Sinoatrial Node Pacemaker Research Articles

Synchronization of Pacemaking in the Sinoatrial Node: A Mathematical Modeling Study

Computational studies using mathematical models of the sinoatrial node (SAN) cardiac action potential (AP) have provided important insight into the fundamental nature of cell excitability, cardiac arrhythmias, and potential therapies. While the impact of ion channel dynamics on SAN pacemaking has been studied, the governing dynamics responsible for regulating spatial and temporal control of SAN synchrony remain elusive. Here we attempt to develop methods to explore cohesion in a network of coupled spontaneously active SAN cells. We present the updated version of a previously published graphical user interface LongQt: a cross-platform, threaded application for advanced cardiac electrophysiology studies that does not require advanced programming skills. We incorporated additional features to the existing LongQt platform that allows users to (1) specify heterogeneous gap junction conductivity across a multicellular grid, and (2) set heterogeneous ion channel conductance across a multicellular grid. We developed two methods of characterizing the synchrony of SAN tissue based on alignment of activation in time and similarity of voltage peaks among clusters of functionally related cells. In pairs and two-dimensional grids of coupled cells, we observed a range of conductivities (0.00014 1/Ω-cm to 0.00033 1/Ω-cm) in which the tissue was more susceptible to developing asynchronous spontaneous pro-arrhythmic behavior (e.g. spiral wave formation). We performed parameter sensitivity analysis to determine the relative impact of ion channel conductances on cycle length, diastolic and peak voltage and synchrony measurements in isolated and coupled cell pairs. We also defined measurements of evaluating synchrony based on peak AP voltage and the rate of wave propagation. Cell-to-cell coupling had a nonlinear effect on the relationship between ion channel conductances, AP properties and synchrony measurements. Our simulations demonstrate that conductivity plays an important role in regulating synchronous firing of heterogeneous SAN tissue, and demonstrate how to evaluate the role of coupling and ion channel conductance in pairs and grids of SAN cells. We anticipate that the approach outlined here will facilitate identification of key cell- and tissue-level factors responsible for cardiac disease.

A coupled-clock system drives the automaticity of human sinoatrial nodal pacemaker cells.

The spontaneous rhythmic action potentials generated by the sinoatrial node (SAN), the primary pacemaker in the heart, dictate the regular and optimal cardiac contractions that pump blood around the body. Although the heart rate of humans is substantially slower than that of smaller experimental animals, current perspectives on the biophysical mechanisms underlying the automaticity of sinoatrial nodal pacemaker cells (SANCs) have been gleaned largely from studies of animal hearts. Using human SANCs, we demonstrated that spontaneous rhythmic local Ca2+ releases generated by a Ca2+ clock were coupled to electrogenic surface membrane molecules (the M clock) to trigger rhythmic action potentials, and that Ca2+-cAMP-protein kinase A (PKA) signaling regulated clock coupling. When these clocks became uncoupled, SANCs failed to generate spontaneous action potentials, showing a depolarized membrane potential and disorganized local Ca2+ releases that failed to activate the M clock. β-Adrenergic receptor (β-AR) stimulation, which increases cAMP concentrations and clock coupling in other species, restored spontaneous, rhythmic action potentials in some nonbeating "arrested" human SANCs by increasing intracellular Ca2+ concentrations and synchronizing diastolic local Ca2+ releases. When β-AR stimulation was withdrawn, the clocks again became uncoupled, and SANCs reverted to a nonbeating arrested state. Thus, automaticity of human pacemaker cells is driven by a coupled-clock system driven by Ca2+-cAMP-PKA signaling. Extreme clock uncoupling led to failure of spontaneous action potential generation, which was restored by recoupling of the clocks. Clock coupling and action potential firing in some of these arrested cells can be restored by β-AR stimulation-induced augmentation of Ca2+-cAMP-PKA signaling.

