiple nodal and nodal-like myocytes contributing to the unique electrophysiology of the AVN in health and diseases. On the other hand, information regarding the contribution of specific ion channels to the function of the AVN remains incomplete. We reason that the identification of AVN-specific ion channels may provide a more direct and rational design of therapeutic target in the control of AVN conduction in atrial flutter/fibrillation, one of the most common arrhythmias seen clinically. In this study, we took advantage of 2 genetically altered mouse models with overexpression or null mutation of 1 of a small conductance Ca2+-activated K+ channel isoform, SK2 channel, and demonstrated robust phenotypes of AVN dysfunction in these experimental models. Overexpression of SK2 channels results in the shortening of the spontaneous action potentials of the AVN cells and an increase in the firing frequency. On the other hand, ablation of the SK2 channel results in the opposite effects on the spontaneous action potentials of the AVN. Furthermore, we directly documented the expression of SK2 channel in mouse AVN using multiple techniques. The new insights may have important implications in providing novel drug targets for the modification of AVN conduction in the treatment of atrial arrhythmias.
Key Words: KCa2.2 channel • SK2 channel • atrioventricular nodes
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Introduction |
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The atrioventricular node (AVN) is a highly specialized pacemaking tissue located at the junction of the right atrium and ventricle. Indeed, it is the only electrical connection between atria and ventricles and provides the critical delay between atrial and ventricular contraction to allow for proper atrial emptying before the start of the ventricular contraction. Pharmacological slowing of impulses across AVN is widely used clinically in atrial flutter/fibrillation to ensure physiological ventricular responses in these conditions. Previous studies have identified the roles of several distinct ion channels in the AVN function,1–5 and recent work has begun to assemble an array of ion channel genes in the pacemaking tissues.6 On the other hand, information regarding contribution of specific ion channels to the function of the AVN remains incomplete. We reason that the identification of AVN-specific ion channels may provide a more direct and rational design of therapeutic target in the control of AVN conduction in atrial flutter/fibrillation, one of the most common arrhythmias seen clinically.
Specifically, we have recently identified several isoforms of Ca2+-activated K+ channels (KCa) in human and mouse cardiac myocytes that we have shown to be critical in sculpting the duration of the cardiac action potential (AP).7,8 KCa channels are highly expressed in atrial compared with ventricular tissues. Moreover, not only does the current play important functional roles in mouse atrial myocytes, it also contributes significantly to the repolarization process in human atria.7,8 KCa channels are present in a wide variety of cells, where they integrate changes in intracellular Ca2+ concentration [Ca2+i] with changes in K+ conductance and membrane potential.9,10 KCa channels can be divided into 3 main subfamilies: the large-conductance Ca2+- and voltage-activated K+ channels (BK, KCa1), the intermediate-conductance Ca2+-activated K+ channels (IK, KCa3), and the small-conductance Ca2+-activated K+ channels (SK or KCa2).9–13 SK channels are encoded by at least 3 distinct genes, namely KCNN1 (SK1), KCNN2 (SK2), and KCNN3 (SK3).9,10,13
Here, we directly document the robust expression of SK2 channel in mouse AVN. Moreover, using genetically altered mouse models with overexpression or null mutation of 1 of the KCa isoforms, SK2 channel, we demonstrate significant changes in the AVN function. The new insights into the functional roles of SK2 channel in AVN may have important implications in providing novel drug targets for the modification of AVN conduction in the treatment of atrial arrhythmias.
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Materials and Methods |
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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
SK2-Null Mutant Mice and Transgenic Mice Overexpressing SK2 Channels
SK2-null mutant (SK2+/
) mice were generated as described previously.14 Transgenic mice overexpressing SK2 channels (SK2+/T) were developed via insertion of a tetracycline-regulatory cassette into the SK2 locus as described previously.15,16 Compared with wild-type (WT) littermate mice, in the absence of doxycycline, the SK2 protein and SK2 mRNA are overexpressed in heterozygotes (SK2+/T) 
10- and 4.5-folds as shown by Western blot and quantitative PCR, respectively. Because the transgenic animals were generated by site-specific insertion of a tetracycline-regulatory cassette into the 5' untranslated region of the SK2 gene, the SK2 channels could be overexpressed without interfering with the normal profile of SK2 expression. Both SK2-null and transgenic mouse lines were backcrossed more than 7 generations onto the C57Bl/6J background.
AVN Recordings
AVN preparation was prepared as described previously.1,6 AVN region was recognized by its anatomic landmarks.
