Mouse Sinoatrial Node Cells Research Articles

Increase in Ca2+-Activated cAMP/PKA Signaling Prevents Hydroxychloroquine-Induced Bradycardia of the Cardiac Pacemaker

Bradycardia or tachycardia are known side effects of drugs that limit their clinical use. The heart pacemaker function which control the heart rate under normal conditions is determined by coupled clock system. Thus, interfering with specific clock mechanism will affect other clock mechanisms through changes in interconnected signaling and can lead to rhythm disturbance. However, upregulation of a different clock components can compensate for this change. We focus here on hydroxychloroquine (HCQ), which has been shown effective in treating COVID-19 patients, however its bradycardic side effect limits its clinical use. We aim to decipher the mechanisms underlying the effect of HCQ on pacemaker automaticity, to identify a potential drug that will eliminate the bradycardia. We used isolated rabbit sinoatrial node (SAN) cells, human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) and mouse SAN cells residing in SAN tissue. Further, we employed SAN cell computational model to suggest mechanistic insights of the effect of HCQ on pacemaker function. HCQ increased mean spontaneous beat interval and variability in all three models in parallel to slower intracellular kinetics. The computational model suggested that HCQ affects the pacemaker (funny) current (If), L-type Ca2+ current (ICa,L), transient outward potassium (Ito) and due to changes in Ca2+ kinetics, the sodium-calcium exchanger current (INCX). Co-application of 3’-isobutylmethylxanthine (IBMX) and HCQ prevented the increase in beat interval and variability in all three experimental models. The HCQ-induced increase in rabbit and mice SAN cell and hiPSC-CM spontaneous beat interval, can be prevented by a phosphodiester inhibitor that restores automaticity due to slower intracellular Ca2+ kinetics.

A novel 3D spatial model of mouse sinoatrial node cells for investigating impact of microstructure changes on pacemaker activity

Spontaneous beating of sinoatrial node (SAN) cells is mediated by a complex coupled system of both membrane- and Ca2+-clocks. Distinct from ventricular myocytes, SAN myocytes typically lack transversal tubules but instead display rich caveolae, i.e., small Ω-shaped invaginations of the plasma membrane. Disruption of caveolae has been associated with dysregulation of caveolar-associated ion channels and transporters and linked to Long QT syndrome and heart failure. However, their functional role in SAN pacemaking remains to be elucidated mechanistically.

Deciphering cellular signals in adult mouse sinoatrial node cells

SummarySinoatrial node (SAN) cells are the pacemakers of the heart. This study describes a method for culturing and infection of adult mouse SAN cells with FRET-based biosensors that can be exploited to examine signaling events. SAN cells cultured in media with blebbistatin or (S)-nitro-blebbistatin retain their morphology, protein distribution, action potential (AP) waveform, and cAMP dynamics for at least 40 h. SAN cells expressing targeted cAMP sensors show distinct β-adrenergic-mediated cAMP pools. Cyclic GMP, protein kinase A, Ca2+/CaM kinase II, and protein kinase D in SAN cells also show unique dynamics to different stimuli. Heart failure SAN cells show a decrease in cAMP and cGMP levels. In summary, a reliable method for maintaining adult mouse SAN cells in culture is presented, which facilitates studies of signaling networks and regulatory mechanisms during physiological and pathological conditions.

Deciphering Cellular Signals in Adult Mouse Sinoatrial Node Cells

The flexibility of manipulating the mouse genome makes cells from this species an ideal model to study cellular signals that control organ function. Genetically-modified mice provide opportunities to decipher how pacemaking sinoatrial node (SAN) cells are regulated by several second messengers and signaling molecules. However, detailed information on the spatiotemporal dynamics of these signaling events in adult mouse SAN cells has been limited due to the challenge of visualizing them in live cells. Here, a method is described for culturing and immediate infection of adult mouse SAN cells with genetically-encoded FRET-based biosensors to examine cellular dynamics of signaling events. SAN cells maintained in culture media containing blebbistatin or (S)-nitro-blebbistatin retain their elongated morphology and action potential (AP) waveform for up to 40 hours. The culturing condition does not change β-adrenergic-mediated cAMP signal as determined in freshly dissociated and cultured SAN cells from a cardiac-specific cAMP reporter mouse. SAN cells expressing cAMP sensors in different cellular compartments show distinct β-adrenergic-mediated cAMP pools. Examination of cyclic GMP (cGMP), protein kinase A (PKA), Ca2+/CaM kinase II (CaMKII) and protein kinase D (PKD) activity with specific FRET biosensors also show unique responses to different stimuli in SAN cells. Isolation and culture of SAN cells from heart failure mice reveal a decrease in cAMP and cGMP signaling. Thus, a reliable and efficient method for culturing adult mouse SAN cells has been developed to facilitate studies of signaling network dynamics during physiological and pathological conditions.

GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells

How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+ (GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαo over Gαi as a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαo or Gαi2 was consistent with these findings. Gαi2 ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαo ablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαo substrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

Increasing T-type calcium channel activity by β-adrenergic stimulation contributes to β-adrenergic regulation of heart rates.

Cav3.1 T-type Ca2+ channel current (ICa-T ) contributes to heart rate genesis but is not known to contribute to heart rate regulation by the sympathetic/β-adrenergic system (SAS). We show that the loss of Cav3.1 makes the beating rates of the heart in vivo and perfused hearts ex vivo, as well as sinoatrial node cells, less sensitive to β-adrenergic stimulation; it also renders less conduction acceleration through the atrioventricular node by β-adrenergic stimulation. Increasing Cav3.1 in cardiomyocytes has the opposite effects. ICa-T in sinoatrial nodal cells can be upregulated by β-adrenergic stimulation. The results of the present study add a new contribution to heart rate regulation by the SAS system and provide potential new mechanisms for the dysregulation of heart rate and conduction by the SAS in the heart. T-type Ca2+ channel can be a target for heart disease treatments that aim to slow down the heart rate ABSTRACT: Cav3.1 (α1G ) T-type Ca2+ channel (TTCC) is expressed in mouse sinoatrial node cells (SANCs) and atrioventricular (AV) nodal cells and contributes to heart rate (HR) genesis and AV conduction. However, its role in HR regulation and AV conduction acceleration by the β-adrenergic system (SAS) is unclear. In the present study, L- (ICa-L ) and T-type (ICa-T ) Ca2+ currents were recorded in SANCs from Cav3.1 transgenic (TG) and knockout (KO), and control mice. ICa-T was absent in KO SANCs but enhanced in TG SANCs. In anaesthetized animals, different doses of isoproterenol (ISO) were infused via the jugular vein and the HR was recorded. The EC50 of the HR response to ISO was lower in TG mice but higher in KO mice, and the maximal percentage of HR increase by ISO was greater in TG mice but less in KO mice. In Langendorff-perfused hearts, ISO increased HR and shortened PR intervals to a greater extent in TG but to a less extent in KO hearts. KO SANCs had significantly slower spontaneous beating rates than control SANCs before and after ISO; TG SANCs had similar basal beating rates as control SANCs probably as a result of decreased ICa-L but a greater response to ISO than control SANCs. ICa-T in SANCs was significantly increased by ISO. ICa-T upregulation by β-adrenergic stimulation contributes to HR and conduction regulation by the SAS. TTCC can be a target for slowing the HR.

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.

Crizotinib inhibits hyperpolarization-activated cyclic nucleotide-gated channel 4 activity

BackgroundSinus bradycardia is frequently observed in patients treated with crizotinib, a receptor tyrosine kinase inhibitor used for the treatment of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC). We investigated whether crizotinib could influence heart rate (HR) through direct cardiac effects.MethodsThe direct effect of crizotinib on HR was studied using ECG analysis of Langendorff-perfused mouse hearts. The whole-cell patch clamp technique was used to measure the effects of crizotinib on the hyperpolarization-activated funny current, If, in mouse sinoatrial node cells (SANCs) and hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) activity in HEK-293 cells stably expressing human HCN4.ResultsCrizotinib resulted in a dose-dependent reduction in HR in isolated intact mouse hearts with a half maximal inhibitory concentration (IC50) of 1.7 ± 0.4 μmol/L. Because ECG analysis revealed that crizotinib (0–5 μmol/L) resulted in significant reductions in HR in isolated mouse hearts without changes in PR, QRS, or QT intervals, we performed whole-cell patch clamp recordings of SANCs which showed that crizotinib inhibited If which regulates cardiac pacemaker activity. Crizotinib resulted in diminished current density of HCN4, the major molecular determinant of If, with an IC50 of 1.4 ± 0.3 μmol/L. Crizotinib also slowed HCN4 activation and shifted the activation curve to the left towards more hyperpolarized potentials.ConclusionsOur results suggest that crizotinib’s effects on HCN4 channels play a significant role in mediating its observed effects on HR.

