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

OBJECTIVE: We have investigated the properties of the hyperpolarization-activated (I(f)) current in pacemaker cells from the mouse sino-atrial node (SAN). METHODS: 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). RESULTS: 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). CONCLUSIONS: 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.     

Role of the 293b-sensitive, slowly activating delayed rectifier potassium current, i(Ks), in pacemaker activity of rabbit isolated sino-atrial node cells.

OBJECTIVES: (i) to characterize the electrophysiological properties of the slowly activating delayed rectifier potassium current, i(Ks), defined as the 293b-sensitive current, during the action potential (AP) of rabbit sino-atrial node (SAN) pacemaker cells; (ii) to evaluate the contribution of i(Ks) to the pacemaker AP under physiological conditions and during beta-adrenergic stimulation. METHODS: Rabbit SAN pacemaker cells were studied using the perforated patch clamp technique in voltage-, AP- and current-clamp modes. RESULTS: Voltage-clamp findings. Block of i(Ks) by 293b is dose-dependent, with an IC(50) (half block) in rabbit SAN cells of 1.35 microM and an IC(80) (sub-maximal block) of 5 microM. Sub-maximal concentrations of 293b have no significant effects on long-lasting and transient inward calcium currents, i(Ca,L) and i(Ca,T), inward hyperpolarization activated current, i(f), and transient outward current, i(to). AP-clamp experiments. The 293b-sensitive current activates near the peak of the SAN pacemaker action potential, reaches a mean maximal current density of 1.0+/-0.3 pA/pF (n=8, cell capacitances 27 to 62 pF, mean 35+/-4.0 pF) during late repolarization, and inactivates towards the end of repolarization. Additionally, in two smaller cells (cell capacitances 15 and 23 pF), no discernible 293b-sensitive current component was detected. Current-clamp data. In spontaneously beating SAN cells under control conditions, sub-maximal block of i(Ks) by 5 microM 293b has negligible effects on action potential characteristics and does not change average cycle length (n=11). In contrast, after pre-treatment with 10 nM isoprenaline to mimic beta-adrenergic stimulation, cells showed a 293b-induced depolarization of maximum diastolic potential by 2.2+/-1%, a decrease in diastolic depolarization rate by 9.9+/-4%, and a slowing of late action potential repolarization by 28.7+/-10.2%, resulting in a prolongation of spontaneous cycle length by 9.8+/-3.0% (P<0.05, n=10; for all parameters). CONCLUSION: Our findings suggest that in rabbit SAN: (i) i(Ks) is activated during the normal pacemaker AP; (ii) the contribution of i(Ks) to beating rate is small under control conditions; and (iii) i(Ks) contributes significantly to spontaneous pacemaker rate during beta-adrenergic stimulation.     

Abnormal pacemaking is modulated by sarcoplasmic reticulum in partially-depolarized myocardium from dilated right atria in humans

Fifty human atrial specimens removed at time of cardiac surgery were studied in vitro. Thirty-four samples were selected as presenting partial cell depolarization and exhibiting slow response action potentials. Twenty of these preparations were automatic whereas 14 were not. Neither mean maximum diastolic potential (-52.8 +/- 1.3 mV and -49.3 +/- 2.2 mV respectively) nor maximum rate of depolarization (Vmax) (1.1 +/- 0.1 V/s and 1.3 +/- 0.8 V/s) significantly differed between these two groups. Abnormal automaticity due to phase 4 depolarization occurred in 12/13 preparations dissected from markedly dilated atria whereas it occurred in only 2/10 preparations sampled from non-dilated atria. A statistically significant relationship between in vitro abnormal pacemaking and atrial dilatations was found. We investigated the effects on abnormal pacemaker depolarization and automaticity of a reduction in the extracellular Na and Ca and of sarcoplasmic reticulum (SR) inhibitors. Abnormal pacemaker depolarization appeared to be much more sensitive to a reduction in the extracellular Na than in the extracellular Ca. Both Sr and Mg slowed the automatic rate. Ryanodine 3 X 10(-6) M, a specific SR inhibitor, irreversibly lengthened the spontaneous basic cycle duration to about 300% of control. Epinephrine up to 10(-4) M was ineffective in accelerating the residual spontaneous rhythm that persists after ryanodine action, although epinephrine markedly enhanced the overshoot and Vmax of the slow responses. It is concluded that, in the human atrial myocardium, abnormal pacemaking that develops at low level of membrane potential: is promoted by chamber dilatation; is strongly modulated by SR-dependent processes.     