Heart automaticity in mice lacking L-type Cav1.3 and T-type Cav3.1 Ca2+ channels: Insights into the cardiac pacemaker mechanism

Sino-atrial node (SAN) pacemaker activity is generated by ion channels of the plasma membrane, such as hyperpolarization-activated “funny” f-(HCN), Ca 2+ channels and ryanodine receptor (RyR) – dependent Ca 2+ release from the sarcoplasmic reticulum (SR). It is currently disputed whether Ca 2+ release from RyRs could sustain viable pacemaker activity provided preserved SR Ca 2+ content. While working myocytes express L-type Ca v 1.2 channels to maintain SR Ca 2+ content, SAN cells express also L-type Ca v 1.3 and T-type Ca v 3.1 channels to generate pacemaking. We used mutant mice carrying concomitant ablation of Ca v 1.3 and Ca v 3.1 (Ca v 1.3 −/− /Ca v 3.1 −/− ) to study the importance of these channels in automaticity. We also investigated the role of f-HCN channels and RyR-dependent Ca 2+ release in residual pacemaker activity of mutant mice. We employed in vivo telemetric recordings of heart rate (HR) in Ca v 1.3 −/− , Ca v 3.1 −/− and Ca v 1.3 −/− /Ca v 3.1 −/− mice. We studied the consequences of pharmacologic inhibition of f-HCN and TTX-sensitive Na + channels in mutant mice using Langendorff perfused hearts or optical mapping (OM) of the pacemaker impulse in intact SAN preparations (SANs). Ca v ablation reduced HR in mice: Ca v 3.1 −/− (−7.6%, n = 11), Ca v 1.3 −/− (−24.4%, n = 8), Ca v 1.3 −/− /Ca v 3.1 −/− (−35%, n = 11). In OM experiments on SANs, concomitant inhibition of f-HCN and Na v 1.1 channels slowed pacemaking in wild-type (−48%, n = 7) and Ca v 3.1 −/− (−37%, n = 7), while arresting automaticity in 4/6 of Ca v 1.3 −/− , 3/6 of Ca v 1.3 −/− /Ca v 3.1 −/− . When present, residual pacemaking was reduced by 82%. Similar results were obtained using isolated Ca v 1.3 −/− /Ca v 3.1 −/− pacemaker cells were automaticity arrested in 5/9 cells tested, or was reduced by 80% in 4/9 cells. Heart automaticity is primarily generated by Ca v 1.3 and f-HCN channels. RyR-dependent Ca 2+ release cannot sustain automaticity following concomitant targeting of Ca v 1.3 and f-HCN channels.

Role of L-type Cav1.3 Ca2+ channels in Ca2+ handling and SAN pacemaker activity altered by external conditions

Membrane currents and Ca 2+ handling generate Sinoatrial node (SAN) automaticity. Several studies showed the negative effect of membrane current impairment, while it is unknown how abnormal Ca 2+ affect pacemaking. To investigate SAN automaticity when Ca 2+ handling is altered by external conditions. We video recorded contraction amplitude and spontaneous rate of SAN cells. In WT, 0.9 mM external Ca 2+ ([Ca 2+ ] 0 ) reduces cell shortening, while 3 mM increased it, compared to control solution (1.8 mM [Ca 2+ ] 0 ). Lowering [Ca 2+ ] 0 to 0.9 mM did not affect WT automaticity, while high [Ca 2+ ] 0 dysregulate it without changing the average rate. L-type channels (LTCCs) are the main Ca 2+ source in SAN cells, with Ca v 1.3 as the dominant isoform and Ca v 1.2 less expressed. To discern their role during abnormal [Ca 2+ ] 0 , we used mice lacking Ca v 1.3 channels (Ca v 1.3 −/− ). As in WT, 0.9 mM [Ca 2+ ] 0 decreased cell shortening in Ca v 1.3 −/− , while only a tendency to increase was determined by 3 mM [Ca 2+ ] 0 . Also, high [Ca 2+ ] 0 significantly reduced spontaneous rate in Ca v 1.3 −/− cells. Stimulation of WT cells with the LTCC dihydropyridine agonist BayK 8644 (BayK) increased cell shortening and spontaneous rate. Under BayK, Ca v 1.3 −/− cells showed similar increase of contraction amplitude but reduced rate acceleration than WT. We repeated this protocol with a mouse where Ca v 1.2 is dihydropyridines insensitive (Cav1.2 DHP−/− ). Cav1.2 DHP−/− SAN cells responded like WT to abnormal [Ca 2+ ] 0 . Instead, BayK caused rate increase equivalent to WT but reduced contraction shortening in Cav1.2 DHP−/− . Conclusion Abnormal increase of Ca 2+ influx dysregulates automaticity in WT cells, likely through hyper-stimulation of Ca 2+ activated currents. Moreover, selective stimulation of Ca v 1.2 and/or Ca v 1.3 with BayK suggests that Ca v 1.2 maintain the bulk of Ca 2+ allowing proper contraction, while Ca v 1.3 mainly carry the influx of Ca 2+ needed to trigger SAN automaticity.

