Cardiovascular Research

Cardiovascular Research 2001 52(1):51-64; doi:10.1016/S0008-6363(01)00370-4
© 2001 by European Society of Cardiology

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Copyright © 2001, European Society of Cardiology

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

Matteo E. Mangoni^* and Joël Nargeot

UPR 1142,CNRS, Institut de Génétique Humaine, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France

^* Corresponding author. Tel.: +33-499-619-939; fax: +33-499-619-901 matteo.mangoni{at}igh.cnrs.fr joel.nargeot{at}igh.cnrs.fr

Received 26 January 2001; accepted 29 May 2001

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
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 µM acetylcholine, and was increased by 10 µM 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.

KEYWORDS Ion channels; Impulse formation; Sinus node

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This article is referred to in the Editorial by T. Opthof (pages 1–4) in this issue.

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The spontaneous activity of pacemaker cells of the sino-atrial node (SAN) underlies the cardiac automaticity in mammals [1]. Automaticity in pacemaker cells is due to the presence of the slow diastolic depolarisation, which drives the membrane potential at the end of an action potential (AP) towards the threshold of the following AP upstroke phase [2]. Different ionic currents with complex reciprocal interactions are involved in the regulation of the diastolic depolarisation (for recent reviews see [3]). Among these, the hyperpolarization-activated current, (I_f) has been proposed to play an important role in the control of automaticity [4], but the physiological impact of I_f in the generation of the diastolic depolarisation is a matter of debate [5].

I_f has been described in the mammalian SAN [6], the frog sinus venosus [7], the latent pacemaker cells of cat right atrium [8], and throughout the heart conduction system [9,10]. The myocardium also expresses I_f upon different physiopathological conditions [11,12]. I_f is activated in hyperpolarization, and is regulated in opposite ways through stimulation by the β-adrenergic, and inhibition by the muscarinic receptors respectively [6,13], a mechanism which depends on direct channel activation by intracellular cyclic-adenosine-monophosphate (cAMP) [14].

A gene family named HCN1-4 has been recently cloned from mouse, human and rabbit, and has been shown to code for cardiac I_f channels [15–18]. Cloned HCN channels open the perspective of investigating the physiopathological role of I_f in pacemaking thanks to specific gene targeting techniques in the mouse. However, the experimental conditions for the isolation of mouse sino-atrial cells have yet to be established, and up to now, there is no available description of I_f, as well as of the other ionic currents regulating the automaticity of mouse pacemaker cell. Here we describe the successful isolation and electrophysiological recording of mouse SAN pacemaker cells. This method has been employed to study the properties of the native I_f current in spontaneously beating pacemaker cells.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
2.1 Mouse sino-atrial cells isolation
Sino-atrial cells used for this study came from 18 C57BL6/J mice (Mus musculus) donors of either sex, aged 4 months, and weighing 20–23 g. The investigation conforms with the Guide for the care and Use of Laboratory Animals published by the US national Institute of Health (NIH Publication No. 85–23, revised 1996), and European directives (86/609/CEE). SAN cells were isolated using an adaptation of the method employed for rabbit SAN cells as described by DiFrancesco and co-workers [6]. Beating hearts were removed under general anesthesia, consisting of 0.01 mg/g of Xylazine (Rompun 2%, Bayer AG, Leverkusen Germany), and 0.1 mg/g of Ketamine (Imalgène, Merial, Bourgelat France). The SAN region (Fig. 1) was then excised in pre-warmed (35°C) Tyrode solution containing (mM/l): NaCl, 140; KCl, 5.4; CaCl₂, 1.8; MgCl₂, 1; Hepes-NaOH, 5; and D-glucose, 5.5; (adjusted to pH=7.4 with NaOH). SAN tissue strips were then transferred into a ‘low-Ca²⁺-low-Mg²⁺’ solution containing (in mM/l): NaCl, 140; KCl, 5.4; MgCl₂, 0.5; CaCl₂, 0.2; KH₂PO₄, 1.2; taurine, 50; D-glucose, 5.5; bovine serum albumin (BSA), 1 mg/ml; Hepes-NaOH, 5; (adjusted to pH=6.9 with NaOH). SAN tissue was digested by adding collagenase type II (229 U/ml, Worthington Biochemical Corporation, Lakewood, NJ, USA), elastase (1.9 U/ml, Boehringer Mannheim, Germany), protease (0.9 U/ml, Sigma, St. Quentin Fallavier, France), BSA 1 mg/ml, and 200 µM CaCl₂. Digestion was carried out for a variable time of 9–13 min at 35°C, under manual mechanical agitation. Tissue strips were then washed, and transferred into a modified ‘Kraftbrühe’ (KB) medium [19] containing (in mM/l): L-glutamic acid, 70; KCl, 20; KOH, 80; (±)D-β-OH-butyric acid, 10; KH₂PO₄, 10; taurine, 10; BSA, 1 mg/ml; and Hepes-KOH, 10; (adjusted to pH=7.4 with KOH). Single sino-atrial cells were isolated by manual agitation in KB solution at 35°C. Every 2 min, small aliquots of the KB solution were inspected for the presence of SAN cells under phase-contrast optics. Cellular automaticity was recovered by re-adapting the cells to a physiological extracellular Ca²⁺concentration by addition of a solution containing (in mM/l): NaCl, 10, CaCl₂, 1.8, and normal Tyrode solution containing BSA (1 mg/ml). The final storage solution contained (mM/l): NaCl, 100; KCl, 35; CaCl₂, 1.3; MgCl₂, 0.7; L-glutamic acid, 14; (±)D-β-OH-butyric acid, 2; KH₂PO₄; 2; taurine, 2; BSA 1 mg/ml; (pH=7.4), and gentamycin (50 µg/ml). All chemicals were from Sigma (St Quentin, Fallavier), except for the (±)D-β-OH-butyric acid that was from Fluka Chemika (Buchs, CH), and the E-4031 that was a gift by Dr. D. Escande (INSERM U533, Nantes).