The cardiac pacemaker current

In mammals cardiac rate is determined by the duration of the diastolic depolarization of sinoatrial node (SAN) cells which is mainly determined by the pacemaker I_f current. f-channels are encoded by four members of the hyperpolarization-activated cyclic nucleotide-gated gene (HCN1-4) family. HCN4 is the most abundant isoform in the SAN, and its relevance to pacemaking has been further supported by the discovery of four loss-of-function mutations in patients with mild or severe forms of cardiac rate disturbances. Due to its selective contribution to pacemaking, the I_f current is also the pharmacological target of a selective heart rate-reducing agent (ivabradine) currently used in the clinical practice. Albeit to a minor extent, the I_f current is also present in other spontaneously active myocytes of the cardiac conduction system (atrioventricular node and Purkinje fibres). In working atrial and ventricular myocytes f-channels are expressed at a very low level and do not play any physiological role; however in certain pathological conditions over-expression of HCN proteins may represent an arrhythmogenic mechanism. In this review some of the most recent findings on f/HCN channels contribution to pacemaking are described.     

Functional role of L-type Cav1.3 Ca2+ channels in cardiac pacemaker activity

The spontaneous activity of pacemaker cells in the sino-atrial node (SAN) controls the heart rhythm and rate under physiological conditions. Pacemaker activity in SAN cells is due to the presence of the diastolic depolarization, a slow depolarization phase that drives the membrane voltage from the end of an action potential to the threshold of a new action potential. SAN cells express a wide array of ionic channels, but we have limited knowledge about their functional role in pacemaker activity and we still do not know which channels play a prominent role in the generation of the diastolic depolarization. It is thus important to provide genetic evidence linking the activity of genes coding for ionic channels to specific alterations of pacemaker activity of SAN cells. Here, we show that target inactivation of the gene coding for alpha(1D) (Ca(v)1.3) Ca(2+) channels in the mouse not only significantly slows pacemaker activity but also promotes spontaneous arrhythmia in SAN pacemaker cells. These alterations of pacemaker activity are linked to abolition of the major component of the L-type current (I(Ca,L)) activating at negative voltages. Pharmacological analysis of I(Ca,L) demonstrates that Ca(v)1.3 gene inactivation specifically abolishes I(Ca,L) in the voltage range corresponding to the diastolic depolarization. Taken together, our data demonstrate that Ca(v)1.3 channels play a major role in the generation of cardiac pacemaker activity by contributing to diastolic depolarization in SAN pacemaker cells.     

SR Ca store refill—a key factor in cardiac pacemaking

This study presents a theoretical analysis of the role of store Ca²⁺ uptake on sinoatrial node (SAN) cell pacemaking. Two mechanisms have been shown to be involved in SAN pacemaking, these being: 1) the membrane oscillator model where rhythm generation is based on the interaction of voltage-dependent membrane ion channels and, 2) the store oscillator model where cyclical release of Ca²⁺ from intracellular Ca²⁺ stores depolarizes the membrane through activation of the sodium-calcium exchanger (NCX). The relative roles of these oscillators in generation and modulation of pacemaker rate have been vigorously debated and have many consequences. The main new outcomes of our study are: 1) uptake of Ca²⁺ by intracellular Ca²⁺ stores increases the maximum diastolic potential (MDP) by reducing the cytosolic Ca²⁺ concentration [Ca²⁺]_c and hence decreasing the NCX current; 2) this hyperpolarization enhances recruitment of key pacemaker currents (e.g. the hyperpolarization-activated HCN current (I_f) and T-type Ca²⁺ current (I_T-Ca)); 3) the resultant enhanced Ca²⁺ entry during the pacemaker depolarization increases [Ca²⁺]_c causing advancement of the store Ca²⁺ release cycle and increased NCX current. In overview, the novel feature of our study is an investigation of the role of store Ca²⁺ uptake on SAN pacemaking. This occurs during the early diastolic period and causes enhanced I_f, I_T-Ca and store release (and hence I_NCX) during the later diastolic period. There is thus a symbiotic interaction between the two pacemaker “clocks” over the entire diastolic period, this providing robust and highly malleable SAN pacemaking. Accounting for store Ca²⁺ uptake also provides insight into hitherto unexplained SAN behaviour, as we exemplify for the sinus bradycardia exhibited in catecholaminergic polymorphic ventricular tachycardia (CPVT).     