Regulation of calcium clock-mediated pacemaking by inositol-1,4,5-trisphosphate receptors in mouse sinoatrial nodal cells.

Inositol-1,4,5-trisphosphate receptors (IP3 Rs) modulate pacemaking in embryonic heart, but their role in adult sinoatrial node (SAN) pacemaking is uncertain. We found that stimulation of IP3 Rs accelerates spontaneous pacing rate in isolated mouse SAN cells, whereas inhibition of IP3 Rs slows pacing. In atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells, where the Ca(2+) clock is uncoupled from the membrane clock, IP3 R agonists and antagonists modulate the rate of spontaneous Ca(2+) waves, suggesting that IP3 R-mediated Ca(2+) release modulates the Ca(2+) clock. IP3 R modulation also regulates Ca(2+) spark parameters, a reflection of ryanodine receptor open probability, consistent with the effect of IP3 signalling on Ca(2+) clock frequency. Modulation of Ca(2+) clock frequency by IP3 signalling in NCX KO SAN cells demonstrates that the effect is independent of NCX. These findings support development of IP3 signalling modulators for regulation of heart rate, particularly in heart failure where IP3 Rs are upregulated. Cardiac pacemaking initiated by the sinus node is attributable to the interplay of several membrane currents. These include the depolarizing 'funny current' (If ) and the sodium-calcium exchanger current (INCX ). The latter is activated by ryanodine receptor (RyR)-mediated calcium (Ca(2+) ) release from the sarcoplasmic reticulum (SR). Another SR Ca(2+) release channel, the inositol-1,4,5-triphosphate receptor (IP3 R), has been implicated in the generation of spontaneous Ca(2+) release in atrial and ventricular cardiomyocytes. Whether IP3 R-mediated Ca(2+) release also influences SAN automaticity is controversial, in part due to the confounding influence of periodic Ca(2+) flux through the sarcolemma accompanying each beat. We took advantage of atrial-specific sodium-calcium exchanger (NCX) knockout (KO) SAN cells to study the influence of IP3 signalling on cardiac pacemaking in a system where periodic intracellular Ca(2+) cycling persists despite the absence of depolarization or Ca(2+) flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca(2+) release in WT and NCX KO SAN cells in the presence or absence of an IP3 R blocker (2-aminoethoxydiphenyl borate, 2-APB), or during block of IP3 production by the phospholipase C inhibitor U73122. 2-APB and U73122 decreased the frequency of spontaneous Ca(2+) transients and waves in WT and NCX KO cells, respectively. Alternatively, increased IP3 production induced by phenylephrine increased Ca(2+) transient and wave frequency. We conclude that IP3 R-mediated SR Ca(2+) flux is crucial for initiating and modulating the RyR-mediated Ca(2+) cycling that regulates SAN pacemaking. Our results in NCX KO SAN cells also demonstrate that RyRs, but not NCX, are required for IP3 to modulate Ca(2+) clock frequency.

Store-operated calcium entry and the localization of STIM1 and Orai1 proteins in isolated mouse sinoatrial node cells.

In many non-excitable and excitable cells, store-operated calcium entry (SOCE) represents an additional pathway for calcium entry upon Ca2+ store depletion. In a previous study, we demonstrated SOCE activity in intact mouse cardiac pacemaker tissue, specifically from sinoatrial node (SAN) tissue. However, store content as a key determinant of SOCE activity is difficult to measure in intact SAN tissue. Therefore, to investigate the interaction between SOCE and store content and its role in cardiac pacemaking, it is necessary to investigate SOCE activity in single cardiac pacemaker cells. Furthermore, recent studies in other tissues have identified two new proteins involved in SOCE, stromal interacting molecule (STIM), which is an ER Ca2+ sensor, and the surface membrane channel Orai, a prototypic gene encoding for SOCE. However, whether STIM and Orai are expressed in native pacemaker cells is still unknown. In this current study, we examined SOCE activity in single firing pacemaker cells isolated from mouse sinoatrial node tissue. We found a significant rise in Ca2+ entry in response to Ca2+ store depletion. SOCE blockers reduced the amplitude and frequency of spontaneous Ca2+ transients and reduced Ca2+ store content. We demonstrated for the first time that STIM and Orai are expressed in pacemaker cells. After store depletion, STIM1 redistributed to the cell periphery and showed increased co-localization with surface membrane located Orai1, indicating a possible involvement of these proteins in SOCE activity in native cardiac pacemaker cells. These results suggest the novel concept that SOCE plays a functional role in regulating intracellular Ca2+ of cardiac pacemaker cells.