APs were recorded from isolated AV nodal preparations using microelectrode techniques with 3 mol/L KCl microelectrodes at 33°C as described previously.17
Single AVN cells were isolated from WT and mutant mice as described previously with some modification.1,17–19 Whole-cell Ca2+-activated K+ current (IK,Ca) was recorded from single AVN cells at room temperature using patch-clamp techniques as described previously.7,20
ECG Recordings
ECG recordings were obtained at 33°C using Bioamplifier (BMA 831, CWE Inc, Ardmore, Pa) as described previously.17
Immunofluorescence Confocal Microscopy and Immunohistochemistry
Immunofluorescence labeling was performed as described previously.7 Immunohistochemistry and antibodies used are described in the online data supplement.
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Results |
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SK2+/T and SK2+/
Mice Show Evidence of Sinoatrial Node and AVN DysfunctionWe documented robust phenotypes of sinoatrial node (SAN) and AVN dysfunction in the SK2+/T and SK2+/
mice. Figure 1A shows ECG recordings from SK2+/T and SK2+/ mice compared with WT animals illustrating significant sinus bradycardia with prolongation of the PR intervals in SK2+/ mice compared with WT animals. In contrast, SK2+/T mice show significant shortening of the RR and PR intervals. Figure 1B further illustrates examples of ECG recordings in SK2/ mice showing complete AV block with AV dissociation. Summary data for PR intervals are shown in Figure 1C (n=10 from each group, *P<0.05). These differences in the RR and PR intervals may represent intrinsic abnormalities in the pacemaking activities in SA and AV nodal cells and/or the His–Purkinje system, respectively, in genetically targeted mice. However, alternative possibilities include altered autonomic input into the SAN and AVN resulting from the overexpression or deletion of the SK2 channel. To rule out this latter possibility, we recorded ECG in the WT and mutant mice using combined intraperitoneal injection of atropine (1 mg/kg) and propranolol (20 mg/kg) at concentrations shown previously to abolish autonomic control of the heart.21,22 Results are presented in Figure 1D. Administration of atropine and propranolol resulted in the prolongation of the PR intervals in the SK2+/T and SK2+/ mice as well as WT animals. More importantly, significant abnormalities in the AVN in SK2+/T and SK2+/ animals remain after treatment with atropine and propranolol compared with the WT controls, suggesting that the defects observed are intrinsic to the pacemaking tissues. On the other hand, the prolongation of the PR interval may be related to either AV node and/or the His–Purkinje system. Moreover, it is well known that the specific conductance-mediated influences on spontaneous pacemaker activity are highly rate-dependent. To further assess the effects of SK2 channels on AV nodes at constant rates, we performed in vivo electrophysiologic studies as described in online data supplement. Figure I in the online data supplement shows the prolongation of the AV node refractory period in SK2+/ and SK2/ compared with WT littermates, consistent with the prolongation of the PR interval as described above.
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To further document whether there is significant alteration within the AVN among the different genetically altered mouse models, we evaluated the spontaneous AP characteristics from isolated AV node preparations.
AVNs Isolated From SK2+/T Mice Show an Increase in the Rate of Firing, Whereas the Opposite Findings Were Observed in SK2+/ Mice
Spontaneous APs were recorded from isolated intact AVN preparation using microelectrode techniques at 33°C to 34°C. Figure 2A shows a photomicrograph of an isolated mouse AVN illustrating the important landmarks used in the identification of the AVN region as described previously.1 Representative spontaneous APs recorded from the regions within the AVN are shown in Figure 2B comparing WT, SK2+/T, and SK2+/. Specifically, APs recorded from within the AVN can be identified by the presence of the slow diastolic depolarization and a very slow upstroke of phase 0. SK2+/T mice show a significant increase in the spontaneous activities of the AVNs compared with age-matched WT controls (Figure 2B and 2C). In contrast, SK2+/ mice show a significant decrease in the firing frequency of the AVNs compared with the age-matched WT controls. Indeed, data obtained from isolated AVN preparations are consistent with the findings from in vivo measurement, as presented in Figure 1, further supporting the notion that the abnormalities observed are intrinsic to the AVN. Moreover, application of apamin (500 pmol/L, a specific blocker of SK2 channel) resulted in a significant decrease in the frequency of firing consistent with data obtained from the SK2+/ mice (see supplemental Figure II).
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Figure 2C shows summary data of cycle length (CL) in ms, the maximum diastolic potential, AP amplitude, maximum upstroke velocity (Vmax), rate of diastolic depolarization (DDR), and AP duration at 50% and 80% repolarization (APD50, APD80). Detailed analysis of the spontaneous AP reveals significant changes in the CL, APD, and DDR in the SK2+/T and SK2+/ compared with the age-matched WT control. Overexpression of the SK2 channel in SK2+/T mice results in a significant shortening of the APD80 compared with WT, whereas APD50 and APD80 were significantly prolonged in SK2+/ mice compared with WT mice. Moreover, SK2+/T mice show a significant increase in the DDR and a corresponding decrease in the CL. The opposite effects were observed in the SK2 knockout mice. There was also a decrease in Vmax in the SK2+/ mice.