Effects of fibroblast-myocyte coupling on the sinoatrial node activity: A computational study.

While the sinoatrial node (SAN) is structurally heterogeneous, most computer simulations of electrical activity take into account SAN pacemaker cells only. Our aim was to investigate how fibroblasts affect the SAN activity. We simulated the rabbit sinoatrial node accounting for differences between central and peripheral pacemaker cells, and for fibroblast-myocyte electrical coupling. We have observed that only if fibroblast-myocyte coupling is taken into account, (1) action potential is initiated in the central part of the SAN (within 1.2mm of the center of simulated tissue); otherwise, leading centers are located on the periphery; (2) few (1 to 6) leading centers initiate action potential in the SAN; otherwise, we observed more than 8 leading centers; (3) acetylcholine superfusion results in a shift of leading centers toward the SAN periphery; and (4) sinus pauses up to 1.9second follow acetylcholine superfusion. We observed negligible effect of fibroblast-myocyte coupling on the period of SAN activation. We conclude that fibroblast-myocyte coupling may explain action potential initiation and propagation from the center of the SAN observed in experimental studies, while atrial load on the peripheral SAN fails to explain this fact.

Cardiac Overexpression of Human Adenylyl Cyclase Type 8 Elicits Wide Spread Adaptations to Limit Adrenergic Signaling in Sinoatrial Nodal Pacemaker Cells that Extend Well Beyond Desensitization of β-Adrenergic Receptors

Cardiac Overexpression of Human Adenylyl Cyclase Type 8 Elicits Wide Spread Adaptations to Limit Adrenergic Signaling in Sinoatrial Nodal Pacemaker Cells that Extend Well Beyond Desensitization of β-Adrenergic Receptors

Effects of Cyclopiazonic Acid on Pacemaker Current of Sinoatrial Nodal Pacemaker Cells

Objective: The sinoatrial node (SAN) automaticity is essential for cardiac rhythm. However, the mechanism underlying the regulation of SAN pacemaker activity remains controversial. We investigated the effect of cyclopiazonic acid (CPA), a specific sarcoplasmic reticulum Ca2+ pump inhibitor, on pacemaker currents (If) in neonatal rat SAN cells.

Mathematical Simulation of the Pacemaker Activity of Rabbit Sinoatrial Node Cells Using Similitude Theory

A mathematical model that reproduces the functioning of the rabbit sinoatrial node is proposed. A similitude theory is applied to single or multiple sinoatrial node pacemaker cells with the goal of significantly simplifying the description of the studied system. This model provides analytical estimates for the general frequency of pacemaker currents in these pacemaker cells and reveals basic stable elements in the pacemaking mechanism of the sinoatrial node.