Action potentials that mimic fibrillation activate sodium current.

Ventricular fibrillation (VF) has brief action potentials (50-70 ms) with short diastolic intervals (10-30 ms). Under these conditions ion channel activity may be grossly different to normal sinus rhythm (NSR). In particular, sodium channel activation may not contribute to the generation and propagation of action potentials during VF. This study determined if sodium channels can be activated when action potentials mimic VF. Isolated chick ventricular myocytes (n=7) were voltage-clamped to quantitate fast inward sodium current. The voltage clamp protocol simulated VF with a 10 pulse train at 10 Hz (100 ms cycle length (CL)) and depolarization interval (action potential duration) ranging from 90 to 20 ms. After each train a test pulse was delivered from holding (-80 mV) in 10-ms steps. The train preceded each step pulse. Peak sodium current for control and each VF protocol occurred at a membrane potential (V(m)) of -10 mV. Sodium current was evident during brief resting intervals as short as 20 ms, albeit 10-20% of baseline. Resting intervals less than 60 ms shifted the sodium conductance activation curve from Vm(0.5)-30 mV to -22 mV membrane potential. Similar findings occurred when resting potential was at -65 mV, although there was less sodium current with all tested protocols. There was significantly less inactivation of sodium current when the prepulse was shorter (100 v 1000 ms). There was approximately 20% greater sodium current when the test pulse followed a short v long depolarized (>-80 mV) prepulse. Although the longer depolarization pulses produce approximately 20% greater sodium current at membrane potentials more negative than -80 mV. Lastly the time for half recovery of sodium current from activation was significantly less when the inactivating prepulse was short v long (45.9+/-9 v 118+/-20 ms, P<0.05). In conclusion, sodium current is evident when the diastolic rest interval is as brief as 10-20 ms. Rest interval, length of membrane depolarization and membrane potential interact to affect sodium channel activation, inactivation and recovery from inactivation. These data demonstrate that the brief action potentials at more depolarized membrane potentials seen during VF allow for inward sodium current upon depolarization, less sodium channel inactivation, and a faster recovery from inactivation, thereby compensating for a short diastolic rest interval. Therefore, it is likely that the inward sodium channel contributes to wave front propagation during ventricular fibrillation.     

Facilitation of the L-type calcium current in rabbit sino-atrial cells: effect on cardiac automaticity

OBJECTIVE: The L-type Ca(2+) current (I(Ca,L)) contributes to the generation and modulation of the pacemaker action potential (AP). We investigated facilitation of I(Ca,L) in sino-atrial cells. METHODS: Facilitation was studied in regularly-beating cells isolated enzymatically from young albino rabbits (0.8-1 kg). We used the whole-cell patch-clamp technique to vary the frequency of the test depolarizations evoked at -10 mV or the conditioning diastolic membrane potential prior to the test pulse. RESULTS: High frequencies (range 0.2-3.5 Hz) slowed the decay kinetics of I(Ca,L) evoked from a holding potential (HP) of -80 mV in 68% of cells resulting in a larger Ca(2+) influx during the test pulse. The amount of facilitation increased progressively between 0.2 and 3.0 Hz. When the frequency was changed from 0.1 to 1 Hz, the averaged increase in the time integral of I(Ca,L) was 27+/-7% (n=22). Application of conditioning voltages between -80 and -50 mV induced similar facilitation of I(Ca,L) in 73% of cells. The maximal increase of Ca(2+) entry occurred between -60 and -50 mV, and was on average 38+/-14% for conditioning prepulses of 5 s in duration (n=15). Numerical simulations of the pacemaker activity showed that facilitation of I(Ca,L) promotes stability of sino-atrial rate by enhancing Ca(2+) entry, thus establishing a negative feedback control against excessive heart rate slowing. CONCLUSION: Facilitation of I(Ca,L) is present in rabbit sino-atrial cells. The underlying mechanism reflects modulation of I(Ca,L) decay kinetics by diastolic membrane potential and frequency of depolarization. This phenomenon may provide an important regulatory mechanism of sino-atrial automaticity.     

