Sinoatrial Node Pacemaker Activity Research Articles

Abstract P3148: Wnt/β-catenin Signaling Inhibits T-type Calcium Current And Causes Sinoatrial Node Dysfunction

Background: The T-type Ca 2+ channels (I Ca,T ) are expressed in the fetal and neonatal cardiomyocytes, but in the adult heart they are exclusively expressed in the cardiac conduction system, including the sinoatrial node (SAN). In contrast, the L-type Ca 2+ channels (I Ca,L ) are expressed ubiquitously in the adult heart and are important for cardiac contraction. The Wnt/β-catenin signaling (Wnt signaling) is activated in various heart disease and we and others have recently demonstrated that Wnt signaling inhibits cardiac Na + current. In the present study, we investigated if calcium channels are also regulated by Wnt signaling. Methods and Results: Because neonatal rat ventricular myocytes (NRVMs) express both I Ca,L and I Ca,T , we investigated the effect of Wnt signaling on NRVMs by treatment with either Wnt3a protein or CHIR-99021 (CHIR) (two different activators of the Wnt signaling). PCR array and RNA sequencing studies showed that Wnt signaling did not affect the mRNA of Cacna1c (encoding the α subunit of I Ca,L , Ca v 1.2), but the mRNA of Cacna1g (encoding the α subunit of I Ca,T , Ca v 3.1) was reduced in NRVMs (p<0.05, n=3) after treatment with either Wnt3a or CHIR. These observations were verified by qRT-PCR which showed dose-dependent reductions (p<0.05, n=4) in Cacna1g mRNA after CHIR treatment. Western blot and patch-clamp also demonstrated reductions (p<0.01, n=5) in channel protein and ionic current density of T-type, but not L-type, Ca 2+ channels after treatment with Wnt3a or CHIR. These observations suggest that Wnt signaling selectively inhibits T-type Ca 2+ channels in cardiomyocytes. Consistent with a critical role of T-type Ca 2+ channels in the pacemaker activity of SAN (the primary pacemaker of the heart), the spontaneous firing rates of isolated rat and mouse SAN tissues were reduced by ML-218 (an I Ca,T blocker) in a dose-dependent manner (p<0.01, n=5). In addition, activation of Wnt signaling in adult rat SAN tissues by CHIR led to slower and irregular beating, indicative of SAN dysfunction (p<0.01, n=5). Conclusions: These observations are consistent with the conclusions that Wnt/β-catenin signaling selectively inhibits T-type calcium current and may play a role in sinoatrial node dysfunction.

Lipopolysaccharide-induced sepsis impairs M2R-GIRK signaling in the mouse sinoatrial node

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely IKACh activation in SAN cells, reduction in Ca2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.

Human Sinoatrial Node Pacemaker Activity: Role of the Slow Component of the Delayed Rectifier K+ Current, IKs.

The pacemaker activity of the sinoatrial node (SAN) has been studied extensively in animal species but is virtually unexplored in humans. Here we assess the role of the slowly activating component of the delayed rectifier K+ current (IKs) in human SAN pacemaker activity and its dependence on heart rate and β-adrenergic stimulation. HEK-293 cells were transiently transfected with wild-type KCNQ1 and KCNE1 cDNA, encoding the α- and β-subunits of the IKs channel, respectively. KCNQ1/KCNE1 currents were recorded both during a traditional voltage clamp and during an action potential (AP) clamp with human SAN-like APs. Forskolin (10 µmol/L) was used to increase the intracellular cAMP level, thus mimicking β-adrenergic stimulation. The experimentally observed effects were evaluated in the Fabbri-Severi computer model of an isolated human SAN cell. Transfected HEK-293 cells displayed large IKs-like outward currents in response to depolarizing voltage clamp steps. Forskolin significantly increased the current density and significantly shifted the half-maximal activation voltage towards more negative potentials. Furthermore, forskolin significantly accelerated activation without affecting the rate of deactivation. During an AP clamp, the KCNQ1/KCNE1 current was substantial during the AP phase, but relatively small during diastolic depolarization. In the presence of forskolin, the KCNQ1/KCNE1 current during both the AP phase and diastolic depolarization increased, resulting in a clearly active KCNQ1/KCNE1 current during diastolic depolarization, particularly at shorter cycle lengths. Computer simulations demonstrated that IKs reduces the intrinsic beating rate through its slowing effect on diastolic depolarization at all levels of autonomic tone and that gain-of-function mutations in KCNQ1 may exert a marked bradycardic effect during vagal tone. In conclusion, IKs is active during human SAN pacemaker activity and has a strong dependence on heart rate and cAMP level, with a prominent role at all levels of autonomic tone.