Characterization of BK Channels Cloned from Mouse Sinoatrial Node Cells

BK currents are well-established modulators of neural and smooth muscle excitability and recently we reported that BK currents alter the excitability of mouse sinoatrial node (SAN) cells, the predominant cardiac pacemaking cells (Lai et al. 2014, AJP- Heart & Circ Physiol). Because of the relatively small amplitude of native BK currents in SANCs, and the inability to record native BK currents under different Ca2+ conditions, we characterized currents produced by two BK channel variants cloned from SAN cells (BKVYR and BKQEERL). BK currents were recorded from inside-out patches from HEK293T cells in symmetrical K+. Analysis of the conductance-voltage relationships revealed similar V1/2s between BKVYR and BKQEERL currents (approximately 142 mV, 55 mV, and −27 mV at 1, 10, and 100 μM Ca2+, respectively). To gain insight on the mechanism underlying the role of BK currents in the SAN action potential, we also characterized BKVYR and BKQEERL currents at nominal, 1 μM, and 50 μM Ca2+ in physiological K+. Under physiological ionic conditions, BKVYR and BKQEERL currents began to activate at voltages near the maximum diastolic potential (MDP), approximately −60 mV, when the intracellular Ca2+ was 50 μM. By comparison, we previously reported that the native SAN cell BK currents activate at potentials positive to the MDP. These data suggest that native SAN BK currents require a significant elevation in intracellular Ca2+ to achieve meaningful BK current activation at physiological membrane potentials, and suggest close functional coupling of BK channels with Ca2+ sources in SAN cells.

Aminopyridine Lengthened the Plateau Phase of Action Potentials in Mouse Sinoatrial Node Cells

Duration of the plateau phase (phase 2) of action potentials in pacemaker cells in mouse sinoatrial node characterized by low upstroke rate during the rapid depolarization (dV/dt max <7 V/sec, phase 0) and in atrial-like cells with dV/dt max <100 V/sec were prolonged by 50% with 4-aminopyridine in concentrations of 0.7 and 0.1 mM, respectively. This blocker did not lengthen the terminal repolarization (phase 3). The above changes were accompanied by a 20-80% decrease in dV/dt max in comparison to the control. It is hypothesized that the short-term (transient) outward current Ito takes a part in the plateau formation and modulates the duration of the rapid depolarization phase of action potentials in mouse sinoatrial node cells.

BK Channel Blockers Regulate Mouse Sinoatrial Node Cell Firing

In the cardiovascular system, BK currents regulate vascular tone and may promote cardioprotection against ischemic injury. Recent experimental results also demonstrate that the BK channel blocker paxilline reduces the heart rate of mice in vivo, and in isolated rat hearts (Imlach, et al. 2010), suggesting a novel role for BK channels in basal heart rate regulation. However, the mechanism underlying this observation is unclear. We hypothesized that BK channel blockers reduce heart rate by altering electrical conduction within the heart. To test this hypothesis, we recorded electrocardiograms from awake, unrestrained mice in the presence of 8mg/kg paxilline, and examined changes in heart rate and cardiac conduction. Electrocardiograms show that the paxilline-induced reduction in heart rate in wild-type mice was associated with a 28.7 ± 4.3% (n = 8, p = 0.0002) elongation of the sinus (P-P) interval, suggesting that BK channels regulate cardiac pacing in vivo. Action potentials were recorded from isolated mouse sinoatrial node cells, and application of 3μM paxilline reduced the average instantaneous firing frequency from 4.81 ± 0.01Hz to 4.50 ± 0.07Hz (n = 8, p < 0.05). The effect of PAX on firing frequency was alleviated by 1μM isoproterenol (firing frequency = 5.28 ± 0.02Hz, n=5). Together, these results suggest that BK channels may influence basal cardiac function through a role in the sinoatrial node.