Influence of postnatal-development on I(f) occurrence and properties in neonatal rat ventricular myocytes.

OBJECTIVE: I(f) is a hyperpolarization-activated current, which plays a key role in determining the spontaneous rate of cardiac pacemaker cells. We have previously shown that I(f) is also expressed in left ventricular myocytes isolated from spontaneously hypertensive rats; in these cells, its occurrence and density is linearly related with the severity of myocardial hypertrophy. Since hypertrophy induces a re-expression of genes encoding fetal proteins, we investigated changes in I(f) properties during post-natal development. METHODS: Fresh ventricular myocytes were enzymatically isolated from the heart of 1-2- to 28-day-old Wistar rats. The whole-cell configuration of the patch-clamp technique was employed to record the action potential and I(f). RESULTS: Membrane capacitance, an index of cell size, progressively increased from 13 +/- 1 pF at 1-2 days to 66 +/- 4 pF at 28 days of age (p < 0.01). At 1-2 days, a cesium-sensitive hyperpolarization-activated inward current (I(f)) was recorded in the majority of tested cells (n = 51). The midpoint of the activation curve (V1/2) was -78 +/- 2 mV (n = 32), and specific current conductance of fully activated I(f) (gf.max) was 60 +/- 11 pS/pF. Reversal potential (Vrev) measured by tail-current analysis was -24 +/- 3 mV (n = 8). Reduction of extracellular Na+ from 140 to 35 mM or extracellular K+ from 25 to 5.4 mM caused a shift of -12 +/- 1 mV (n = 3) or -11 +/- 2 mV (n = 5) of Vrev, respectively. Occurrence of I(f) decreased with aging, being present in 64%, 48% and 32% of cells at 10, 15 and 28 days, respectively. When present, I(f) density was significantly smaller than at 1-2 days (p < 0.05), reaching a value of 8 +/- 2 pS/pF at 28 days. However, V1/2 did not change in the older rats, being -80 +/- 2, -83 +/- 4 and -85 +/- 3 mV at 10, 15 and 28 days, respectively. Vrev at 10 and 15 days was -27 and -28 mV, respectively, thus suggesting that channel selectivity did not change. CONCLUSIONS: The pacemaker current, I(f), is expressed in ventricular myocytes from neonatal rats and progressively disappears; when present, it shows electrophysiological properties similar to I(f) re-expressed in hypertrophied adult rat ventricular myocytes. Thus, it is likely that the occurrence of I(f) in ventricular myocytes of hypertrophied and failing hearts is due to the re-expression of a fetal gene.     

Deep bradycardia and heart block caused by inducible cardiac-specific knockout of the pacemaker channel gene Hcn4

Cardiac pacemaking generation and modulation rely on the coordinated activity of several processes. Although a wealth of evidence indicates a relevant role of the I(f) ("funny," or pacemaker) current, whose molecular constituents are the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and particularly HCN4, work with mice where Hcn genes were knocked out, or functionally modified, has challenged this view. However, no previous studies used a cardiac-specific promoter to induce HCN4 ablation in adult mice. We report here that, in an inducible and cardiac-specific HCN4 knockout (ciHCN4-KO) mouse model, ablation of HCN4 consistently leads to progressive development of severe bradycardia (～50% reduction of original rate) and AV block, eventually leading to heart arrest and death in about 5 d. In vitro analysis of sinoatrial node (SAN) myocytes isolated from ciHCN4-KO mice at the mean time of death revealed a strong reduction of both the I(f) current (by ～70%) and of the spontaneous rate (by ～60%). In agreement with functional results, immunofluorescence and Western blot analysis showed reduced expression of HCN4 protein in SAN tissue and cells. In ciHCN4-KO animals, the residual I(f) was normally sensitive to β-adrenergic receptor (β-AR) modulation, and the permanence of rate response to β-AR stimulation was observed both in vivo and in vitro. Our data show that cardiac HCN4 channels are essential for normal heart impulse generation and conduction in adult mice and support the notion that dysfunctional HCN4 channels can be a direct cause of rhythm disorders. This work contributes to identifying the molecular mechanism responsible for cardiac pacemaking.     

Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers

We tested the ability of human mesenchymal stem cells (hMSCs) to deliver a biological pacemaker to the heart. hMSCs transfected with a cardiac pacemaker gene, mHCN2, by electroporation expressed high levels of Cs+-sensitive current (31.1+/-3.8 pA/pF at -150 mV) activating in the diastolic potential range with reversal potential of -37.5+/-1.0 mV, confirming the expressed current as I(f)-like. The expressed current responded to isoproterenol with an 11-mV positive shift in activation. Acetylcholine had no direct effect, but in the presence of isoproterenol, shifted activation 15 mV negative. Transfected hMSCs influenced beating rate in vitro when plated onto a localized region of a coverslip and overlaid with neonatal rat ventricular myocytes. The coculture beating rate was 93+/-16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161+/-4 bpm when hMSCs were expressing both EGFP+mHCN2 (P<0.05). We next injected 10(6) hMSCs transfected with either control plasmid or mHCN2 gene construct subepicardially in the canine left ventricular wall in situ. During sinus arrest, all control (EGFP) hearts had spontaneous rhythms (45+/-1 bpm, 2 of right-sided origin and 2 of left). In the EGFP+mHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rate=61+/-5 bpm; P<0.05). Moreover, immunostaining of the injected regions demonstrated the presence of hMSCs forming gap junctions with adjacent myocytes. These findings demonstrate that genetically modified hMSCs can express functional HCN2 channels in vitro and in vivo, mimicking overexpression of HCN2 genes in cardiac myocytes, and represent a novel delivery system for pacemaker genes into the heart or other electrical syncytia.     

Modulation of the hyperpolarization-activated current (I(f)) by calcium and calmodulin in the guinea-pig sino-atrial node

The aim of this study was to investigate possible regulation of the hyperpolarization-activated current (I(f)) by cytosolic calcium in guinea-pig sino-atrial (SA) node cells. Isolated SA node cells were superfused with physiological saline solution (36 degrees C) and the perforated patch voltage-clamp technique used to record I(f) activated by hyperpolarizing voltage steps. A 10-min loading of SA node cells with the calcium chelator BAPTA (using 10 microM BAPTA-AM) significantly reduced the amplitude of I(f) at all potentials studied (69+/-8% at -80 mV, n=6). BAPTA loading also shifted the voltage of half-activation (V(h)) of the conductance from -83+/-2 mV in control to -93+/-2 mV in BAPTA (n=6) without significantly altering the slope of activation. The calmodulin antagonists W-7 (10 microM), calmidazolium (25 microM) and ophiobolin A (20 microM) caused similar reductions in I(f) amplitude (73+/-4, 86+/-9 and 59+/-6% at -80 mV, n=6, 5 and 4, respectively) and shifts in V(h) (11+/-3, 14+/-3 and 8+/-2 mV). In cells pre-treated with W-7, exposure to BAPTA caused no further reduction in current amplitude (n=6). I(f) current amplitude was unaffected by the calmodulin dependent kinase (CaMKII) inhibitor KN-93 (1 microM) although this CaMKII inhibition did reduce L-type calcium by 48+/-19% at 0 mV (n=3). These results are consistent with a role for calcium and calmodulin in the regulation of I(f), via a mechanism that is independent of CaMKII. Alterations in intracellular calcium during the cardiac cycle may be involved in fine tuning the voltage-dependent properties of I(f) and may thus determine its relative contribution to pacemaking in the SA node.     

Contribution of sodium channel mutations to bradycardia and sinus node dysfunction in LQT3 families

One variant of the long-QT syndrome (LQT3) is caused by mutations in the human cardiac sodium channel gene. In addition to the characteristic QT prolongation, LQT3 carriers regularly present with bradycardia and sinus pauses. Therefore, we studied the effect of the 1795insD Na+ channel mutation on sinoatrial (SA) pacemaking. The 1795insD channel was previously characterized by the presence of a persistent inward current (Ipst) at -20 mV and a negative shift in voltage dependence of inactivation. In the present study, we first additionally characterized Ipst over the complete voltage range of the SA node action potential (AP) by measuring whole-cell Na+ currents (INa) in HEK-293 cells expressing either wild-type or 1795insD channels. Ipst for 1795insD channels varied between 0.8+/-0.2% and 1.9+/-0.8% of peak INa. Activity of 1795insD channels during SA node pacemaking was confirmed by AP clamp experiments. Next, Ipst and the negative shift were implemented into SA node AP models. The -10-mV shift decreased sinus rate by decreasing diastolic depolarization rate, whereas Ipst decreased sinus rate by AP prolongation, despite a concomitant increase in diastolic depolarization rate. In combination, moderate Ipst (1% to 2%) and the shift reduced sinus rate by approximately 10%. An additional increase in Ipst could result in plateau oscillations and failure to repolarize completely. Thus, Na+ channel mutations displaying an Ipst or a negative shift in inactivation may account for the bradycardia seen in LQT3 patients, whereas SA node pauses or arrest may result from failure of SA node cells to repolarize under conditions of extra net inward current.     