Abstract 24075: Heart Failure Induced Upregulations of MicroRNAs in the Human Sinoatrial Node Associated with Pacemaker Dysfunction

Background: Heart failure (HF), a leading cause of morbidity and mortality, involves significant dysfunction of the sinoatrial node (SAN). MicroRNAs (miRs) are abundant, non-coding RNAs that ultimately regulate protein expression at the post-transcriptional level and have been implicated in HF. However, nothing is known about the distribution and expression of miRs in the human SAN as well as their roles in regulating pacemaker channels and SAN dysfunction (SND) in HF. Methods: The human SAN was isolated and cryopreserved from failing hearts with implantable pacemakers (n=5) or non-diseased donor hearts (n=3) that were cardioplegically arrested in the surgery room. Utilizing immunohistochemistry, the intact 3D intramural SAN structure was precisely identified as the fibrotic region around the SAN artery containing Connexin43-negative pacemaker cardiomyocytes (Figure). Small biopsies from the central SAN compartment were used to characterize the expression pattern of 14 miRs and their associated pacemaker channels. Ingenuity software (Qiagen) was used to predict the relationship between miRs and their targeted mRNA of SAN ion channels Results: Out of 14 studied miRs, 3 miRs (mir-370-3p miR-133-3a and miR-1) were significantly upregulated in SAN, but not in atria, during HF. We found that mir-370-3p was predominantly expressed in the human SAN, but not atria. Whereas, both miR-133-3a and miR-1 were highly expressed in atria vs SAN. All three miRs were predicted to regulate pacemaker HCN1 and/or HCN4 channel expression. RT-PCR showed that HCN1 and HCN4 mRNA were downregulated in the HF SAN. Conclusions: This is the first study to explore the miRs profiles in healthy and diseased human SAN with SND. We demonstrate that miR-370, miR-133a and miR-1, thought to regulate SAN pacemaker HCN channel expression, are selectively upregulated in human HF SAN. We propose that understanding the function of miRs in human SAN might lead to novel SND treatments.

Modeling pacemaker deterioration with age.

New JGP study models how sinoatrial node pacemaker activity changes in aged hearts.

Mechanism underlying impaired cardiac pacemaking rhythm during ischemia: A simulation study.

Ischemia in the heart impairs function of the cardiac pacemaker, the sinoatrial node (SAN). However, the ionic mechanisms underlying the ischemia-induced dysfunction of the SAN remain elusive. In order to investigate the ionic mechanisms by which ischemia causes SAN dysfunction, action potential models of rabbit SAN and atrial cells were modified to incorporate extant experimental data of ischemia-induced changes to membrane ion channels and intracellular ion homeostasis. The cell models were incorporated into an anatomically detailed 2D model of the intact SAN-atrium. Using the multi-scale models, the functional impact of ischemia-induced electrical alterations on cardiac pacemaking action potentials (APs) and their conduction was investigated. The effects of vagal tone activity on the regulation of cardiac pacemaker activity in control and ischemic conditions were also investigated. The simulation results showed that at the cellular level ischemia slowed the SAN pacemaking rate, which was mainly attributable to the altered Na+-Ca2+ exchange current and the ATP-sensitive potassium current. In the 2D SAN-atrium tissue model, ischemia slowed down both the pacemaking rate and the conduction velocity of APs into the surrounding atrial tissue. Simulated vagal nerve activity, including the actions of acetylcholine in the model, amplified the effects of ischemia, leading to possible SAN arrest and/or conduction exit block, which are major features of the sick sinus syndrome. In conclusion, this study provides novel insights into understanding the mechanisms by which ischemia alters SAN function, identifying specific conductances as contributors to bradycardia and conduction block.

CaV1.3 L-type Ca2+ channel contributes to the heartbeat by generating a dihydropyridine-sensitive persistent Na+ current

