Dual role of miR-1 in the development and function of sinoatrial cells.

miR-1, the most abundant miRNA in the heart, modulates expression of several transcription factors and ion channels. Conditions affecting the heart rate, such as endurance training and cardiac diseases, show a concomitant miR-1 up- or down-regulation. Here, we investigated the role of miR-1 overexpression in the development and function of sinoatrial (SAN) cells using murine embryonic stem cells (mESC). We generated mESCs either overexpressing miR-1 and EGFP (miR1OE) or EGFP only (EM). SAN-like cells were selected from differentiating mESC using the CD166 marker. Gene expression and electrophysiological analysis were carried out on both early mES-derived cardiac progenitors and SAN-like cells and on beating neonatal rat ventricular cardiomyocytes (NRVC) over-expressing miR-1. miR1OE cells increased significantly the proportion of CD166+ SAN precursors compared to EM cells (23% vs 12%) and the levels of the transcription factors TBX5 and TBX18, both involved in SAN development. miR1OE SAN-like cells were bradycardic (1,3 vs 2 Hz) compared to EM cells. In agreement with data on native SAN cells, EM SAN-like cardiomyocytes show two populations of cells expressing either slow- or fast-activating If currents; miR1OE SAN-like cells instead have only fast-activating If with a significantly reduced conductance. Western Blot and immunofluorescence analysis showed a reduced HCN4 signal in miR-1OE vs EM CD166+ precursors. Together these data point out to a specific down-regulation of the slow-activating HCN4 subunit by miR-1. Importantly, the rate and If alterations were independent of the developmental effects of miR-1, being similar in NRVC transiently overexpressing miR-1. In conclusion, we demonstrated a dual role of miR-1, during development it controls the proper development of sinoatrial-precursor, while in mature SAN-like cells it modulates the HCN4 pacemaker channel translation and thus the beating rate.

Cell-specific Dynamic Clamp analysis of the role of funny If current in cardiac pacemaking.

We used the Dynamic Clamp technique for i) comparative validation of conflicting computational models of the hyperpolarization-activated funny current, If, and ii) quantification of the role of If in mediating autonomic modulation of heart rate. Experimental protocols based on the injection of a real-time recalculated synthetic If current in sinoatrial rabbit cells were developed. Preliminary results of experiments mimicking the autonomic modulation of If demonstrated the need for a customization procedure to compensate for cellular heterogeneity. For this reason, we used a cell-specific approach, scaling the maximal conductance of the injected current based on the cell's spontaneous firing rate. The pacemaking rate, which was significantly reduced after application of Ivabradine, was restored by the injection of synthetic current based on the Severi-DiFrancesco formulation, while the injection of synthetic current based on the Maltsev-Lakatta formulation did not produce any significant variation. A positive virtual shift of the If activation curve, mimicking the Isoprenaline effects, led to a significant increase in pacemaking rate (+17.3 ± 6.7%, p < 0.01), although of lower magnitude than that induced by real Isoprenaline (+45.0 ± 26.1%). Similarly, a negative virtual shift of the activation curve significantly lowered the pacemaking rate (-11.8 ± 1.9%, p < 0.001), as did the application of real Acetylcholine (-20.5 ± 5.1%). The Dynamic Clamp approach, applied to the If study in cardiomyocytes for the first time and rate-adapted to manage intercellular variability, indicated that: i) the quantitative description of the If current in the Severi-DiFrancesco model accurately reproduces the effects of the real current on rabbit sinoatrial cell pacemaking rate and ii) a significant portion (50-60%) of the physiological autonomic rate modulation is due to the shift of the If activation curve.

Properties of ivabradine-induced block of HCN1 and HCN4 pacemaker channels.

Ivabradine is a 'heart rate-reducing' agent able to slow heart rate, without complicating side-effects. Its action results from a selective and specific block of pacemaker f-channels of the cardiac sinoatrial node (SAN). Investigation has shown that block by ivabradine requires open f-channels, is use dependent, and is affected by the direction of current flow. The constitutive elements of native pacemaker channels are the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, of which four isoforms (HCN1-4) are known; in rabbit SAN tissue HCN4 is expressed strongly, and HCN1 weakly. In this study we have investigated the blocking action of ivabradine on mouse (m) HCN1 and human (h) HCN4 channels heterologously expressed in HEK 293 cells. Ivabradine blocked both channels in a dose-dependent way with half-block concentrations of 0.94 microm for mHCN1 and 2.0 microm for hHCN4. Properties of block changed substantially for the two channels. Block of hHCN4 required open channels, was strengthened by depolarization and was relieved by hyperpolarization. Block of mHCN1 did not occur, nor was it relieved, when channels were in the open state during hyperpolarization; block required channels to be either closed, or in a transitional state between open and closed configurations. The dependence of block upon current flow was limited for hHCN4, and not significant for mHCN1 channels. In summary our results indicate that ivabradine is an 'open-channel' blocker of hHCN4, and a 'closed-channel' blocker of mHCN1 channels. The mode of action of ivabradine on the two channels is discussed by implementing a simplified version of a previously developed model of f-channel kinetics.