View larger version (55K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 The isolated mouse SAN preparation. The dotted line indicates the experimental limits of SAN. Solid lines delimit the position of tissue strips used for cell isolation. Figure labelling is as follows: SVC, superior vena cava, IVC, inferior vena cava, IAS, interatrial septum, CT, crista terminalis, RA right atrium, RSARB, right branch of the sino-atrial ring bundle, LSARB, left branch of the sino-atrial ring bundle.

 
2.2 Electrophysiological recordings
For electrophysiological recordings, aliquots of the cell suspension were harvested in 35-mm Petri dishes, mounted on the recording chamber of an inverted microscope (Nikon, ELWD 03), and then continuously superfused with normal Tyrode solution. To allow comparison between the kinetics of native mouse I_f and that of cloned HCN genes, the recording temperature was set to 26°C. Cell imaging was performed by connecting the output of a CCD camera (Hitachi, model FP-C1E), to a digital video/audio tape (Panasonic DVCPRO, model AJD640). The whole-cell patch-clamp technique [20] was used to record I_f and cellular automaticity, by employing an Axopatch 200A (Axon Instruments, Foster USA) patch-clamp amplifier, connected to the ground by an agar bridge filled with 150 mM KCl. Recording electrodes were fabricated from borosilicate glass, by employing a Flaming-Brown microelectrode puller (Sutter, Novato CA, USA), and had a resistance of about 5.5 M, when filled with the recording solution containing (in mM/l): KCl, 135; MgCl₂, 1; ATP-Mg²⁺ salt, 4; GTP Na⁺ salt, 0.1; EGTA-KOH, 5; Hepes-KOH, 5; (adjusted to pH=7.2 with KOH). The cell AP was recorded by using an intracellular solution containing (mM/l): K⁺-aspartate, 130; NaCl, 10; ATP-Na⁺ salt, 2; creatine phosphate, 6.6; GTP-Mg²⁺, 0.1; CaCl₂, 0.04 (pCa=7); Hepes-KOH, 10; (adjusted to pH=7.2 with KOH). Seal resistances were in the range of 2–5 G.