The spontaneous activity of sinoatrial node (SAN) pacemaker cells is generated by a functional interplay between the activity of ionic currents of the plasma membrane and intracellular Ca2+ dynamics. The molecular correlate of a dihydropyridine (DHP)-sensitive sustained inward Na+ current (Ist), a key player in SAN automaticity, is still unknown. Here we show that Ist and the L-type Ca2+ current (ICa,L) share CaV1.3 as a common molecular determinant. Patch-clamp recordings of mouse SAN cells showed that Ist is activated in the diastolic depolarization range, and displays Na+ permeability and minimal inactivation and sensitivity to ICa,L activators and blockers. Both CaV1.3-mediated ICa,L and Ist were abolished in CaV1.3-deficient (CaV1.3−/−) SAN cells but the CaV1.2-mediated ICa,L current component was preserved. In SAN cells isolated from mice expressing DHP-insensitive CaV1.2 channels (CaV1.2DHP−/−), Ist and CaV1.3-mediated ICa,L displayed overlapping sensitivity and concentration–response relationships to the DHP blocker nifedipine. Consistent with the hypothesis that CaV1.3 rather than CaV1.2 underlies Ist, a considerable fraction of ICa,L was resistant to nifedipine inhibition in CaV1.2DHP−/− SAN cells. These findings identify CaV1.3 channels as essential molecular components of the voltage-dependent, DHP-sensitive Ist Na+ current in the SAN.

55Role of L-type Cav1.3 Ca2+ channels in Ca2+ handling and sinoatrial node pacemaker activity altered by external conditions

55Role of L-type Cav1.3 Ca2+ channels in Ca2+ handling and sinoatrial node pacemaker activity altered by external conditions

105Contribution of small conductance K+ channels to sinoatrial node pacemaker activity of control and atrial-specific Na+/Ca2+ exchange knock out mice

105Contribution of small conductance K+ channels to sinoatrial node pacemaker activity of control and atrial-specific Na+/Ca2+ exchange knock out mice

Contribution of small conductance K+ channels to sinoatrial node pacemaker activity: insights from atrial-specific Na+ /Ca2+ exchange knockout mice.

Repolarizing currents through K+ channels are essential for proper sinoatrial node (SAN) pacemaking, but the influence of intracellular Ca2+ on repolarization in the SAN is uncertain. We identified all three isoforms of Ca2+ -activated small conductance K+ (SK) channels in the murine SAN. SK channel blockade slows repolarization and subsequent depolarization of SAN cells. In the atrial-specific Na+ /Ca2+ exchanger (NCX) knockout mouse, cellular Ca2+ accumulation during spontaneous SAN pacemaker activity produces intermittent hyperactivation of SK channels, leading to arrhythmic pauses alternating with bursts of pacing. These findings suggest that Ca2+ -sensitive SK channels can translate changes in cellular Ca2+ into a repolarizing current capable of modulating pacemaking. SK channels are a potential pharmacological target for modulating SAN rate or treating SAN dysfunction, particularly under conditions characterized by abnormal increases in diastolic Ca2+ . Small conductance K+ (SK) channels have been implicated as modulators of spontaneous depolarization and electrical conduction that may be involved in cardiac arrhythmia. However, neither their presence nor their contribution to sinoatrial node (SAN) pacemaker activity has been investigated. Using quantitative PCR (q-PCR), immunostaining and patch clamp recordings of membrane current and voltage, we identified all three SK isoforms (SK1, SK2 and SK3) in mouse SAN. Inhibition of SK channels with the specific blocker apamin prolonged action potentials (APs) in isolated SAN cells. Apamin also slowed diastolic depolarization and reduced pacemaker rate in isolated SAN cells and intact tissue. We investigated whether the Ca2+ -sensitive nature of SK channels could explain arrhythmic SAN pacemaker activity in the atrial-specific Na+ /Ca2+ exchange (NCX) knockout (KO) mouse, a model of cellular Ca2+ overload. SAN cells isolated from the NCX KO exhibited higher SK current than wildtype (WT) and apamin prolonged their APs. SK blockade partially suppressed the arrhythmic burst pacing pattern of intact NCX KO SAN tissue. We conclude that SK channels have demonstrable effects on SAN pacemaking in the mouse. Their Ca2+ -dependent activation translates changes in cellular Ca2+ into a repolarizing current capable of modulating regular pacemaking. This Ca2+ dependence also promotes abnormal automaticity when these channels are hyperactivated by elevated Ca2+ . We propose SK channels as a potential target for modulating SAN rate, and for treating patients affected by SAN dysfunction, particularly in the setting of Ca2+ overload.