Whole-Cell IK,Ca Recorded From Isolated AVN Cells
To document that there are differences in the expression of the SK2 channels in SK2+/T and SK2+/ mice, we directly assessed the SK2 current density measured as apamin-sensitive IK,Ca in single isolated AVN cells from transgenic and knockout animals compared with age-matched WT controls. Shown in Figure 3A are examples of the whole-cell current density elicited from a holding potential of –55 mV to various voltage steps, as shown at baseline and after application of apamin (500 pmol/L). Apamin-sensitive currents were obtained using digital subtraction and are shown to the right. Summary data in Figure 3B show a significant increase in the apamin-sensitive current density in AVN cells isolated from SK2+/T and a significant decrease in the current density in the SK2+/ mice compared with WT littermates.
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Immunohistochemistry and Immunofluorescence Confocal Microscopy
We performed immunofluorescence confocal laser-scanning microscopy using isolated single AVN cells to document the expression of the SK2 channel in AVN as shown in Figure 4. Figure 4A shows bright-field images of single isolated AVN cells (Figure 4Aa and 4Ab) compared with atrial (Figure 4Ac) and ventricular myocytes (Figure 4Ad). In Figure 4B and 4D, Cav3.1 and neurofilament (NF)-160 were further used as specific AVN cell markers. It has been demonstrated previously that NF-160 is a marker of the pacemaker and conduction system.23–25 Cav3.1 was only expressed in AVN cells (Figure 4Ba) and not atrial or ventricular myocytes (Figure 4Bc and 4Bd), consistent with previously published literature.5 Figure 4C and 4D illustrates positive SK2 staining in AVN (Figure 4Ca and 4Da), as well as atrial (Figure 4Cd and 4Dd) and ventricular myocytes (Figure 4Ce and 4De). In contrast, NF-160 was only expressed in AVN (Figure 4Da), further documenting the specific cell types used in our study. To further document the specificity of the anti-SK2 antibody used in our study, we performed control experiments as shown in Figure 4Cb, 4Cc, 4Db, and 4Dc. Anti-SK2 antibody was preincubated with antigenic peptide which eliminated the positive staining (Figure 4Cb and 4Db). Figure 4Cc and 4Dc shows lack of staining with secondary antibodies only. Finally, experiments were repeated using homozygous null mutant mice (SK2/) showing lack of staining in the atrial myocytes (Figure 4Cf).
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Immunohistochemistry of Histologic Section Through Mouse AVN
Expression of SK2 channel protein in the mouse AVN was further evaluated using immunostaining of cardiac section through AVN. Figure 5A and 5A' shows photomicrographs of histologic sections of AVN and working myocardium from WT mice at low and high magnification using hematoxylin/eosin. Masson’s trichrome (Figure 5B and B') was used to stain fibrous tissue to view the AVN, demonstrating compact node cells next to the central fibrous body and the ventricular septum. Expression of SK2 protein in the AVN and working myocardium in WT animals was documented as peroxidase-positive staining (Figure 5C and C'). Absence of SK2 protein expression in the AVN and working myocardium in SK2/ mice is shown in Figure 5D and 5D'. Treatment with secondary antibody only was used as negative control (Figure 5E and E').
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Discussion |
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The 2 gene-targeted mouse models used in our study with overexpression and knockout of SK2 channel offer us the unique opportunities to directly test the functional roles of SK2 channel in the whole animals and isolated AVN preparations, as well as single isolated AVN cells. Moreover, the 2 mouse models were backcrossed onto the same genetic background, allowing for the direct comparison between these 2 models. Indeed, we documented robust phenotypes of SAN and AVN abnormalities in the 2 mouse models, consistent with a critical role of SK2 channel in the pacemaking activities of both SAN and AVN. Here, we have focused our efforts on the AVN and have documented the existence of SK2 channel in AVN cells using multiple techniques. Overexpression of SK2 channels results in the shortening of the APs of the AVN cells associated with an increase in the firing frequency. On the other hand, SK2 channel knockout results in the opposite effects on the spontaneous APs of the AVN. Specifically, SK2+/T mice show a significant increase in the DDR and a corresponding decrease in the CL. The opposite effects were observed in the SK2 knockout mice. There was also a decrease in Vmax in the SK2+/
mice, and the finding may result from changes in the Ca2+ current. Indeed, we have documented previously that SK2 channels are functionally coupled to L-type Ca2+ channels.26 However, detailed analyses of the Ca2+ current in these mouse models are beyond the scope of the present study.
