Rabbit Sinoatrial Node Research Articles

Mechanistic Links Between Na + Channel (SCN5A) Mutations and Impaired Cardiac Pacemaking in Sick Sinus Syndrome

Familial sick sinus syndrome (SSS) has been linked to loss-of-function mutations of the SCN5A gene, which result in decreased inward Na(+) current, I(Na). However, the functional role of I(Na) in cardiac pacemaking is controversial, and mechanistic links between mutations and sinus node dysfunction in SSS are unclear. To determine mechanisms by which the SCN5A mutations impair cardiac pacemaking. Action potential (AP) models for rabbit sinoatrial node (SAN) cells were modified to incorporate experimentally reported I(Na) changes induced by 2 groups of SCN5A gene mutations (affecting the activation and inactivation of I(Na), respectively). The cell models were incorporated into an anatomically detailed 2D model of the intact SAN-atrium. Effects of the mutations and vagal nerve activity on cardiac pacemaking at the single-cell and tissue levels were studied. Multielectrode extracellular potential recordings of activation pattern from intact SAN-atrium preparations were performed to test predictions of the models. At the single-cell level, the mutations slowed down pacemaking rates in peripheral, but not in central SAN cells that control the heart rhythm. However, in tissue simulations, the mutations not only slowed down pacemaking, but also compromised AP conduction across the SAN-atrium, leading to a possible SAN exit block or sinus arrest, the major features of SSS. Simulated vagal nerve activity amplified the bradycardiac effects of the mutations. Two groups of SCN5A mutations showed subtle differences in impairing the ability of the SAN to drive the surrounding atrium, primarily attributable to their differential effects on atrial excitability and conduction safety. Experimental data with tetrodotoxin and carbachol confirmed the simulation outcomes. Our study substantiates the causative link between SCN5A gene mutations and SSS and illustrates mechanisms by which the mutations impair the driving ability of the SAN.

Large conductance Ca 2+-activated K + channels inhibit vagal acetylcholine release at the rabbit sinoatrial node

Although large conductance Ca 2+-activated K + (BK) channels play an important role in determining vascular tone, their role in the efferent cardiac vagal system remains to be elucidated. In anesthetized rabbits ( n = 9), acetylcholine (ACh) was measured at the right atrium near the sinoatrial node by a cardiac microdialysis technique, and the ACh release in response to electrical stimulation of the cervical preganglionic vagal nerves was examined. Local administration of a BK channel blocker iberiotoxin (2 μM) through a dialysis fiber increased the stimulation-induced ACh release from 7.6 ± 2.7 to 9.0 ± 3.2 nM ( P < 0.05). Addition of intravenous administration of iberiotoxin (0.11 mg/body) did not increase the stimulation-induced ACh release further (10.8 ± 4.4 nM). These results indicate that the BK channels play an inhibitory role in the vagal ACh release to the sinoatrial node.

Roles of hyperpolarization-activated current If in sinoatrial node pacemaking: insights from bifurcation analysis of mathematical models

To elucidate the roles of hyperpolarization-activated current (I(f)) in sinoatrial node (SAN) pacemaking, we theoretically investigated 1) the effects of I(f) on stability and bifurcation during hyperpolarization of SAN cells; 2) combined effects of I(f) and the sustained inward current (I(st)) or Na(+) channel current (I(Na)) on robustness of pacemaking against hyperpolarization; and 3) whether blocking I(f) abolishes pacemaker activity under certain conditions. Bifurcation analyses were performed for mathematical models of rabbit SAN cells; equilibrium points (EPs), periodic orbits, and their stability were determined as functions of parameters. Unstable steady-state potential region determined with applications of constant bias currents shrunk as I(f) density increased. In the central SAN cell, the critical acetylcholine concentration at which bifurcations, to yield a stable EP and quiescence, occur was increased by smaller I(f), but decreased by larger I(f). In contrast, the critical acetylcholine concentration and conductance of gap junctions between SAN and atrial cells at bifurcations progressively increased with enhancing I(f) in the peripheral SAN cell. These effects of I(f) were significantly attenuated by eliminating I(st) or I(Na), or by accelerating their inactivation. Under hyperpolarized conditions, blocking I(f) abolished SAN pacemaking via bifurcations. These results suggest that 1) I(f) itself cannot destabilize EPs; 2) I(f) improves SAN cell robustness against parasympathetic stimulation via preventing bifurcations in the presence of I(st) or I(Na); 3) I(f) dramatically enhances peripheral cell robustness against electrotonic loads of the atrium in combination with I(Na); and 4) pacemaker activity of hyperpolarized SAN cells could be abolished by blocking I(f).