Sinus node dysfunction in heart failure is characterized by reduced CaMKII signaling

Heart failure (HF) is a complex syndrome in which death rates are over 50%. The main cause of death among HF patients is pump failure and ventricular arrhythmias, but severe bradycardia is also a common cause of sudden cardiac death, pointing to sinoatrial node (SAN) dysfunction. SAN pacemaker activity is regulated by voltage-clock and Ca2+-clock mechanisms and, although voltage-clock dysfunction in SAN has been largely proved in HF, Ca2+-clock dysfunction mechanisms in SAN remains undiscovered. Here, we used a HF model in mice with transverse aortic constriction (TAC) and using telemetry saw slower heart rhythm under autonomic nervous system blockade. Then, using confocal microscopy we analyzed Ca2+ handling in HF SAN tissue and found that intracellular Ca2+ transient rates were slower in addition to less frequency of Ca2+ sparks than in SHAM SAN tissue. Next, we studied protein expression of key excitation-contraction coupling proteins and found reduced expression of the Na+/Ca2+ exchanger and reduced phosphorylated status of ryanodine receptor and phospholamban in the CaMKII sites for the SAN in TAC mice. Finally, the application of the CaMKII inhibitor KN93 caused less effect in slowing the Ca2+ transient rates in HF SAN tissue, confirming the reduced CaMKII activation. In conclusion, our data demonstrate a reduction in CaMKII activation in the Ca2+-clock function of the SAN tissue in a mouse model of HF.

Sinus node dysfunction in heart failure is characterized by reduced CaMKII signaling

Heart failure (HF) is a complex syndrome in which death rates are >50%. The main causes of death among HF patients are pump failure and ventricular arrhythmias, but severe bradycardia is also a common cause of sudden cardiac death, pointing to sinoatrial node (SAN) dysfunction. SAN pacemaker activity is regulated by voltage-clock and Ca2+-clock mechanisms and, although voltage-clock dysfunction in SAN has been largely proved in HF, Ca2+-clock dysfunction mechanisms in SAN remains unraveled. Here, we used an HF model in mice with transverse aortic constriction (TAC) and, using telemetry, saw slower heart rhythm under autonomic nervous system blockade. Then, by confocal microscopy, we analyzed Ca2+ handling in HF SAN tissue and found that intracellular Ca2+ transients rate were slower together with less frequency of Ca2+ sparks than in SHAM SAN tissue. Next, we studied protein expression of key excitation-contraction coupling proteins and found reduced expression of the Na+/Ca2+ exchanger and reduced phosphorylated status of ryanodine receptor and phospholamban in the CaMKII sites for the SAN in TAC mice. Finally, the application of the CaMKII inhibitor, KN93, caused less effect in slowing the Ca2+ transient rates in HF SAN tissue, confirming the reduced CaMKII activation. In conclusion, our data demonstrates a reduction in CaMKII activation in the Ca2+-clock function of the SAN tissue in a mouse model of HF.