AVN Electrophysiology
Since the first description of the anatomical AVN by Tawara in 1906,27 a large number of studies have provided insights into the heterogeneity of the structure as well as a repertoire of ion channel proteins that govern this complex conduction pathway between the atria and ventricles.1–5,28–32 These studies have revealed the intricate organization of multiple nodal and nodal-like myocytes contributing to the unique electrophysiology of the AVN in health and diseases, including slow conduction and dual pathways of the AVN. Indeed, reentrant tachycardia within the AVN represents the most common type of paroxysmal supraventricular tachycardia in patient population. In addition, previous studies have documented several distinct ion channel proteins that contribute to the pacemaking nature of the AVN as well as distribution of the specific connexin within the AVN structure.1–6,30,31 For example, we and others have documented previously the important roles of Cav1.3, in addition to Cav1.2 isoform of the L-type Ca2+ channels in pacemaking tissues.4,17,22 In addition, disruption of the gene encoding for Cav3.1 abolishes T-type Ca2+ current in SAN and AVN in mice. On the other hand, no T-type Ca2+ current was detected in the atria of the WT or knockout animals.5
Presence of KCa Channels in the Heart
Even though the diversity of the Ca2+-independent voltage-gated K+ channels in cardiac myocytes has been well documented, there has been some uncertainty with regard to the existence of KCa channels in the hearts. In general, to ensure stable recording conditions, whole-cell currents are recorded using relatively high concentrations of Ca2+ chelators, which would mask the Ca2+-activated currents. Nonetheless, the presence of IK,Ca has been suggested previously in rabbit myocytes.33 On the other hand, a previous study has argued that apamin at a high concentration (50 nmol/L) may have an irreversible effect on the delayed rectifier K+ current (IK,s) in guinea pig ventricular myocytes.34 To resolve some of this uncertainty, we have used previously a combination of techniques to demonstrate the presence of several isoforms of SK channels in human and mouse cardiac myocytes.7,8 Moreover, we have documented the important functional roles of the SK channels in the heart as well as the more prominent expression in atria compared with ventricular tissues.7,8 However, the functional roles of the SK channels in pacemaking tissues have not been investigated to date.
SK channels have been shown to play an important role in setting the tonic firing frequency of neurons.35 Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the frequency of repetitive APs. The increase in intracellular Ca2+ evoked by AP firing decays slowly, allowing SK channel activation to generate a long-lasting hyperpolarization, termed the slow afterhyperpolarization. This spike frequency adaptation protects the cell from the deleterious effects of continuous tetanic activity. In contrast to the hyperpolarization effects of SK channels in neuron, in atrial myocytes, the current contributes markedly toward the late phase of the cardiac repolarization.7,8 Nonetheless, contrary to the atrial myocytes, the maximum diastolic potential in AV nodes is approximately –60 mV, and a significant hyperpolarization may be expected. Therefore, the observed AP shortening with no associated changes in the maximum diastolic potentials in the SK2+/T model may not be entirely attributable to modulation of a Ca2+-activated K+ current. Additional studies are required to further assess possible changes in other ionic currents which may contribute to the above findings.
Pharmacology of SK Channels
The bee venom peptide toxin apamin is an 18-aa peptide with 2 internal disulfide bridges.36 Two residues, an aspartic acid and an asparagine, that reside on opposite sides of the deep pore have been shown to be essential for apamin sensitivity. No other class of K+ channels is blocked by this drug, and among the cloned K+ channels, the residues that endow sensitivity are present at those positions in only SK2 and SK3 channels. These data suggest that apamin is a very specific blocker for SK channels and that the SK channels may represent the sole class of apamin receptors in the body.37 In our voltage-clamp experiments, a relatively low dose of apamin (500 pmol/L) was used to ensure the specificity of the toxin in our study.
Physiological Significance and Implication for Human Diseases
Here, we demonstrate that overexpression of SK2 channel in the heart increases the frequency of firing in pacemaking tissues. Therefore, in cardiovascular system, SK2 channels may serve a distinct role to potentiate the increase in AVN conduction during exercise or heightened sympathetic responses under normal physiologic conditions. On the other hand, an increase in intracellular Ca2+ under certain pathologic conditions may produce profound changes in AVN conduction. For example, during atrial fibrillation, the rapid depolarization may increase intracellular Ca2+ and potentiate the IK,Ca and AVN conduction. Hence, SK2 channel may represent an attractive target to modulate atrial conduction during atrial arrhythmias.
Limitations
Even though the gene-targeted mouse models offer unique advantage for our study to directly probe the functional roles of SK2 channel in AVN, we recognize the fact that there are well-documented differences between small animal models and human.38,39 Additional studies will need to be performed using large animal models.
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Acknowledgments |
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We are indebted to Dr E.N. Yamoah for helpful suggestions and comments.
Sources of Funding
Supported by NIH/National Heart, Lung, and Blood Institute grants R01 HL075274 and HL085844 (to N.C.) and a Veterans Affairs Merit Review grant (to N.C.).
Disclosures
None.
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Footnotes |
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Original received May 21, 2007; resubmission received August 13, 2007; revised resubmission received November 13, 2007; accepted December 6, 2007.
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