Localization of pacemaker channels in lipid rafts regulates channel kinetics

Lipid rafts are discrete membrane subdomains rich in sphingolipids and cholesterol. In ventricular myocytes a function of caveolae, a type of lipid rafts, is to concentrate in close proximity several proteins of the beta-adrenergic transduction pathway. We have investigated the subcellular localization of HCN4 channels expressed in HEK cells and studied the effects of such localization on the properties of pacemaker channels in HEK and rabbit sinoatrial (SAN) cells. We used a discontinuous sucrose gradient and Western blot analysis to detect HCN4 proteins in HEK and in SAN cells, and found that HCN4 proteins localize to low-density membrane fractions together with flotillin (HEK) or caveolin-3 (SAN), structural proteins of caveolae. Lipid raft disruption by cell incubation with methyl-beta-cyclodextrin (MbetaCD) impaired specific HCN4 localization. It also shifted the midpoint of activation of the HCN4 current in HEK cells and of I(f) in SAN cells to the positive direction by 11.9 and 10.4 mV, respectively. These latter effects were not due to elevation of basal cyclic nucleotide levels because the cholesterol-depletion treatment did not alter the current response to cyclic nucleotides. In accordance with an increased I(f), MbetaCD-treated SAN cells showed large increases of diastolic depolarization slope (87%) and rate (58%). We also found that the kinetics of HCN4- and native f-channel deactivation were slower after lipid raft disorganization. In conclusion, our work indicates that pacemaker channels localize to lipid rafts and that disruption of lipid rafts causes channels to redistribute within the membrane and modifies their kinetic properties.     

A hyperpolarization-activated inward current in human myocardial cells.

Normally-polarized tissue from the human atrial myocardium usually exhibits a diastolic depolarization phase which can be suppressed reversibly by Cs+ or enhanced by inhibiting the inward rectifier K+ current, iK1, with Ba2+. (Escande et al., 1986). Because the suppression of the diastolic slope by Cs+ leads to a hyperpolarization of the cell membrane at the end of the diastolic phase, it was suggested that Cs+ might inhibit an inward current responsible for diastolic depolarization. Among the ionic mechanisms underlying the diastolic depolarization phase of cardiac tissues, the hyperpolarization-activated inward current, if, fits well to explain the small diastolic slope of human atrial fibres. In other preparations, this inward current carried both by Na+ and K+ ions is rapidly deactivated during the action potential and entirely blocked by millimolar concentrations of Cs+ (DiFrancesco 1981; DiFrancesco, et al., 1986; Kokubun et al., 1982; Callewaert et al., 1984; Denyer and Brown, 1990). Such a current in human myocardial cells has not been characterized so far although its existence in human atrial trabeculae was previously reported in an abstract (Carmeliet, 1984). In the present study, we describe an inward current which activates upon hyperpolarization in patch-clamped single human atrial cells and shares similar characteristics with the if pacemaker current described in unicellular and intact preparations of mammalian cardiac tissues.     

Effect of 5-HT4 receptor stimulation on the pacemaker current I(f) in human isolated atrial myocytes