HCN4 current during human sinoatrial node-like action potentials

BackgroundDespite the many studies carried out over the past 40 years, the contribution of the HCN4 encoded hyperpolarization-activated ‘funny’ current (If) to pacemaker activity in the mammalian sinoatrial node (SAN), and the human SAN in particular, is still controversial and not fully established. ObjectiveTo study the contribution of If to diastolic depolarization of human SAN cells and its dependence on heart rate, cAMP levels, and atrial load. MethodsHCN4 channels were expressed in human cardiac myocyte progenitor cells (CMPCs) and HCN4 currents assessed using perforated patch-clamp in traditional voltage clamp mode and during action potential clamp with human SAN-like action potential waveforms with 500–1500 ms cycle length, in absence or presence of forskolin to mimic β-adrenergic stimulation and a −15 mV command potential offset to mimic atrial load. ResultsForskolin significantly increased the fully-activated HCN4 current density at −140 mV by 14% and shifted the steady-state activation curve by +7.4 mV without affecting its slope. In addition, forskolin significantly accelerated current activation but slowed deactivation. The HCN4 current did not completely deactivate before the subsequent diastolic depolarization during action potential clamp. The amplitude of HCN4 current increased with increasing cycle length, was significantly larger in the presence of forskolin at all cycle lengths, and was significantly increased upon the negative offset to the command potential. ConclusionsIf is active during a human SAN action potential waveform and its amplitude is modulated by heart rate, β-adrenergic stimulation, and diastolic voltage range, such that If is under delicate control.

Peculiarities of Autonomic Regulation of the Heart in Experimental Normobaric Hypoxia of Different Genesis.

We compared autonomic regulation of the heart in Wistar rats with acute exogenous hypoxic and chronic normobaric hypoxia against the background of experimental chronic obstructive pulmonary disease. In animals subjected to normobaric hypoxia, dysfunction of the autonomic regulation of the heart developed; it manifested in a significant limitation of the effects of the parasympathetic and sympathetic nervous systems on the pacemaker activity of the sinoatrial node. Disturbances in autonomic regulation of the heart in acute exogenous hypoxic normobaric hypoxia were characterized by "rigid" heart rhythm and bradycardia in the presence of systolic-diastolic arterial hypotension. Centralization of heart rhythm control did not provide compensation for hemodynamic disturbances. In case of chronic normobaric hypoxia, an increase in arterial hypoxemia was associated with protective "denervation" of the heart against the background of increased activity of the humoral regulation, which manifested in tachycardia, isolated systolic arterial hypertension, and overstrain of regulatory systems.

Na/Ca exchange in the atrium: Role in sinoatrial node pacemaking and excitation-contraction coupling

Na/Ca exchange is the dominant calcium (Ca) efflux mechanism in cardiac myocytes. Although our knowledge of exchanger function (NCX1 in the heart) was originally established using biochemical and electrophysiological tools such as cardiac sarcolemmal vesicles and the giant patch technique [1–4], many advances in our understanding of the physiological/pathophysiological roles of NCX1 in the heart have been obtained using a suite of genetically modified mice. Early mouse studies focused on modification of expression levels of NCX1 in the ventricles, with transgenic overexpressors, global NCX1 knockout (KO) mice (which were embryonic lethal if homozygous), and finally ventricular-specific NCX1 KO [5–12]. We found, to our surprise, that ventricular cardiomyocytes lacking NCX1 can survive and function by engaging a clever set of adaptations to minimize Ca entry, while maintaining contractile function through an increase in excitation-contraction (EC) coupling gain [5,6,13]. Having studied ventricular NCX1 ablation in detail, we more recently focused on elucidating the role of NCX1 in the atria through altering NCX1 expression. Using a novel atrial-specific NCX1 KO mouse, we found unexpected changes in atrial cell morphology and calcium handling, together with dramatic alterations in the function of sinoatrial node (SAN) pacemaker activity. In this review, we will discuss these findings and their implications for cardiac disease.