Spontaneous Beating of Rabbit Sinoatrial Node Cells Requires Basal Protein Kinase C Activity

Cardiac rhythm is governed by the spontaneous rhythmic excitations of rabbit sinoatrial node cells (SANC), a crucial component of which is cAMP-mediated, PKA-dependent local subsarcolemmal Ca2+ releases (LCRs) from ryanodine receptors (RyR). LCRs activate inward Na+-Ca2+ exchange current during late diastolic depolarization (DD) leading to the increase in the DD rate and spontaneous SANC beating rate. Here we show that in addition to PKA-dependent phosphorylation, spontaneous SANC beating requires basal PKC activity. In freshly isolated SANC the PKC inhibitor GF109203X, 10 μmol/L, suppressed spontaneous SANC beating rate, recorded using perforated patch by ∼2.5-fold (from 154±7 to 60±14 beat/min, n=6). To study whether IP3 receptor-mediated Ca2+ release is involved in the PKC-dependent modulation of the spontaneous SANC beating, IP3 receptors were inhibited by 2 μmol/L 2-APB. However, 2-APB produced no significant changes in the spontaneous SANC beating rate suggesting no direct involvement of IP3-mediated Ca2+ release in the spontaneous SANC beating. Confocal imaging of isolated SANC loaded with the Ca2+indicator, Fluo-3, demonstrated that PKC inhibition markedly decreased LCR size from (from 8.6±0.5 to 4.0± 0.4 μm, n=3, P<0.01); LCR number per each spontaneous cycle (from 1.5± 0.3 to 0.6± 0.2, n=3, P<0.05), and prolonged the LCR period, the time from AP-induced Ca2+ transient to the time of LCR appearance (from 406 ± 7 to 836 ± 80 ms, n=3, P<0.05). The increase in the LCR period accompanied the increase in the spontaneous SANC cycle length, strongly suggesting that LCRs could be the major target of the basal PKC activity. Thus, our data show for the first time that the basal PKC-dependent activity regulates spontaneous SANC beating rate through modulation of LCRs from RyR, while IP3 receptors make no substantial contribution in this effect.

A Reduction of Spontaneous Beating Rate of Adult Rabbit Pacemaker Cells in Culture is Reversed by RGS2 Overexpression, Gi Inhibition or β-Ar Stimulation

Genetic manipulation of signaling proteins is an important tool to study signaling mechanisms. While rabbit sinoatrial node cells (SANC) are an excellent model for the study of autonomic signaling, genetic manipulation of freshly isolated rabbit SANC (f-SANC) is not possible. Here we report important characteristics of a cultured rabbit SANC model (c-SANC) that is suitable for manipulation of gene expression. C-SANC generate regular and rhythmic APs at 34±0.5oC, and beat spontaneously at a lower rate (1.35±0.02Hz, n=803) than f-SANC (2.79±0.04Hz, n=203 p<0.001).

Multiple effects of 4-aminopyridine on feline and rabbit sinoatrial node myocytes and multicellular preparations