For recording calcium currents (I_CaT, I_Ca,L), we replaced KCl in the intracellular solution, with an equal amount of CsCl. The extracellular solution contained (in mM/l): tetraetylammonium-chloride (TEA-Cl), 130; CaCl₂, 2; MgCl₂, 1; 4-amino-pyridine, 10; Hepes, 25; (adjusted to pH=7.4 with TEAOH). The fast component of the delayed rectifier (I_Kr) was recorded in Tyrode, after addition of 10 µM tetrodotoxin (TTX), and 0.2 µM isradipine. The I_f current was routinely recorded in Tyrode solution containing 1 mM BaCl₂, and 2 mM MnCl₂ to block the inward rectifier (I_Kir) and I_Ca currents [6]. 10 mM 4-AP, and 10 µM TTX were also added when measuring the I_f reversal potential, and fully activated current-to-voltage relation [21]. Cells that showed regular activity for several minutes were used for recordings. Data acquisition was performed by using the pClamp software (ver. 6.2, Axon Instruments). Analysis was performed by employing the Origin software (ver. 6.0, Microcal, Northampton MA, USA).

2.3 Data analysis
The AP parameters were calculated accordingly to Honjo et al. [22], and Rocchetti et al. [23]. The following AP parameters were calculated (Fig. 3a): the cycle length (CL), the AP duration (APD), the duration of the diastolic depolarisation (DI), the maximum diastolic potential (E_MDP), the rate of the diastolic depolarisation (DDR), the AP threshold (E_th), and the voltage jump between the maximum diastolic potential and the AP threshold (V_th). The peak of the first derivative of the AP waveform was taken as the overshoot velocity (MOV, Fig. 3b). The cell membrane capacitance was monitored by applying brief (10 ms) voltage steps of ±10 mV amplitude from a holding potential (HP) of –35 mV.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Cellular automaticity (a), and first derivative of the AP cycle (b) in a mouse SAN cell. Reference dotted lines for the calculations of the AP parameters are shown. See the Methods section for definition of abbreviations.

 
For I_f current analysis, we applied the general set of equations described by Noble and co-workers [24]. The I_f fully-activated current-to-voltage relation (I) was fitted according to the equation

(1)

where V is the membrane voltage, g_max is the current maximal conductance, and V_rev is the current reversal potential. The I_f activation curve was fitted according to the equation

(2)

where V_1/2 is the voltage for current half activation, and v is the slope factor.

The voltage-dependency of I_f activation–deactivation time constants has been fitted according to equation:

(3)

where ₀, β₀ are opening and closing rates at zero voltage.

Activation, and deactivation time constants were calculated by fitting experimental traces according to the single-exponential equation:

(4)

where A is a fixed factor, t is the time, and t₀ sets the onset of current activation–deactivation.

Results are presented as means±the standard error of the mean (S.E.M, number of cells), or the standard deviation (S.D.), when stated. For calculating the level of significance, the one-way ANOVA test has been employed. When testing statistical differences, results were considered not significant with P>0.05, and significant with 0.0001<P<0.05.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 Cellular automaticity and ionic currents in mouse SAN cells
Isolated mouse SAN cells showed were similar to cells that have been previously described by other authors in SAN of the rabbit and guinea pig [6,25,26]. Also, atrial cells that have been reported in SAN [27] were identified, and will be referred to as ‘atrial-like’ cells (Fig. 2d). Upon 1675 visually identified SAN cells in KB solution 28% were of the ‘spindle’ type (Fig. 2a), 6% were of the ‘elongated spindle’ (Fig. 2b), 3% were of the ‘spider’ type (Fig. 2c), and 67% were atrial-like cells (Fig. 2d). Other cells were round-shaped. Round cells were not further investigated.

View larger version (65K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Different cellular morphologies are observed after enzymatic digestion of mouse SAN tissue. (a) Spindle-shaped cell, (b) elongated spindle-shaped cell, (c) spider cell. All cells from (a) to (c) were beating spontaneously. A cell showing atrial-like phenotype is also shown in (d).