A mathematical model of action potentials of mouse sinoatrial node cells with molecular bases

Genetically modified mice are popular experimental models for studying the molecular bases and mechanisms of cardiac arrhythmia. A postgenome challenge is to classify the functional roles of genes in cardiac function. To unveil the functional role of various genetic isoforms of ion channels in generating cardiac pacemaking action potentials (APs), a mathematical model for spontaneous APs of mouse sinoatrial node (SAN) cells was developed. The model takes into account the biophysical properties of membrane ionic currents and intracellular mechanisms contributing to spontaneous mouse SAN APs. The model was validated by its ability to reproduce the physiological exceptionally short APs and high pacing rates of mouse SAN cells. The functional roles of individual membrane currents were evaluated by blocking their coding channels. The roles of intracellular Ca2+-handling mechanisms on cardiac pacemaking were also investigated in the model. The robustness of model pacemaking behavior was evaluated by means of one- and two-parameter analyses in wide parameter value ranges. This model provides a predictive tool for cellular level outcomes of electrophysiological experiments. It forms the basis for future model development and further studies into complex pacemaking mechanisms as more quantitative experimental data become available.

Local control of Ca2+-induced Ca2+ release in mouse sinoatrial node cells

Emerging evidence from large animal models implicates Ca2+ regulation, particularly intracellular sarcoplasmic reticulum (SR) Ca2+ release, as essential for sinoatrial node (SAN) automaticity. However, despite the apparent importance of SR Ca2+ release to SAN cell function it is uncertain how SR Ca2+ release is controlled in SAN cells from mouse. Understanding mouse SAN SR Ca2+ release mechanism will allow improved understanding of results in studies on SAN from genetic mouse models of Ca2+ homeostatic proteins. Here we investigated the functional relationship between sarcolemmal Ca2+ influx and SR Ca2+ release at the level of single SAN cell, using simultaneous patch-clamp current recording and high resolution confocal Ca2+ imaging techniques. In mouse SAN cells, both Ca2+ channel currents and triggered SR Ca2+ transients displayed bell-shaped, graded function with the membrane potential. Moreover, the gain function for Ca2+-induced Ca2+ release (CICR) displayed a monotonically decreasing function with strong voltage dependence, consistent with a “local control” mechanism for CICR. In addition, we observed numerous discrete Ca2+ sparks at the voltage range of diastolic depolarization, in sharp contrast to the much lower frequency of sparks observed at resting potentials. We concluded that the “local control” mechanism of CICR is responsible for both local Ca2+ release during diastolic depolarization and the synchronized Ca2+ transients observed during action potential in SAN cells.

TRPM4, a Ca2+-activated nonselective cation channel in mouse sino-atrial node cells

A calcium-activated nonselective cation channel (NSC(Ca)) has been recently described in several cardiac preparations. This channel is over-expressed in models of ventricular hypertrophy showing electrophysiological perturbations of heart activity, including occurrence of spontaneous activity. While these perturbations are currently attributed to a modification of the pacemaker I(f) current activity, arguments are also in favor of participation of an NSC(Ca). Similarly, the NSC(Ca) may be expressed in specialized pacemaker cells, i.e. sino-atrial node (SAN) cells. The aim of the present study was to detect such current in mouse pacemaker cells. The inside-out configuration of the patch-clamp technique was used in freshly isolated SAN cells from adult mice. Also, RT-PCR and Western-blotting studies were used to probe for TRPM4 mRNA and protein expression. In these cells, an NSC(Ca) activity was detected. The channel is voltage dependant with a conductance of 20.9+/-0.5 pS (n = 11). It is equally permeable for Na+ and K+ but does not conduct Ca2+. It is activated by rise in intracellular calcium concentrations and blocked by intracellular ATP (0.5 mmol/L). Also, as a new property in cardiac cells, the channel is activated by internal application of phosphatidylinositol 4,5-bisphosphate (10 microM). It is reversibly inhibited by flufenamic acid and glibenclamide. This channel shows the hallmarks of the TRPM4 molecule, a member of the TRP melastatin subfamily. We confirm the expression of this TRP channel on SAN cells by Western blotting and RT-PCR and validate that TRPM4 is glibenclamide sensitive. TRPM4 is functionally expressed in SAN cells and may be a key player in the generation and/or perturbation of heart rhythm.