124 - Contribution of Small K+ channels to Sinoatrial Node pacemaking of control and atrial-specific Na+/Ca2+ exchange knock out mice

124 - Contribution of Small K+ channels to Sinoatrial Node pacemaking of control and atrial-specific Na+/Ca2+ exchange knock out mice

123 - Role of L-type Cav1.3 Ca2+ channels in Ca2+ handling and SAN pacemaker activity altered by external conditions

Introduction Membrane currents and Ca2+ handling generate Sinoatrial node (SAN) automaticity. Several studies showed the negative effect of membrane current impairment, while it is unknown how abnormal Ca2+ affect pacemaking. Purpose To investigate SAN automaticity when Ca2+ handling is altered by external conditions. Methods We video recorded contraction amplitude and spontaneous rate of SAN cells. Results In WT, 0.9 mM external Ca2+ ([Ca2+]0) reduces cell shortening, while 3 mM increased it, compared to control solution (1.8 mM [Ca2+]0). Lowering [Ca2+]0 to 0.9 mM did not affect WT automaticity, while high [Ca2+]0 dysregulate it without changing the average rate. L-type channels (LTCCs) are the main Ca2+ source in SAN cells, with Cav1.3 as the dominant isoform and Cav1.2 less expressed. To discern their role during abnormal [Ca2+]0, we used mice lacking Cav1.3 channels (Cav1.3−/−). As in WT, 0.9 mM [Ca2+]0 decreased cell shortening in Cav1.3−/−, while only a tendency to increase was determined by 3 mM [Ca2+]0. Also, high [Ca2+]0 significantly reduced spontaneous rate in Cav1.3−/− cells. Stimulation of WT cells with the LTCC dihydropyridine agonist BayK 8644 (BayK) increased cell shortening and spontaneous rate. Under BayK, Cav1.3−/− cells showed similar increase of contraction amplitude but reduced rate acceleration than WT. We repeated this protocol with a mouse where Cav1.2 is dihydropyridines insensitive (Cav1.2DHP−/−). Cav1.2DHP−/− SAN cells responded like WT to abnormal [Ca2+]0. Instead, BayK caused rate increase equivalent to WT but reduced contraction shortening in Cav1.2DHP−/−. Conclusion Abnormal increase of Ca2+ influx dysregulates automaticity in WT cells, likely through hyper-stimulation of Ca2+ activated currents. Moreover, selective stimulation of Cav1.2 and/or Cav1.3 with BayK suggests that Cav1.2 maintain the bulk of Ca2+ allowing proper contraction, while Cav1.3 mainly carry the influx of Ca2+ needed to trigger SAN automaticity.

Computational analysis of the human sinus node action potential: model development and effects of mutations.

We constructed a comprehensive mathematical model of the spontaneous electrical activity of a human sinoatrial node (SAN) pacemaker cell, starting from the recent Severi-DiFrancesco model of rabbit SAN cells. Our model is based on electrophysiological data from isolated human SAN pacemaker cells and closely matches the action potentials and calcium transient that were recorded experimentally. Simulated ion channelopathies explain the clinically observed changes in heart rate in corresponding mutation carriers, providing an independent qualitative validation of the model. The model shows that the modulatory role of the 'funny current' (If ) in the pacing rate of human SAN pacemaker cells is highly similar to that of rabbit SAN cells, despite its considerably lower amplitude. The model may prove useful in the design of experiments and the development of heart-rate modulating drugs. The sinoatrial node (SAN) is the normal pacemaker of the mammalian heart. Over several decades, a large amount of data on the ionic mechanisms underlying the spontaneous electrical activity of SAN pacemaker cells has been obtained, mostly in experiments on single cells isolated from rabbit SAN. This wealth of data has allowed the development of mathematical models of the electrical activity of rabbit SAN pacemaker cells. The present study aimed to construct a comprehensive model of the electrical activity of a human SAN pacemaker cell using recently obtained electrophysiological data from human SAN pacemaker cells. We based our model on the recent Severi-DiFrancesco model of a rabbit SAN pacemaker cell. The action potential and calcium transient of the resulting model are close to the experimentally recorded values. The model has a much smaller 'funny current' (If ) than do rabbit cells, although its modulatory role is highly similar. Changes in pacing rate upon the implementation of mutations associated with sinus node dysfunction agree with the clinical observations. This agreement holds for both loss-of-function and gain-of-function mutations in the HCN4, SCN5A and KCNQ1 genes, underlying ion channelopathies in If , fast sodium current and slow delayed rectifier potassium current, respectively. We conclude that our human SAN cell model can be a useful tool in the design of experiments and the development of drugs that aim to modulate heart rate.