Reduced Basal Heart Rate Variability in a Calcineurin Aβ Deficient Mouse

PurposeHeart rate and heart rhythm, quantified by heart rate variability (HRV) in vivo, are both determined by intrinsic pacemaker activity of sinoatrial node (SAN) pacemaker cells (SANC), and input to SAN from the autonomic nervous system (ANS). The spontaneous action potential (AP) firing rate and rhythm of SANC are governed, at least in part, by phosphorylation of proteins that regulate their intracellular calcium cycling and ion channel functions. Calcineurin Aβ (CnAβ), the catalytic subunit of a calcium‐activated serine/threonine phosphatase, influences SANC function protein phosphorylation. Increased calcium cycling and ion channel phosphorylation leads to an increase in SANC AP firing rate and reduced AP firing interval variability. CnAβ likely impacts SANC AP firing rate and AP interval variability, making CnAβ a prime candidate of in vivo HR and HRV regulation. Thus, we hypothesized the role of CnAβ in CnAβ−/− mice, in which cardiac Cn activity is decreased by 80%, would manifest an increased in vivo HR and reduced HRV.MethodsElectrocardiograms (EKG) were measured (via telemetry) in awake, unrestrained, 3‐month‐old male CnAβ−/− mice (n = 9) and wildtype littermates (n = 9). Four 1.5‐hour basal recordings were obtained at least 24 hours apart. A segment of at least 2048 beats with minimal noise were selected for each EKG time series recording and analyzed for different types of HRV parameters (LabChart). HRV parameters in the time domain quantify variance of unordered beat‐to‐beat intervals, frequency domain parameters measure how variance of ordered beat‐to‐beat intervals embedded within an EKG time series distribute as a function of frequency, and non‐linear domain parameters detect variation in fractals buried within the heart rhythm. Statistical analyses were performed using a linear mixed effect model (R) accounting for the four repeated measures.ResultsC.F. table. Mean basal HR was substantially higher in CnAβ−/− than in WT, and time, non‐linear, and most frequency domain HRV descriptors were markedly reduced in CnAβ−/− than WT.ConclusionBecause CnAβ regulates intrinsic SANC function to enable increased calcium cycling and ion channel phosphorylation, our results suggest intrinsic SANC function is a factor that contributes to HR and HRV in vivo. Whether the altered HR and HRV in the CnAβ−/− heart are driven solely by CnAβ dependent mechanisms intrinsic to SANC or, in addition, reflect changes in ANS input to the heart require further studies.Support or Funding InformationFunded by the National Institute of Health/National Institute on Aging Intramural Research Program. Average^ Basal HRV Values in CnAβ−/− vs WT Mice Parameter WT CnAβ−/− Mean HR (bpm) 460 ± 10.8 507 ± 9.6* TIME DOMAIN SD NN (ms) 9.87 ±0.8 5.39 ± 0.4** Range NN (ms) 47.77 ± 3.7 26.68 ± 2.4** Coefficient of Variance (%) 7.22 ± 0.5 4.34 ± 0.3** FREQUENCY DOMAIN Total Power (Power Spectral Density) 284.93 ± 23.5 184.21 ± 19.0* High Frequency (PSD) 70.28 ± 6.2 66.42 ± 9.5 Low Frequency (PSD) 62.58 ± 5.2 40.09 ± 4.7* Very Low Frequency (PSD) 152.07 ± 19.8 77.70 ± 10.5* NON‐LINEAR DOMAIN Beta Slope −2.15 ± 0.1 −2.84 ± 0.1** SDl(ms) 5.72 ± 0.4 2.80 ± 0.7* SD2 (ms) 12.57 ± 0.9 7.00 ± 0.6** Multiscale Entropy 1.57 ± 0.1 0.93 ± 0.1** Values are mean ± SE p<0.05 p<0.01 Average of 4 basal recordings over separate days This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long QT Syndrome and Sinus Bradycardia-A Mini Review.