4-aminopyridine (4-AP) is commonly used to block the transient outward potassium current, I(to), in cardiac and noncardiac tissues. In the present work, we found that 4-AP inhibited the rapid component of the delayed rectifier potassium current, I(Kr), in rabbit-isolated sinoatrial node myocytes by 25% (1 mM) and 51% (5 mM) and inhibited the slow component of the delayed rectifier potassium current, I(Ks), in cat- isolated sinoatrial node myocytes by 39% (1 mM) and 62% (5 mM). In cat- and rabbit-isolated sinoatrial node myocytes, 4-AP activated muscarinic receptors in a voltage-dependent manner to increase the acetylcholine-activated potassium current, I(KACh). In multicellular preparations of the central region of the sinoatrial node from nonreserpinized rabbits, 4-AP produced an increase in action potential overshoot, frequency, and rate of diastolic depolarization. In the presence of the beta-adrenergic antagonist propranolol, 4-AP produced a marked increase in duration and a marked decrease in maximum diastolic potential and eventually, cessation of the spontaneous activity in preparations from the sinoatrial central region. In multicellular preparations from reserpinized rabbits, 4-AP produced similar effects to those observed in the presence of propranolol. We conclude that 4-AP inhibits multiple cardiac K(+) currents, including I(to), I(Kr), and I(Ks), and that these activities mask I(KACh) activation. In addition, in multicellular preparations, 4-AP produces neurotransmitter release from the autonomic nerve terminals. These multiple effects need to be considered when using 4-AP as a "specific" I(to) blocker.

Effects of muscarinic receptor stimulation on Ca2+ transient, cAMP production and pacemaker frequency of rabbit sinoatrial node cells

We investigated the contribution of the intracellular calcium (Cai2+) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Cai2+ transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE®). Our data show that the Cai2+ transient, like the hyperpolarization-activated “funny current” (I f) and the ACh-sensitive potassium current (I K,ACh), is an important determinant of ACh-mediated pacemaker slowing. When I f and I K,ACh were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 μM ACh was still able to reduce pacemaker frequency by 72%. In these I f and I K,ACh-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Cai2+ transient decay (r 2 = 0.98) and slow diastolic Cai2+ rise (r 2 = 0.73). Inhibition of the Cai2+ transient by ryanodine (3 μM) or BAPTA-AM (5 μM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Cai2+ transient and reduced the sarcoplasmic reticulum (SR) Ca2+ content, all in a concentration-dependent fashion. At 1 μM ACh, the spontaneous activity and Cai2+ transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 μM) and I K,ACh was inhibited by tertiapin (100 nM). Also, inhibition of the Cai2+ transient by ryanodine (3 μM) or BAPTA-AM (25 μM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Cai2+ affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Cai2+ transient via a cAMP-dependent signaling pathway. Inhibition of the Cai2+ transient contributes to pacemaker slowing and inhibits Cai2+-stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Cai2+ transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.

Modulation of rabbit sinoatrial node activation sequence by acetylcholine and isoproterenol investigated with optical mapping technique

Changes in the rabbit sinoatrial node (SAN) activation sequence with the cholinergic and adrenergic factors were studied. The correlation between the sinus rhythm rate and the leading pacemaker site shift was determined. The hypothesis concerning the cholinergic suppression of nodal cell excitability as one of the mechanisms associated with pacemaker shift was tested. A high-resolution optical mapping technique was used to register beat-to-beat changes in the SAN activation pattern under the influence of the cholinergic and adrenergic factors. Acetylcholine (10 microm) and strong intramural parasympathetic nerve stimulation caused a pacemaker shift as well as rhythmic slowing and the formation of an inexcitable region in the central part of SAN. In this region the generation of action potentials was suppressed. The slowing of the sinus rhythm (which exceeded 12.8 +/- 3.1% of the rhythm control rate) always accompanied the pacemaker shift. Isoproterenol (10, 100 nm, 1 microm) and sympathetic postganglionic nerve stimulation also evoked a pacemaker shift but without formation of an inexcitable zone. The acceleration of the sinus rhythm, which exceeded 10.5 +/- 1.3% of the control rate of the rhythm, always accompanied the shift. Both cholinergic and adrenergic factors cause pacemaker shifts in the rabbit SAN. While modest changes in the sinus rhythm do not coincide with the pacemaker shift, greater changes always accompany the shift and may be caused by it, according to one hypothesis. The formation of an inexcitable zone at the place where the leading pacemaker is situated is one of the mechanisms associated with pacemaker shift.