OBJECTIVE: 5-HT4 receptors are present in human atrial cells and their stimulation has been implicated in the genesis of atrial arrhythmias including atrial fibrillation. An I(f)-like current has been recorded in human atrial myocytes, where it is modulated by beta-adrenergic stimulation. In the present study, we investigated the effect of serotonin (5-hydroxytryptamine, 5-HT) on I(f) electrophysiological properties, in order to get an insight into the possible contribution of I(f) to the arrhythmogenic action of 5-HT in human atria. METHODS: Human atrial myocytes were isolated by enzymatic digestion from samples of atrial appendage of patients undergoing coeffective cardiac surgery. Patch-clamped cells were superfused with a modified Tyrode's solution in order to amplify I(f) and reduce overlapping currents. RESULTS AND CONCLUSIONS: A time-dependent, cesium-sensitive increasing inward current, that we had previously described having the electrophysiological properties of the pacemaker current I(f), was elicited by negative steps (-60 to -130 mV) from a holding potential of -40 mV. Boltzmann fit of control activation curves gave a midpoint (V1/2) of -88.9 +/- 2.6 mV (n = 14). 5-HT (1 microM) consistently caused a positive shift of V1/2 of 11.0 +/- 2.0 mV (n = 8, p < 0.001) of the activation curve toward less negative potentials, thus increasing the amount of current activated by clamp steps near the physiological maximum diastolic potential of these cells. The effect was dose-dependent, the EC50 being 0.14 microM. Maximum current amplitude was not changed by 5-HT. 5-HT did not increase I(f) amplitude when the current was maximally activated by cAMP perfused into the cell. The selective 5-HT4 antagonists, DAU 6285 (10 microM) and GR 125487 (1 microM), completely prevented the effect of 5-HT on I(f). The shift of V1/2 caused by 1 microM 5-HT in the presence of DAU 6285 or GR 125487 was 0.3 +/- 1 mV (n = 6) and 1.0 +/- 0.6 mV (n = 5), respectively (p < 0.01 versus 5-HT alone). The effect of 5-HT4 receptor blockade was specific, since neither DAU 6285 nor GR 125487 prevented the effect of 1 microM isoprenaline on I(f). Thus, 5-HT4 stimulation increases I(f) in human atrial myocytes; this effect may contribute to the arrhythmogenic action of 5-HT in human atrium.     

Thyroid hormone increases pacemaker activity in rat neonatal atrial myocytes

The effects of thyroid hormone (3,3',5-triiodo- L -thyronine, T3) on pacemaker activity were studied with electrophysiological and pharmacological approaches using spontaneously beating neonatal atrial myocytes cultured from 2-day-old rats. Treatment with T3 (10(-8)m) for 24-48 h led to a positive chronotropic effect. The beating rate of T3-treated cells was 244+/-19 beats/min and for control cells it was 122+/-10 beats/min (P<0.05). Action potentials were recorded and showed that the predominant effect of T3 was to increase the diastolic depolarization rate (99.5+/-9.8 in T3-treated group v 44.0+/-7.8 mV/s in untreated group). Some cells that exhibited pacemaker activity lacked a pacemaker current (I(f)) under voltage clamp conditions I(f)was recorded in 5 of 12 spontaneously active control cells and in 6 of 10 T3-treated cells. In those cells exhibiting the pacemaker current, the I(f)density was significantly larger in T3-treated cells (-7.9+/-2.6 pA/pF v-1.8+/-0.5 pA/pF in control). The L-type Ca2+ current density was similar in the two groups (at -7 mV, -7.5+/-1.5 in treated group v-8.6+/-1.0 pA/pF in control). In the presence of T3, the Na+-Ca2+ exchanger current (I(Na/Ca)) density was larger (e.g. at +60 mV, it was 4.8+/-0.5 v 3.5+/-0.2 pA/pF in control cells, P<0.05). As intracellular Ca2+ is extruded from the cell, the electrogenic Na+-Ca2+ exchanger causes a declining inward current, which may contribute to the pacemaker potential-this declining inward current was demonstrated using the action potential voltage clamp technique and was shown to be larger in T3-treated myocytes. Our data demonstrate that thyroid hormone enhances pacemaker activity and that this may be due in part to an increased Na+-Ca2+ exchanger activity.     

Sinoatrial node pacemaker activity requires Ca(2+)/calmodulin-dependent protein kinase II activation