Congenital long-QT syndrome (LQTS) is an inherited cardiac disorder characterized by the prolongation of ventricular repolarization, susceptibility to Torsades de Pointes (TdP), and a risk for sudden death. Various types of congenital LQTS exist, all due to specific defects in ion channel-related genes. Interestingly, almost all of the ion channels affected by the various types of LQTS gene mutations are also expressed in the human sinoatrial node (SAN). It is therefore not surprising that LQTS is frequently associated with a change in basal heart rate (HR). However, current data on how the LQTS-associated ion channel defects result in impaired human SAN pacemaker activity are limited. In this mini-review, we provide an overview of known LQTS mutations with effects on HR and the underlying changes in expression and kinetics of ion channels. Sinus bradycardia has been reported in relation to a large number of LQTS mutations. However, the occurrence of both QT prolongation and sinus bradycardia on a family basis is almost completely limited to LQTS types 3 and 4 (LQT3 and Ankyrin-B syndrome, respectively). Furthermore, a clear causative role of this sinus bradycardia in cardiac events seems reserved to mutations underlying LQT3.

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 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.

Modeling pacemaker deterioration with age.

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

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.

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.

SERCA2a was serotonylated and may regulate sino-atrial node pacemaker activity

The modulation of sino-atrial node (SAN) automaticity is an essential mechanism of heart rate generation that is still not completely understood. Recent studies highlighted the importance of protein serotonylation by intracellular 5-HT during varies physiological actions. Nevertheless, the functional role of protein serotonylation in controlling SAN automaticity is largely unexplored. In this study, we screened the cardiomyocytes proteins and found that sarco(endo)plasmic reticulum Ca ATPase type 2a (SERCA2a) can be serotonylated. Simulation studies using mathematical SAN cell model showed that variation of Ca2+ affinity of SERCA2a pump cause either tachycardia or bradycardia.

0128 : Regulation of pacemaker activity by PDE4 isoforms

Numerous epidemiological and clinical studies have revealed a positive correlation between heart rate (HR) and cardiovascular morbimortality. The autonomic nervous system is the major extracardiac determinant of HR. During sympathetic stimulation, the activation of β- adrenergic receptors (βAR) induces an increase in cAMP levels, leading to a chronotopic effect. Among the 5 cAMP-PDE families expressed in the heart, PDE4 is critical for controlling excitation-contraction coupling (ECC) during βAR stimulation in atrial and ventricular cells. PDE4 may also be important for automaticity. 3 genes encode for cardiac PDE4s : pde4a, pde4b and pde4d . Their respective contribution to the regulation of pacemaker activity remains ill-defined. The total enzymatic PDE activity was determined in mouse sinoatrial node (SAN) tissue as the cAMP hydrolytic activity measured in the absence of PDE inhibitor and the fraction corresponding to PDE4 activity was assessed by including the PDE4 inhibitor Ro-20-1724. The protein expression of PDE4A, 4B and 4D was evaluated by Western blot using specific antibodies for each variant. The in vitro pacemaker activity was assessed by measuring the spontaneous Ca 2+ transients in Fluo4-loaded-SAN intact tissue. Images were obtained using confocal microscopy. Inhibition of PDE4 by Ro-20-1724 increased the beating rate of intact mouse SAN. PDE4 inhibition also altered Ca 2+ homeostasis, as shown by the presence of irregular Ca 2+ transients and increased PKA-phosphorylation of key ECC actors (ryanodine receptor, phospholamban) after treatment with Ro-20-1724. PDE4 enzymatic activity was found to account for 60% of the total cAMP-PDE activity in SAN. The 3 isoforms PDE4A, 4B and 4D were found to be expressed in mouse SAN. Our preliminary results reveal the involvement of PDE4 in the control of pacemaker activity in SAN. The characterization of pacemaker activity in PDE4B and PDE4D-null mice is under investigation. The author hereby declares no conflict of interest