 
Fig. 3 shows a representative example of the spontaneous activity observed in mouse SAN cells. In three independent cells investigated, averaged CL, MOV, DI, and E_MDP values were 322±2 ms, 25±3 V/s, 125±5 ms, and –60±3 mV, respectively. The mean capacitance in spontaneously beating cells was 24±2 pF (n=23).

In Tyrode solution, the fast sodium current (I_Na) was evoked in depolarisation from a HP of –80 mV (Fig. 4a). I_Na started to activate at –50 mV, peaked at –20 mV (Fig. 4b), and was blocked by 10 µM TTX by 88±3% (n=7). The density of the net TTX-sensitive current was 118±17 pA/pF (n=7). In extracellular solution containing TEA-Cl (see Methods), depolarising steps from a HP of –80 mV, elicited both I_Ca,T and I_Ca,L [28] (Fig. 4c). I_Ca,T was activated from a threshold of –50 mV, peaked at –30 mV and was completely inactivated when the HP was set to –60 mV (n=4, data not shown). From a HP of –60 mV I_Ca,L was activated from –50 mV, and peaked at –20, or –10 mV (Fig. 4d). I_Ca,L density was 2.6±2 pA/pF (n=9) at –10 mV. From a HP of –40 mV, the fast component of the delayed rectifier (I_Kr) [29] was elicited in depolarisation (Fig. 4e). Three µM of the class III antiarrythmic agent E-4031 completely blocked decaying tail currents upon deactivation to –40 mV (Fig. 4e, inset). From a test potential of +20 mV, the density of the net E-4031-sensitive tail current measured upon deactivation to –40 mV was 1.3±0.2 pA/pF (n=5).

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Ionic currents in spontaneously beating mouse SAN cells. (a) Typical INa is recorded from a HP of –80 mV, by applying depolarising steps lasting 80-ms in 10-mV increments. (b) Corresponding current-to-voltage relation of INa in Tyrode (open circles), and upon addition of 10 µM TTX (filled circles). (c) Depolarising steps from a HP of –80 mV elicited both ICaL and ICa,T. Experimental protocol as in (a). (d) Example of the current-to-voltage relation of total ICa (filled circles), ICa,T (open squares) and ICa,L (open circles). The ICa,L current-to-voltage relation has been recorded from a HP of –60 mV. The ICa,T current-to-voltage relation has been calculated by subtracting ICa,L from total ICa (e) IKr is elicited by applying 1s depolarising steps from a HP of –40 mV in 10-mV increments. The inset shows a block of IKr decaying tail by 3 µM E-4031. The dotted line indicate the zero current level. (f) Current-to-voltage relation of IKr in the absence (filled circles), and in the presence (open circles) of E-4031. Data points represent the peak tail current at –40 mV, upon deactivation from the test potential indicated. (g) Application of 2-s hyperpolarising steps from a HP of –35 mV at the voltages indicated, activate inward current. The corresponding membrane current-to-voltage relation measured after 200 ms from the step onset is shown in (h).

 
3.2 Identification of the I_f current
From a HP of –35 mV, hyperpolarising steps elicited inward current (Fig. 4g,h). To isolate I_f from the total current we added 1 mM Ba²⁺ and 2 mM Mn²⁺ to the Tyrode solution (see Methods). In Fig. 5a the instantaneous and time-dependent membrane currents elicited at –100 mV (Fig. 5, open circle) were slightly reduced by superfusing the cell with 1 mM Ba²⁺, and 2 mM Mn²⁺ (filled circle), an effect which is consistent with a block by Ba²⁺ of I_Kir. Five mM Cs⁺ (Fig. 5a, open square) blocked I_f by 96±2% (n=9) in a reversible way (Fig. 5a, filled square). Upon deactivation at +5 mV I_f was blocked by Cs⁺ by 33±4% (n=9), the difference in the percentage of Cs⁺ block upon hyperpolarization and depolarisation indicating voltage-dependency of I_f block (P<0.0001). We have then used the I_f sensitivity to extracellular Cs⁺ to construct the I_f current-to-voltage relation (Fig. 5b). Fig. 5c shows the I_f sensitivity to application of the β-adrenergic agonist noradrenaline and the muscarinic agonist acetylcholine, as reported for native I_f in rabbit SAN cells [6,13]. At –90 mV, application of 10 µM noradrenaline stimulated I_f by 21%, while application of 5 µM acetylcholine, inhibited I_f by 37%.