Glycyrretinic acid blocks cardiac sodium channels expressed in Xenopus oocytes

Ethnopharmacological relevance Licorice has been used to treat many ailments including cardiovascular disorders in China for long time . Recent studies have shown that the cardiac actions of licorice have been attributed to its active component, glycyrretinic acid (GA). However, its mechanism remains poorly understood. Aim of the Study The effects of GA on the cardiac sodium currents ( I Na), L-type calcium currents ( I Ca,L) and hyperpolarization-activated inward currents ( I f) were investigated. Materials and methods Human isoforms of wild-type and ΔKPQ-mutant type sodium channels were expressed in Xenopus oocytes, and the resulting currents (peak and late I Na) were recorded using a two-microelectrode voltage-clamp technique. A perforated patch clamp technique was employed to record I Ca,L and I f from isolated rabbit sinoatrial node pacemaker cells. Results GA inhibited peak I Na (33% at 90 μM) and late I Na (72% at 90 μM), but caused no significant effects on I Ca,L and I f. Conclusion GA blocked cardiac sodium currents, particularly late I Na. Our findings might help to understand the traditional use of licorice in the treatment of cardiovascular disorders, because reduction of sodium currents (particularly late I Na) would be expected to provide protection from Na +-induced Ca 2+ overload and cell damage.

Stochastic vagal modulation of cardiac pacemaking may lead to erroneous identification of cardiac “chaos”

Fluctuations in the time interval between two consecutive R-waves of electrocardiogram during normal sinus rhythm may result from irregularities in the autonomic drive of the pacemaking sinoatrial node (SAN). We use a biophysically detailed mathematical model of the action potentials of rabbit SAN to quantify the effects of fluctuations in acetylcholine (ACh) on the pacemaker activity of the SAN and its variability. Fluctuations in ACh concentration model the effect of stochastic activity in the vagal parasympathetic fibers that innervate the SAN and produce varying rates of depolarization during the pacemaker potential, leading to fluctuations in cycle length (CL). Both the estimated maximal Lyapunov exponent and the noise limit of the resultant sequence of fluctuating CLs suggest chaotic dynamics. Apparently chaotic heart rate variability (HRV) seen in sinus rhythm can be produced by stochastic modulation of the SAN. The identification of HRV data as chaotic by use of time series measures such as a positive maximal Lyapunov exponent or positive noise limit requires both caution and a quantitative, predictive mechanistic model that is fully deterministic.

Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node

Several studies of the pacemaker mechanisms in mammalian cells, most of which were carried out in cells isolated from the rabbit sinoatrial node (SAN), have highlighted the role of the I f current. While the distribution of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, the molecular correlates of f-channels, is known at the mRNA level, the identification of f-channel proteins in this tissue is still undetermined. Here we investigate HCN protein expression in the rabbit pacemaker region. We found that HCN4 is the main isoform, and set therefore to analyze its distribution within the SAN and surrounding areas with the aim of correlating protein expression and pacemaking function. The analysis was carried out in tissue slices and single cells of the intercaval area, which includes the crista terminalis (CT), the SAN, and the septum interatrialis (SI). Immunolabeling, in situ hybridization, qRT-PCR analysis, and electrophysiological recordings identified the SAN as a region characterized by high HCN4 signal and current levels, while the expression in the CT and in the SI was either negligible or absent. Detailed analysis of the central SAN area showed that cells are predominantly distributed in islets interconnected by cell prolongations, and single-cell HCN4 labeling suggested sites of channel clustering. Our data indicate that in the rabbit SAN, HCN4 proteins are major constituents of native f-channels, and their distribution matches closely the SAN as defined morphologically and electrophysiologically. Until recently, the SAN was identified as the region where Cx43 and atrial natriuretic peptide are not expressed; we propose here that expression of HCN4 is an appropriate tool to map and identify the cardiac SAN pacemaker region.