Bradycardic and Proarrhythmic Properties of Sinus Node Inhibitors

Sinus node inhibitors reduce the heart rate presumably by blocking the pacemaker current If in the cardiac conduction system. This pacemaker current is carried by four hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels. We tested the potential subtype-specificity of the sinus node inhibitors cilobradine, ivabradine, and zatebradine using cloned HCN channels. All three substances blocked the slow inward current through human HCN1, HCN2, HCN3, and HCN4 channels. There was no subtype-specificity for the steady-state block, with mean IC50 values of 0.99, 2.25, and 1.96 microM for cilobradine, ivabradine, and zatebradine, respectively. Native If, recorded from mouse sinoatrial node cells, was slightly more efficiently blocked by cilobradine (IC50 value of 0.62 microM) than were the HCN currents. The block of I(f) in sinoatrial node cells resulted in slower and dysrhythmic spontaneous action potentials. The in vivo action of these blockers was analyzed using telemetric ECG recordings in mice. Each compound reduced the heart rate dose-dependently from 600 to 200 bpm with ED50 values of 1.2, 4.7, and 1.8 mg/kg for cilobradine, ivabradine, and zatebradine, respectively. beta-Adrenergic stimulation or forced physical activity only partly reversed this bradycardia. In addition to bradycardia, all three drugs induced increasing arrhythmia at concentrations greater than 5 mg/kg for cilobradine, greater than 10 mg/kg for zatebradine, or greater than 15 mg/kg for ivabradine. This dysrhythmic heart rate is characterized by periodic fluctuations of the duration between the T and P wave, resembling a form of sick sinus syndrome in humans. Hence, all available sinus node inhibitors possess an as-yet-unrecognized proarrhythmic potential.

A rapidly activating delayed rectifier K+ current regulates pacemaker activity in adult mouse sinoatrial node cells.

We have investigated the physiological role of the "rapidly activating" delayed rectifier K+ current (IKr) in pacemaker activity in isolated sinoatrial node (SAN) myocytes and the expression of mouse ether-a-go-go (mERG) genes in the adult mouse SAN. In isolated, voltage-clamped SAN cells, outward currents evoked by depolarizing steps (greater than -40 mV) were strongly inhibited by the class III methanesulfonanilide compound E-4031 (1-2.5 microM), and the deactivation "tail" currents that occurred during repolarization to a membrane potential of -45 mV were completely blocked. E-4031-sensitive currents (IKr) reached a maximum at a membrane potential of -10 mV and showed pronounced inward rectification at more-positive membrane potentials. Activation of IKr occurred at -40 to 0 mV, with half-activation at about -24 mV. The contribution of IKr to action potential repolarization and diastolic depolarization was estimated by determining the E-4031-sensitive current evoked during voltage clamp with a simulated mouse SAN action potential. IKr reached its peak value (approximately 0.6 pA/pF) near -25 mV, close to the midpoint of the repolarization phase of the simulated action potential, and deactivated almost completely during the diastolic interval. E-4031 (1 microM) slowed the spontaneous pacing rate of Langendorff-perfused, isolated adult mouse hearts by an average of 36.5% (n = 5). Expression of mRNA corresponding to three isoforms coded by the mouse ERG1 gene (mERG1), mERG1a, mERG1a', and mERG1b, was consistently found in the SAN. Our data provide the first detailed characterization of IKr in adult mouse SAN cells, demonstrate that this current plays an important role in pacemaker activity, and indicate that multiple isoforms of mERG1 can contribute to native SAN IKr.

Properties of the hyperpolarization-activated current (I(f)) in isolated mouse sino-atrial cells.

We have investigated the properties of the hyperpolarization-activated (I(f)) current in pacemaker cells from the mouse sino-atrial node (SAN). The I(f) current was studied in cells isolated enzymatically from the SAN region of adult C57BL6/J mice. The whole-cell variation of the patch-clamp technique was employed to investigate the basic properties of I(f). In mouse SAN cells, the I(f) current density at -120 mV was 18+/-2 pA/pF (n=23). I(f) was not detected in cells showing atrial-like morphology that were also found in SAN preparations (n=7). I(f) was blocked by 5 mM Cs(+), was inhibited by application of 5 microM acetylcholine, and was increased by 10 microM noradrenaline. The I(f) current reversal potential was -31+/-2 mV under physiological concentration of Na(+) and K(+) ions. Lowering the extracellular Na(+) concentration reduced I(f) amplitude, while increased when the extracellular K(+) concentration was augmented. I(f) voltage for half activation was -87+/-1 mV (n=6). We conclude that the native I(f) current in mouse SAN cells shows functional properties that are similar to I(f) described in rabbit SAN tissue. This study opens the possibility of investigating the involvement of I(f) in the regulation of heart rate in genetically modified mice.