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 If is activated by applying a hyperpolarising step for 1.6 s, of a HP of –35 to –100 mV, and then deactivated by a 360-ms depolarising step at +5 mV, as indicated. Membrane current in Tyrode solution (open circles), after the addition of 1 mM BaCl2, and 2 mM MnCl2 (filled circle), in the presence of 5 mM Cs+ (open square), and after Cs+ washout (filled square). (b). Current-to voltage relation of If, measured as the net Cs+-sensitive current component at the end of 1.6 s hyperpolarising steps. Data are from n=5 cells. (c) Effect of bath applied noradrenaline (NA), and acetylcholine (ACh). If was activated by stepping to –90 mV in control conditions (Cont). ACh was superfused, and then washed out (not shown) before application of NA.

 
In spontaneously beating cells, the I_f current density at –120 mV was 18±9 S.D. pA/pF (n=23, Fig. 6a). This value is significantly higher (P<0.001) to that reported for rabbit SAN cells by Wilders et al. [30] (10±4.8 S.D. n=23), and by Honjo et al. [22] (7±2 S.D. n=12, P<0.001). The I_f current density was poorly correlated with the cellular capacitance (r=0.1 P>0.5, Fig. 6c). In atrial-like cells the membrane capacitance was 45±7 pF (n=7), a value which is significantly higher to that of spontaneously beating cells (P<0.01). Almost no detectable I_f was found in atrial-like cells. (Fig. 6b). Indeed, the net Cs⁺-sensitive component at –120 mV was 0.04±0.02 pA/pF, a value which is significantly lower than that calculated in spontaneously-beating cells (P<0.001).

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 If in spontaneously beating (a), and in atrial-like cells (b). If was activated in hyperpolarization for 2s, at the test potentials indicated. Traces obtained after subtraction by 5 mM extracellular Cs+ are shown. (c). In this plot, each point represents the net Cs+-sensitive If measured at the end of the hyperpolarising pulse at –120 mV, from a given cell. The solid line shows the linear regression fitting of data. The dotted line has been extracted from Honjo and co-workers [22], and is shown for comparison.

 
3.3 Ionic properties of I_f
We next examined the I_f current reversal potential in mouse SAN cells (Fig. 7a). The apparent reversal potential of I_f tail currents was between –30 and –20 mV (n=7). Precise quantification of I_f reversal potential was obtained by measuring the fully activated I_f current-to-voltage relation (Fig. 7b). Best fit of data points (Fig. 7b, dotted lines) yielded parameters: V_rev=–31±2 mV, and g_max=4±1 nS (n=4). The fully activated current-to-voltage relation was linear, indicating that measurements of I_f tail currents were not contaminated by residual I_Kir in depolarisation.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 If current reversal potential in mouse SAN cells. In (a), If has been activated by a 3.2 s hyperpolarising step to –120 mV, and then deactivated in depolarisation for 960 ms, from –100 to +40 mV, in incremental 10 mV steps. In (b,c) the fully-activated current-to-voltage relation of If is shown. We used a previously described protocol [6] consisting of pairs of conditioning hyperpolarising (to the full activation range), and depolarising (to the full deactivation range), followed by steps to the same test voltage. In the cell shown here, conditioning steps were applied to –120 mv in hyperpolarization, and to –15 mV in depolarisation, and test potentials spanned the voltage range between –100 and +40 mV. Conditioning, and test potential duration were the same as in (a). Pairs of traces (indicated by arrows) at –100, –80, and +0 mV are shown. The difference current between each pair of traces has been plotted with respect to the corresponding test potential in (c).