Inhomogeneous distribution of action potential characteristics in the rabbit sino-atrial node revealed by voltage imaging

The sino-atrial node (SAN) is the natural pacemaker of the heart. Mechanisms of the leading pacemaker site generation and dynamic pacemaker shifts in the SAN have been so far studied with an electrophysiological technique, but the detailed spatial distribution of action potential characteristics in the SAN has not been analyzed due to the limited number of simultaneously recorded sites in microelectrode recording. To elucidate the mechanism of leading pacemaker site generation in the SAN, we applied a voltage imaging technique and analyzed the spatial distribution of action potential characteristics in the rabbit SAN. Action potential parameters, i.e., action potential duration at 50% repolarization level, the slope of upstroke, and the slope of the linearly depolarizing early phase of pacemaker activity (phase-4), were calculated from optical signals. Action potential parameter values derived from intracellular recording with a microelectrode and those from optical recording were significantly correlated. The leading pacemaker site occurred in the region of either globally or locally maximum phase-4 slope in 7 of 12 preparations, however, it did not coincide with the region of the early maximum phase-4 slope in the other 5 preparations. Carbenoxolone, a gap junction blocker, changed action potential properties and caused pacemaker shifts. Model simulation, assuming an inhomogeneous distribution of intrinsic properties of SAN cells, reproduced the experimental results. We conclude that the functional structure of the SAN is more inhomogeneous than that dictated by previous models. Besides intrinsic cellular properties, cell-to-cell interaction through gap junctions influences action potential characteristics and leading pacemaker site generation.

Lidocaine Inhibition of HCN1 Channels is Fast, Voltage-dependent and Reversible

Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) channels underlie the funny current or the hyperpolarization-activated current (If or Ih), which is important in regulating excitability in the neurons of the central nervous system and in the conduction tissue of the heart. There are four mammalian isoforms of HCN channels (HCN1-4), each exhibiting different kinetics, voltage dependence, and amounts of inward, time-dependent current (If). Lidocaine, a local anesthetic and antiarrhythmic drug, has been shown to inhibit HCN-mediated currents in the rabbit sinoatrial (SA) node, which expresses various HCN isoforms. Previously, we showed that lidocaine, at concentrations ranging from 20 to 200 μM, inhibits mouse HCN1 channels, but the rate and extent of both inhibition and drug washout, were not determined. Here, these issues are examined using Chinese hamster ovary (CHO) cells, transiently transfected with mouse HCN1 DNA, measurement of resulting currents using the whole-cell patch clamp technique, and a fast perfusion system for the application of lidocaine. We found that the onset of lidocaine action is fast (∼6 seconds) with maximum inhibition occurring at approximately 20 seconds, and that its effects are partially reversible. With 200 μM lidocaine perfused directly onto the cell under study, If elicited in response to -85mV and -100mV test pulses was reduced by 18.4±3.6 % (n= 6 cells) and 8.3±1.0 % (n= 7 cells), respectively, with average current return of 49.1±10.9 % and 73.9±9.4 % to the level prior to lidocaine addition. The fact that currents did not return completely may be attributed to a phenomenon called current rundown, or to incomplete washout of lidocaine. The significant difference in current reduction between -85mV and -100mV suggests that the effect of lidocaine on HCN1 channels depends on voltage, with greater If inhibition at less negative voltages.

Electrophysiological Evaluation of Novel Blockers of If Current

In the sino-atrial node (SAN) a major role in rhythm generation is played by f-channels. They mediate a sodium-potassium inward current (If) activated upon hyperpolarization and encoded by HCN genes (HCN1-4). If is overexpressed in cardiac diseases at ventricular level, where it may contribute to the increased propensity for arrhythmias. Selective f-channel blockers have a potential therapeutic use as bradycardic and antiarrhythmic agents. Zatebradine and ivabradine act as f-channel blockers but they lack cardiac selectivity, blocking the neuronal HCN isoforms. As a consequence an unmet need exists to develop new blockers selective for the mammalian SAN channel isoform, HCN4. Zatebradine analogues (C1-C5) were synthesized and the effect on If was measured on patch-clamped HEK293 cells expressing mHCN1, mHCN2 and hHCN4 and native guinea-pig and rabbit SAN cells. At 10 μM concentration all compounds reduced maximal If amplitude; however, potencies (defined by EC50) differed considerably. Ivabradine, taken as reference compound, showed no isoform selectivity; C1 and C4 were more potent on HCN1, the ratio being 4.3 (HCN2/HCN1) and 7.5 (HCN4/HCN1). C2 was more potent on HCN4, the ratio being 6 and 17 vs. HCN1 and HCN2, respectively. C3 was equipotent on HCN1 and HCN4 and C5 had low activity on all isoforms. Blockade was concentration-dependent, did not reverse upon drug removal and did not change current activation properties. Finally, data obtained in SAN cells show that effects on native If resemble those obtained on HCN4 isoform, in accordance with the hypothesis that HCN4 has a major contribution in SAN cells. Present results indicate that drug interaction with different HCN isoforms has diverse structural requirements. Current investigations are aimed to characterize the pharmacological profile of the new f-channel blockers and improve their isoform selectivity (Supported by EU - LSH M/CT/2006/018676, Normacor).