From funny current to HCN channels: 20 years of excitation.

The "funny" (pacemaker) current has unusual characteristics, including activation on hyperpolarization, permeability to K(+) and Na(+), modulation by internal cAMP, and a tiny, single-channel conductance. In cardiac cells and neurons, pacemaker channels control repetitive activity and excitability. The recent cloning of HCN subunits provides new insight into the molecular basis for the funny channel properties.

C terminus-mediated control of voltage and cAMP gating of hyperpolarization-activated cyclic nucleotide-gated channels.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) family of "pacemaker" channels includes 4 isoforms, the kinetics and cAMP-induced modulation of which differ quantitatively. Because HCN isoforms are highly homologous in the central region, but diverge more substantially in the N and C termini, we asked whether these latter regions could contribute to the determination of channel properties. To this aim, we analyzed activation/deactivation kinetics and the response to cAMP of heterologously expressed isoforms mHCN1 and rbHCN4 and verified that mHCN1 has much faster kinetics and lower cAMP sensitivity than rbHCN4. We then constructed rbHCN4 chimeras by replacing either the N or the C terminus, or both, with the analogous domains from mHCN1. We found that: 1) replacement of the N terminus (chimera N1-4) did not substantially modify either the kinetics or cAMP dependence of wild-type channels; 2) replacement of the C terminus, on the contrary, resulted in a chimeric channel (4-C1), the kinetics of which were strongly accelerated compared with rbHCN4, and that was fully insensitive to cAMP; 3) replacement of both N and C termini led to the same results as replacement of the C terminus alone. These results indicate that the C terminus of rbHCN4 contributes to the regulation of voltage- and cAMP-dependent channel gating, possibly through interaction with other intracellular regions not belonging to the N terminus.

Single-channel properties of the sinoatrial node Na+ current in the newborn rabbit.

We have reported previously that the sinoatrial node (SAN) in the newborn rabbit expresses a Na+ current (INa) with properties similar to the neuronal type-I isoform and that this current contributes to the net inward current flowing during diastolic depolarization. To characterize this current further we conducted cell-attached single-channel experiments in isolated newborn SAN myocytes. The Na+ channel was sensitive to divalent cation block and had a single-channel conductance of 25.6 pS in the absence of divalent cations. Kinetic compatibility between single-channel and previous whole-cell data was confirmed by measuring the time constant of current decay. At pacemaker potentials, time constants were of the order of tens of milliseconds. Additional experiments indicated that this slow inactivation arises because the Na+ channels expressed in the neonatal SAN tend to re-open frequently at potentials in the pacemaker range. We suggest that this is the mechanism by which a small tetrodotoxin (TTX)-sensitive current contributes to the total inward current flowing during slow diastolic depolarization in neonatal (but not adult) pacemaker myocytes.

Integrated allosteric model of voltage gating of HCN channels.

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.

Na(+) current contribution to the diastolic depolarization in newborn rabbit SA node cells.

Isolated newborn, but not adult, rabbit sinoatrial node (SAN) cells exhibit spontaneous activity that (unlike adult) are highly sensitive to the Na(+) current (I(Na)) blocker TTX. To investigate this TTX action on automaticity, cells were voltage clamped with ramp depolarizations mimicking the pacemaker phase of spontaneous cells (-60 to -20 mV, 35 mV/s). Ramps elicited a TTX-sensitive current in newborn (peak density 0.89 +/- 0.14 pA/pF, n = 24) but not adult (n = 5) cells. When depolarizing ramps were preceded by steplike depolarizations to mimic action potentials, ramp current decreased 54.6 +/- 8.0% (n = 3) but was not abolished. Additional experiments demonstrated that ramp current amplitude depended on the slope of the ramp and that TTX did not alter steady-state holding current at pacemaker potentials. This excluded a steady-state Na(+) window component and suggested a kinetic basis, which was investigated by measuring TTX-sensitive I(Na) during long step depolarizations. I(Na) exhibited a slow but complete inactivation time course at pacemaker voltages (tau = 33.9 +/- 3.9 ms at -50 mV), consistent with the rate-dependent ramp data. The data indicate that owing to slow inactivation of I(Na) at diastolic potentials, a small TTX-sensitive current flows during the diastolic depolarization in neonatal pacemaker myocytes.

Effects of dronedarone on acetylcholine-activated current in rabbit SAN cells.