 
Lowering the extracellular Na⁺ concentration from 135 to 50 mM diminished I_f throughout its voltage range of activation (Fig. 8A). Increasing the K⁺ concentration from 5 to 35 mM significantly increased I_f (Fig. 8B). Note that I_f amplitude at –125 mV (Fig. 8Bb,c) was enhanced by 110% despite the net sum of Na⁺ and K⁺ concentrations being diminished with respect to control conditions (Fig. 8Ba).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Dependence of If upon the extracellular concentration of Na+(A), and Na+ and K+ (B) on If. In (Aa), If was activated throughout its complete range of activation for 2 s, and then deactivated at +5 mV for 350 ms. Switching the extracellular concentration of Na+ from 135 to 50 mM, diminished If (Ab). Data points were measured at the end of each hyperpolarising step. In another cell (Ba), the result of extracellular superfusion with a solution containing 50 mM Na+, and 35 mM K+ was a net augmentation of If (Bb). Corresponding current-to-voltage relations are shown in (Ac) and (Bc). In each experiment, NaCl has been substituted with appropriate amounts of choline chloride (80 mM in A and 50 mM in B, respectively).

 
3.4 I_f Kinetics in mouse SAN cells
The I_f activation curve was calculated according to a protocol first described by DiFrancesco and Mangoni [31] (Fig. 9). This consisted of a hyperpolarising ramp from –35 to –140 mV (Fig. 9a). Ramp duration was set to 60 s. The activation curve was measured as the ratio between the steady-state current, and the fully activated current-to-voltage relation (Fig. 9b). Best fit of activation curves by employing Eq. (2) gave parameter values of V_1/2=–87±1, and v=12±1 (n=6).

View larger version (9K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 9 If activation curve in mouse SAN cells. A hyperpolarising ramp from –35 to –140 mV of 60 s duration was applied to the cell as shown in (a). After linear leakage correction, the total membrane current recorded during ramp application has been plotted in (b). The averaged activation curve from n=6 cells is shown in (c), together with its corresponding best fit (open circles).

 
The I_f activation time course showed variability in mouse SAN cells. Experimentally, we could distinguish between cells in which the current steady state was reached in about 6s (Fig. 10a), and cells where steps of 10s or longer were required (Fig. 10b). In the two groups of cells, I_f activation time constants were 1.54±0.23 s (n=6, Fig. 10a), and 3.11±0.12 s (n=4, Fig. 10b) at –90 mV; and 1.01±0.069 s, and 1.87±0.16 s at –120 mV. Time constants measured in cells showing kinetics as in Fig. 10b were significantly higher (P<0.001 at –90 mV and P<0.005 at –120 mV), reflecting slower activation kinetics at both voltages.

View larger version (8K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 10 Variability of If kinetics in mouse SAN cells. Representative records of If in cells showing faster (a), and slower (b) activation kinetics. In each panel, If was activated by applying hyperpolarising steps lasting 10s at –90, and –120 mV as indicated, and then deactivated at +5 mV.

 
Comparison of the kinetic behaviour of I_f in mouse, to that of rabbit SAN and cloned HCN channels is shown in Fig. 11. The I_f activation (Fig. 11a,c) and deactivation (Fig. 11b,d) rate constants in mouse SAN cells were strongly voltage-dependent, with more negative voltages resulting in faster activation (Fig. 11c), and positive voltages resulting in faster deactivation (Fig. 11d). Data were averaged to generate a bell-shaped curve (Fig. 11e, solid line), that was fitted according to Eq. (3), to give the parameters values ₀=0.00196 s^–1, β₀=13.26129 s^–1, V₀=19.58724 mV. Consistently with the calculated V_1/2, the solid curve in Fig. 11e peaked at –86 mV, indicating that the exponential model correctly interpreted experimental data. Taking the averaged time constants at –90 and –120 mV as references, our data were not significantly different (P>0.05), to that reported in rabbit SAN cells at both voltages. In contrast, the kinetic behaviour of expressed hHCN2 was significantly faster (P<0.001) at both voltages. Finally, the activation kinetics of the expressed hHCN4 was significantly slower (P<0.001) than both native I_f, and hHCN2.