Beating to time: calcium clocks, voltage clocks, and cardiac pacemaker activity

the heartbeat arises in the sinoatrial node (SAN) and then spreads throughout the heart. SAN function is therefore essential for normal cardiac physiology. The function of the SAN decreases with age, and this has been correlated with changes of ion channel expression ([12][1]). It is therefore of

Regional Difference in Dynamical Property of Sinoatrial Node Pacemaking: Role of Na + Channel Current

To elucidate the regional differences in sinoatrial node pacemaking mechanisms, we investigated 1), bifurcation structures during current blocks or hyperpolarization of the central and peripheral cells, 2), ionic bases of regional differences in bifurcation structures, and 3), the role of Na + channel current ( I Na) in peripheral cell pacemaking. Bifurcation analyses were performed for mathematical models of the rabbit sinoatrial node central and peripheral cells; equilibrium points, periodic orbits, and their stability were determined as functions of parameters. Structural stability against applications of acetylcholine or electrotonic modulations of the atrium was also evaluated. Blocking L-type Ca 2+ channel current ( I Ca,L) stabilized equilibrium points and abolished pacemaking in both the center and periphery. Critical acetylcholine concentration and gap junction conductance for pacemaker cessation were higher in the periphery than in the center, being dramatically reduced by blocking I Na. Under hyperpolarized conditions, blocking I Na, but not eliminating I Ca,L, abolished peripheral cell pacemaking. These results suggest that 1), I Ca,L is responsible for basal pacemaking in both the central and peripheral cells, 2), the peripheral cell is more robust in withstanding hyperpolarizing loads than the central cell, 3), I Na improves the structural stability to hyperpolarizing loads, and 4), I Na-dependent pacemaking is possible in hyperpolarized peripheral cells.

Fibroblasts: modulating the rhythm of the heart

Cardiac fibroblasts have been implicated in arrhythmia initiation and maintenance affecting electrical propagation through slow, discontinuous conduction. Fibroblasts are unexcitable cells, their resting membrane potential is more depolarized than that of myocytes and their membrane resistance is higher. These characteristics suggest that, if coupled to myocytes, fibroblasts may function as current sinks and/or sources for electrical charge and as shortand long-range conductors. Abnormal accumulation of fibroblasts occurs in the heart under pathological conditions and ageing, which may alter normal cardiac function. Most clinical, experimental and numerical studies have regarded fibroblasts and fibrosis as electrically insulating obstacles. However, recent suggest that when coupled to cardiac myocytes, fibroblasts could influence impulse propagation not only by acting as passive transducers but also by influencing excitability of neighbouring myocytes (Miragoli et al. 2007). Compared to the atria and ventricles, the sinoatrial (SA) node contains a relatively higher amount of fibroblasts, which can be electrically coupled to pacemaker cells. Electrical coupling through gap junctions in the SA node is important for achieving pacemaker synchronization (Jalife, 1984). Pacemaker cells in the intact SA node beat at a common frequency but when isolated they show large variations in intrinsic beating frequency. Functional electrical coupling of SA nodal pacemaker cells through connexins forming gap junctions has been demonstrated in several species. However, the connexin isotypes expressed in the SA node vary somewhat depending on the species. Cx43, the most abundant connexin in the heart, has also been found in the SA node of rabbit, hamster and guinea pig although it is less abundant than other connexins. Interestingly, Cx43 was not detected in rat, cow or human nodal tissue. Recently, Kreuzberg et al. (2005) demonstrated that the mouse SA node expresses Cx30.2. Further studies using Cx30.2-knock-out mice suggest that Cx30.2 provides higher intercellular resistance and slower conductance due to a relatively small unitary conductance (∼9 pS). Nevertheless, the role of gap junctions in heterocellular tissue in the SA node remains unknown. The study by Fahrenbach et al. (2007) provides indirect evidence toward addressing this question. They investigated the mechanism through which non-excitable cells influence the spontaneous activity of pacemaker cells using heterocellular monolayers of HL-1 cells, a cell line derived from murine atrial myocyte tumour lineage AT-1, and fibroblasts that were isolated from neonatal rat and mouse ventricular tissue. The influence of fibroblasts on frequency of spontaneous activity, conduction velocity and interbeat interval variability were studied by co-culturing fibroblasts and myocytes at varying ratios.