Cardiac beating arises from the spontaneous rhythmic excitation of sinoatrial (SA) node cells. Here we report that SA node pacemaker activity is critically dependent on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In freshly dissociated rabbit single SA node cells, inhibition of CaMKII by a specific peptide inhibitor, autocamtide-2 inhibitory peptide (AIP, 10 micromol/L), or by KN-93 (0.1 to 3.0 micromol/L), but not its inactive analog, KN-92, depressed the rate and amplitude of spontaneous action potentials (APs) in a dose-dependent manner. Strikingly, 10 micromol/L AIP and 3 micromol/L KN-93 completely arrested SA node cells, which indicates that basal CaMKII activation is obligatory to the genesis of pacemaker AP. To understand the ionic mechanisms of the CaMKII effects, we measured L-type Ca(2+) current (I(Ca, L)), which contributes both to AP upstroke and to pacemaker depolarization. KN-93 (1 micromol/L), but not its inactive analog, KN-92, decreased I:(Ca, L) amplitude from 12+/-2 to 6+/-1 pA/pF without altering the shape of the current-voltage relationship. Both AIP and KN-93 shifted the midpoint of the steady-state inactivation curve leftward and markedly slowed the recovery of I(Ca, L) from inactivation. Similar results were observed using the fast Ca(2+) chelator BAPTA, whereas the slow Ca(2+) chelator EGTA had no significant effect, which suggests that CaMKII activity is preferentially regulated by local Ca(2+) transients. Indeed, confocal immunocytochemical imaging showed that active CaMKII is highly localized beneath the surface membrane in the vicinity of L-type channels and that AIP and KN-93 significantly reduced CaMKII activity. Thus, we conclude that CaMKII plays a vital role in regulating cardiac pacemaker activity mainly via modulating I(Ca, L) inactivation and reactivation, and local Ca(2+) is critically involved in these processes.     

Calcium influx through hyperpolarization-activated cation channels (I(h) channels) contributes to activity-evoked neuronal secretion

The hyperpolarization-activated cation channels (I(h)) play a distinct role in rhythmic activities in a variety of tissues, including neurons and cardiac cells. In the present study, we investigated whether Ca(2+) can permeate through the hyperpolarization-activated pacemaker channels (HCN) expressed in HEK293 cells and I(h) channels in dorsal root ganglion (DRG) neurons. Using combined measurements of whole-cell currents and fura-2 Ca(2+) imaging, we found that there is a Ca(2+) influx in proportion to I(h) induced by hyperpolarization in HEK293 cells. The I(h) channel blockers Cs(+) and ZD7288 inhibit both HCN current and Ca(2+) influx. Measurements of the fractional Ca(2+) current showed that it constitutes 0.60 +/- 0.02% of the net inward current through HCN4 at -120 mV. This fractional current is similar to that of the low Ca(2+)-permeable AMPA-R (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) channels in Purkinje neurons. In DRG neurons, activation of I(h) for 30 s also resulted in a Ca(2+) influx and an elevated action potential-induced secretion, as assayed by the increase in membrane capacitance. These results suggest a functional significance for I(h) channels in modulating neuronal secretion by permitting Ca(2+) influx at negative membrane potentials.     

Localization of f-channels to caveolae mediates specific beta2-adrenergic receptor modulation of rate in sinoatrial myocytes

beta(1)- and beta(2)-adrenergic receptors (ARs) coexist in different regions of the heart. The beta(2)/beta(1) expression ratio is higher in the sinoatrial node (SAN) than in atria and ventricles, but the specific contribution of either type of receptor to rate modulation is still not well established. We have recently demonstrated that pacemaker ("funny") f-channels are located in lipid rafts of the rabbit SAN. Since in ventricular myocytes beta(2)-, but not beta(1)-ARs, localize to caveolae, we hypothesized that modulation of f-channels and of pacemaker activity in SAN myocytes is controlled mainly by beta(2)-AR activation. To address this point, we investigated the caveolar localization of proteins by co-immunoprecipitation and immunocytochemistry, and found that f-channels interact with caveolin 3. We also recorded I(f) current and spontaneous activity from SAN myocytes, and found that beta-AR activation by the non-selective agonists isoproterenol and fenoterol shifted the I(f) activation curve similarly (by 6.3 and 5.3 mV) and increased similarly spontaneous rate (by 23.1% and 21.6%, respectively). Specific beta(2) stimulation had similar effects (4.9 mV shift of the activation curve and 16.9% rate increase), but specific beta(1) stimulation was less effective (1.7 mV shift and 7.2% rate increase). However, after caveolar disorganization by MbetaCD (2%), stimulation of beta(1)-ARs was as effective as non-specific beta-AR stimulation. These data show that specific stimulation of beta(2)-ARs is the main mechanism by which heart rate is modulated through a positive shift of the I(f) activation curve and that this mechanism requires specific membrane compartmentation.