Loss of Caveolin-3 Causes Heart Rhythm Abnormalities by Affecting Ca2+-Voltage Coupling in the Mouse Sinoatrial Node

Caveolin-3 (Cav3) is a myocyte-specific scaffolding protein within caveolar membranes that compartmentalize a number of ion channels and transporters involved in sinoatrial node (SAN) pacemaking. These channels and transporters are coupled with intracellular Ca2+ cycling (Ca2+-voltage coupling) through rhythmic local Ca2+ releases (LCRs) from ryanodine receptors to initiate an action potential. We hypothesize that disruption of caveolae-associated macromolecular signaling complexes alters SAN automaticity leading to SAN dysfunction. In vivo ECG telemetry, in vitro high-resolution optical mapping, and confocal imaging of intracellular Ca2+ cycling were performed in wild type (WT, n=8) and conditional tamoxifen-induced α-MHC-controlled Cav3 knockout (Cav3-/-, n=8) mice and isolated SAN myocytes. Both in vivo and in vitro,Cav3-/- mice exhibited SAN pacemaking abnormalities characterized by alternating periods of tachycardia-bradycardia rhythm (cycle length varied from 123.6±2.6 ms to 464.4±130 ms), significant beat-to-beat cycle length variability (3.8±1.1 ms in WT vs. 19.5±3.6 ms in Cav3-/-, p<0.01) and shift of the leading pacemaker from the SAN to ectopic foci. In SAN myocytes, Cav3 deficiency led to irregular spontaneous rate with significant cycle length variability (30.9±5.2 ms in WT vs. 644.7±256.4 ms in Cav3-/-, p<0.01). Because LCRs are critically important for the Na+/Ca2+ exchanger mediated component of slow diastolic depolarization of transmembrane potential, we measured the period from LCRs to Ca2+ transient initiation in order to evaluate Ca2+-voltage coupling. Cav3-/- SAN myocytes showed significant prolongation (73.9±6.7 ms in WT vs. 130.6±15.0 ms in Cav3-/-, p<0.01) of this period with large beat-to-beat variability (15.5±2.6 ms in WT vs. 71.6±17.4 ms in Cav3-/-, p<0.01). Our findings demonstrate that Cav3 plays a crucial role in supporting functional integrity of the SAN by synchronizing Ca2+-voltage coupling and regulating rhythm of LCRs.

SK4 Ca2+-Activated K+ Channels Regulate Sinoatrial Node Firing Rate and Cardiac Pacing In Vivo

The sinoatrial node (SAN) controls the heart rhythm under physiological conditions. We recently showed that SK4 calcium-activated potassium channels (SK4) are important for automaticity of cardiomyocytes derived from human embryonic stem cells. Here we tested whether SK4 are expressed in adult mouse SAN and play a role in pacemaker function. TRAM-34, a selective blocker of SK4, significantly reduced the firing rate and depolarized the maximal diastolic potential in SAN cells. Western blots revealed the presence of an SK4 protein in mouse SAN. In vivo ECG recording in mice showed that intraperitoneal injection of SK4 blockers produced bradycardia and prolonged the PR interval. Mathematical modeling predicted that addition of SK4 to the model increases SAN firing rate, while its removal decreases pacemaker frequency. This work shows that SK4 play a role in SAN pacemaker function by acting at late repolarization and that they are potential therapeutic targets for treating cardiac arrhythmias.