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 11 Analysis of If activation and deactivation time course in mouse SAN cells. If was activated by 10-s hyperpolarising steps at voltages indicated in (a). Then, If was fully activated at –120 mV and subsequently deactivated by 11-s depolarising steps at the voltages indicated in (b). In (a) and (b) sample traces are shown together with exponential fittings generated by employing the Eq. (4) (open circles). Corresponding voltage-dependence of activation (c, filled circles), and deactivation (d, open circles) are also shown. (e). Plot of time constants mean±S.E.M. in n=10 cells. Time constants for activation and deactivation collected at the same voltages were pooled. Experimental data points (filled balls), were fitted according to Eq. (3). For comparison, the kinetics of native If in rabbit SAN ([36] open square), and that of hHCN2 channel ([35] open circle) are shown for comparison. Also for comparison, the activation time constants of hHCN4 channel [16] are presented (filled square).

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
To the best of our knowledge, our work represents the first electrophysiological description of mouse SAN cells. Four lines of evidence indicate that cells used in this study are in fact, pacemaker SAN cells. Firstly, they were all spontaneously beating upon visual inspection. Secondly, their anatomical location, their morphology, and their electrical capacitance are all consistent with previous reports on pacemaker cells from rabbit SAN (see for example Verheijck et al., [32]). Thirdly, spontaneously beating cells did show spontaneous action potentials. Finally, together with I_f, other currents that have been previously reported in other SAN preparations [3] have been consistently found here (Fig. 4).

I_f recorded in mouse SAN cells is similar to rabbit SAN I_f. Indeed, the insensitivity of I_f to Ba²⁺, the voltage-dependent block by Cs⁺, the current reversal potential, and the dependency from extracellular Na⁺ and K⁺, are all hallmarks of this current. [33]. These properties are consistent to that observed in cloned murine, rabbit, and human HCN channels [16,17,34,35].

In our experimental conditions, the I_f current was found to be restricted to spontaneously beating cells (Fig. 6). The possibility that I_f could also be functionally expressed in atrial-like cells cannot be completely ruled out, in the hypothesis that the current voltage-dependency is shifted to very negative voltages, as reported in adult ventricular myocytes [11].

The averaged voltage for half activation of I_f that we report here is –87 mV. This value is consistent with the one reported in mouse embryonic ventricular myocytes [21]. Analysis of the time course of activation and deactivation indicates that I_f in mouse SAN cells has the same kinetic behaviour of I_f described in rabbit SAN at the same recording temperature [36] (see Fig. 11e). Comparison of our data with the current available literature shows that none of the cloned HCN channels, by itself, match the kinetics of native I_f in mouse SAN. This view is reinforced by the existence of cells having significantly slower activation kinetics than others (Fig. 10b). Heterogeneity of I_f kinetics was not due to a negative shift of the I_f activation, since we found almost no variability in the voltage for half activation, and only a negative shift of about 30 mV in cells showing slower kinetics would account for our results (see Fig. 9c). Our data thus suggest that the native SAN I_f is constituted by a heterogeneous composition of channel transcripts. Consistently with this hypothesis, analysis of transcripts from rabbit SAN has shown the coexistence of mRNAs belonging to at least two different HCN genes [37].

In our study employing mouse SAN cells, the averaged I_f density was higher than that reported for rabbit SAN. Enhanced expression of I_f in mouse SAN cells, could be related to the fast heart rate in the mouse, which is reported to vary between 450 and 650 beats per minute in vivo [38]. However, fast rate implies that only a small fraction of I_f would be activated in the diastolic range. Indeed, in this voltage interval, the fractional activation of I_f varies between 0.02 and 0.1 (Fig. 9c). Accordingly, the densities of the net Cs⁺-sensitive I_f at 100 ms upon hyperpolarization were 0, 0.7± 0.2, and 1.3±0.3 pA/pF (n=5) at –40, –50, and –60, respectively. As a comparison, I_Ca,L densities were 1.1±0.3 and 0.25±0.2 pA/pF at –40 at –50 mV, respectively. Taken together, our data suggest that automaticity in mouse SAN cells is likely to be generated by different ionic channels; the decaying I_Kr (Fig. 4e); the activating I_f and I_Ca,L and possibly I_Ca,T, depending on the relative channel availability close to the MDP (Fig. 4). The possibility that the I_st current [39] could also be involved cannot be excluded, since our recording conditions were not adapted for recording I_st in mouse SAN cells. The hypothesis that automaticity in mouse SAN involves several ionic channels is consistent with the observation that in null mice in which the genes coding for I_K(ACh) and I_Ca,L have been inactivated show specific patterns of SAN rhythm dysfunction [38,40]. These reports also indicate that different ionic channels may underlie distinct physiological roles in the regulation of automaticity. To this respect, our data suggest that I_f could participate in the generation of the diastolic depolarisation by supplying inward current close to the MDP, thus contributing to its proper setting. Accordingly, the regulation of I_f by neurotransmitters would have higher physiological impact near the MDP.