Ca2+-stimulated Basal Adenylyl Cyclase Activity Localization in Membrane Lipid Microdomains of Cardiac Sinoatrial Nodal Pacemaker Cells

Spontaneous, rhythmic subsarcolemmal local Ca(2+) releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca(2+) releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca(2+) and cAMP/PKA regulatory components of automaticity are coupled via Ca(2+) activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca(2+)-activated AC isoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca(2+) over the entire physiological Ca(2+) range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca(2+) cycling proteins drive sarcoplasmic reticulum Ca(2+) releases, which, in turn, activate ACs. This feed forward "fail safe" system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells.

Constitutive Phosphodiesterase Activity Restricts Spontaneous Beating Rate of Cardiac Pacemaker Cells by Suppressing Local Ca 2+ Releases

Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMP-mediated, protein kinase A-dependent local subsarcolemmal ryanodine receptor Ca(2+) releases (LCRs). LCRs activated an inward Na(+)/Ca(2+) exchange current that increases the terminal diastolic depolarization rate and, therefore, the spontaneous SANC beating rate. Basal cAMP in SANCs is elevated, suggesting that cAMP degradation by phosphodiesterases (PDEs) may be low. Surprisingly, total suppression of PDE activity with a broad-spectrum PDE inhibitor, 3'-isobutylmethylxanthine (IBMX), produced a 9-fold increase in the cAMP level, doubled cAMP-mediated, protein kinase A-dependent phospholamban phosphorylation, and increased SANC firing rate by approximately 55%, indicating a high basal activity of PDEs in SANCs. A comparison of specific PDE1 to -5 inhibitors revealed that the specific PDE3 inhibitor, milrinone, accelerated spontaneous firing by approximately 47% (effects of others were minor) and increased amplitude of L-type Ca(2+) current (I(Ca,L)) by approximately 46%, indicating that PDE3 was the major constitutively active PDE in the basal state. PDE-dependent control of the spontaneous SANC firing was critically dependent on subsarcolemmal LCRs, ie, PDE inhibition increased LCR amplitude and size and decreased LCR period, leading to earlier and augmented LCR Ca(2+) release, Na(+)/Ca(2+) exchange current, and an increase in the firing rate. When ryanodine receptors were disabled by ryanodine, neither IBMX nor milrinone was able to amplify LCRs, accelerate diastolic depolarization rate, or increase the SANC firing rate, despite preserved PDE inhibition-induced augmentation of I(Ca,L) amplitude. Thus, basal constitutive PDE activation provides a novel and powerful mechanism to decrease cAMP, limit cAMP-mediated, protein kinase A-dependent increase of diastolic ryanodine receptor Ca(2+) release, and restrict the spontaneous SANC beating rate.

Dynamic imaging analysis of the pacemaker activity within the rabbit sino-atrial node

Dynamic imaging analysis of the pacemaker activity within the rabbit sino-atrial node