1. The effect of the antiarrhythmic drug dronedarone on the Acetylcholine-activated K(+) current (I(K(ACh))) was investigated in single cells isolated from sinoatrial node (SAN) tissue of rabbit hearts. 2. Externally perfused dronedarone (0.001 - 1 microM) caused a potent, voltage independent block of I(K(ACh)). Fitting of the dose response curve of I(K(ACh)) block yielded an IC(50) value of 63 nM, a value over one order of magnitude lower than those reported for dronedarone block of other cardiac currents. 3. I(K(ACh)) block was not due to an inhibitory action of dronedarone on the muscarinic M2 receptor activation, since the drug was effective on I(K(ACh)) constitutively activated by intracellular perfusion with GTP-gammaS. 4. External cell perfusion with dronedarone inhibited the activity of I(K(ACh)) channels recorded from cell-attached patches by reducing the channel open probability (from 0.56 to 0.11) without modification of the single-channel conductance. 5. These data suggest that dronedarone blocks I(K(ACh)) channels either by disrupting the G-protein-mediated activation or by a direct inhibitory interaction with the channel protein.

Kinetic and ionic properties of the human HCN2 pacemaker channel.

Human cDNA coding for the hyperpolarization-activated "pacemaker" channel HCN2 was expressed in Phoenix cells and yielded an inward current (IhHCN2) activated on hyperpolarization. The average IhHCN2 was half-activated at -83.1 mV and its kinetics could be described by second-order Hodgkin-Huxley gating. The time constant curve was bell-shaped and peaked at -82.2 mV. With 115 mM external Na+ and 30 mM external K+, IhHCN2 reversed at -17.1 mV, and had a mean conductance of 5.6 nS. Reducing the external K+ or Na+ concentration led to a concentration-dependent reduction of the IhHCN2 conductance and to a hyperpolarizing shift of reversal potential. External Cs+ ions (5 mM) blocked IhHCN2 in a voltage-dependent way according to a Woodhull-type block model, at an electrical distance of 0.66 from the external membrane surface, and with a dissociation constant of 15 mM at 0 mV. Increasing cytoplasmic cAMP using forskolin increased IhHCN2 by shifting the current activation curve to more positive voltages (11.7 mV). Exposure of the intracellular side of inside-out macro-patches to cAMP led to a depolarizing shift of the channel open probability curve (15.2 mV with 10 microM cAMP). These results indicate that although hHCN2 channels share several properties with native cardiac f-channels, differences also exist in permeability and block properties, suggesting that native channels may not be composed simply of homomeric constructs.

Action of internal pronase on the f-channel kinetics in the rabbit SA node.

1. The hyperpolarization-activated If current was recorded in inside-out macropatches from sino-atrial (SA) node myocytes during exposure of their intracellular side to pronase, in an attempt to verify if cytoplasmic f-channel domains are involved in both voltage- and cAMP-dependent gating. 2. Superfusion with pronase caused a quick, dramatic acceleration of channel opening upon hyperpolarization and slowing, rapidly progressing into full blockade, of channel closing upon depolarization; these changes persisted after wash off of pronase and were irreversible, indicating proteolytic cleavage of channel regions which contribute to gating. 3. If recorded from patches normally responding to cAMP became totally insensitive to cAMP following pronase treatment, indicating partial or total removal of channel regions involved in the cAMP-dependent activation. 4. The fully activated I-V relationship was not modified by pronase, indicating that internal proteolysis did not affect the f-channel conductance. 5. The changes in If kinetics induced by pronase were due to a large depolarizing shift of the f-channel open probability curve (56.5 +/- 1.1 mV, n = 7). 6. These results are consistent with the hypothesis that cytoplasmic f-channel regions are implicated in dual voltage- and cAMP-dependent gating; also, since pronase does not abolish hyperpolarization-activated opening, an intrinsic voltage-dependent gating mechanism must exist which is inaccessible to proteolytic cleavage. A model scheme able to account for these data thus includes an intrinsic gating mechanism operating at depolarized voltages, and a blocking mechanism coupled to cAMP binding to the channel.

Activation of f-channels by cAMP analogues in macropatches from rabbit sino-atrial node myocytes.

1. The action of the two diastereometric phosphorothioate derivatives of cAMP, Rp-cAMPs and Sp-cAMPs, was investigated on hyperpolarization-activated 'pacemaker' current (i(f)) recorded in inside-out macropatches from rabbit sino-atrial (SA) node myocytes. 2. When superfused on the intracellular side of f-channels at the concentration of 10 microM, both cAMP derivatives accelerated i(f) activation; their action was moderately less pronounced than that due to the same concentration of cAMP. 3. The measurement of the i(f) conductance-voltage relation by voltage ramp protocols indicated that both cAMP analogues shift the activation curve of i(f) to more positive voltages with no change in maximal (fully activated) conductance. 4. Dose-response relationships of the shift of the i(f) activation curve showed that both Rp-cAMPs and Sp-cAMPs act as agonists in the cAMP-dependent direct f-channel activation. Fitting data to the Hill equation resulted in maximal shifts of 9.6 and 9.5 mV, apparent dissociation constants of 0.82 and 5.4 microM, and Hill coefficients of 0.82 and 1.12 for Sp-cAMPs and Rp-cAMPs, respectively. 5. The activating action of Rp-cAMPs, a known antagonist of cAMP in the activation of cAMP-dependent protein kinase, confirms previously established evidence that f-channel activation does not involve phosphorylation. These results also suggest that the cAMP binding site of f-channels may be structurally similar to the cyclic nucleotide binding site of olfactory receptor channels.

The newborn rabbit sino-atrial node expresses a neuronal type I-like Na+ channel.

1. Newborn rabbit sino-atrial node (SAN) myocytes were recently found to express a tetrodotoxin (TTX)-sensitive Na+ current. We now report that the dose-response relation indicates that this SAN Na+ channel has unusually high TTX sensitivity, with half-maximal inhibition (26 +/- 5 nM) which is more typical of neuronal than cardiac tissue. 2. Additional characterization used mu-conotoxin GIIIA and Cd2+ as relatively selective blockers of the skeletal and cardiac isoforms, respectively. mu-Conotoxin GIIIA had no effect on the current recorded from SAN myocytes, but the Cd2+ sensitivity was unexpectedly high for a neuronal isoform (half-maximal inhibition = 185 +/- 8 microM). 3. Analysis of the time constant of inactivation did not reveal evidence of multiple inactivation processes, with the data well fitted by a single, relatively rapid exponential (inactivation time constant = 0.58 +/- 0.03 ms at 0 mV). 4. In situ hybridization with anti-sense cDNA probes was used to test for expression of neuronal type I, II and III Na+ channel isoforms. Myocardial cells in newborn SAN tissue exhibited clear hybridization to the type I, but not the type II or III probes. No hybridization was observed in adult SAN tissue with any of the three probes. 5. It is concluded that the newborn SAN expresses a neuronal type I-like Na+ channel isoform, and that this probably accounts for the unusual characteristic of high sensitivity to both TTX and Cd2+.

Adenovirus-mediated expression of 5-HT1B receptors in cardiac ventricle myocytes; coupling to inwardly rectifying K+ channels.

The 5-HT1B receptor is expressed on nerve terminals where it inhibits neurotransmitter release. When expressed ectopically in fibroblasts, the 5-HT1B receptor inhibits adenylyl cyclase. However, in the central nervous system, the effect of this receptor on neurotransmitter release appears to be cAMP-independent. We therefore investigated alternative effector systems that might be activated by the 5-HT1B receptor. We constructed a recombinant adenovirus that allows expression of high levels of the 5-HT1B receptor in a variety of cells. We chose cardiac ventricle myocytes because they express a muscarinic-gated, inwardly rectifying K+ channel (i[KACh]). In infected ventricle cells, both 5-HT and the muscarinic receptor agonist, carbachol, elicited a similar inwardly rectifying K+ current. The currents elicited by these agonists were pertussis-toxin sensitive and were not additive. These results suggest a common signal transduction pathway for 5-HT1B and muscarinic receptors in ventricle cells.

A TTX-sensitive inward sodium current contributes to spontaneous activity in newborn rabbit sino-atrial node cells.

1. Single cells were isolated from the sinus node region of rabbits (2 days old to adult) to study the age-dependent contribution of the sodium current (iNa) to pacemaker activity. 2. Experiments were conducted in 50 mM Na(+)-Ca(2+)-free solution. All newborn cells (2-19 days) exhibited a TTX-sensitive, Mn(2+)-insensitive fast inward Na+ current (peak current density 115.5 +/- 11.9 pA pF-1 at 0 mV). Fifty per cent of young cells (20-40 days) possessed the current, but only one in ten adult cells. Current density decreased with development independently of cell capacitance. 3. Newborn cells exhibited a noticeable window current. With development, the position of the activation curve was shifted in the positive direction, while the inactivation was unaltered, resulting in reduced overlap of the two curves and hence less window current. 4. In newborn cells, 3 microM TTX significantly reduced all measured parameters of spontaneous action potentials, slowing rate by 63%. In contrast, there was no significant effect of TTX on rate or most of the same parameters in adult cells. 5. These results indicate that cells of the sinus node region exhibit a substantial TTX-sensitive current at birth. With development, both the density and frequency of occurrence of this current within the sinus node decrease, as does its contribution to automaticity.