In conclusion, the isolation of spontaneously beating mouse SAN cells opens the way to gain new insights in the role of ionic channels in automaticity thanks to the electrophysiological characterisation of pacemaker cells from genetically modified mice. Particularly, the similarity between the kinetics of native mouse I_f and that of the rabbit demonstrates that our preparation would be appropriate to study the consequences of I_f gene invalidation on pacemaking. Furthermore, new information about the physiological role of I_f in the autonomic regulation of heart rate could be obtained from mouse strains where genes coding for channels such as I_Ca,L and I_K, channels that are involved in the cAMP-dependent regulation of the diastolic depolarisation [3] have been selectively invalidated or modified.

Time for primary review 31 days.

	Acknowledgements

 
We thank the ‘Association Française contre les Myophaties’ (AFM) for financial support. We are grateful to A. Cohen-Solal, and A. Delalbre for technical assistance with the mice. We are also grateful to D. Clapham for reading the manuscript. We thank P. Lory, J. Chemin, E. Bourinet, S. Dubel, and C. Altier for helpful discussion and critical reading of the manuscript. We are also indebted to M. Andreo, G. Roua and P. Atger for setting up cellular imaging.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

1. Bouman L.N., Jongsma H.J. Structure and function of the sino-atrial node: a review. Eur. Heart J. (1986) 7:94–104.[Free Full Text]
2. Weidmann S. The slow inward current and cardiac arrhythmias. Zipes D.P., Bayley J.C., Elharrar V., eds. (1980) The Hague: Nijoff.
3. Boyett M.R., Honjo H., Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovasc. Res. (2000) 47:658–687.[Abstract/Free Full Text]
4. DiFrancesco D. The pacemaker current (I_f) plays an important role in regulating SA node pacemaker activity. Cardiovasc. Res. (1995) 30:307–308.[Free Full Text]
5. Vassalle M. The pacemaker current (I_f) does not play an important role in regulating SA node pacemaker activity. Cardiovasc. Res. (1995) 30:309–310.[Free Full Text]
6. DiFrancesco D., Ferroni A., Mazzanti M., Tromba C. Properties of the hyperpolarizing-activated current (I_f) in cells isolated from the rabbit sino-atrial node. J. Physiol. (1986) 377:61–88.[Abstract/Free Full Text]
7. Champigny G., Lenfant J. Block and activation of the hyperpolarization-activated inward current by Ba and Cs in frog sinus venosus. Pflügers Arch. (1986) 407:684–690.[CrossRef][Web of Science][Medline]
8. Zhou Z., Lipsius S.L. Properties of the pacemaker current (I_f) in latent pacemaker cells isolated from cat right atrium. J. Physiol. (1992) 453:503–523.[Abstract/Free Full Text]
9. Noma A., Irisawa H., Kokobun S., Kotake H., Nishimura M., Watanabe Y. Slow current systems in the A–V node of the rabbit heart. Nature (1980) 285:228–229.[CrossRef][Medline]
10. DiFrancesco D. A new interpretation of the pace-maker current in calf Purkinje fibres. J. Physiol. (1981) 314:359–376.[Abstract/Free Full Text]
11. Yu H., Chang F., Cohen I.S. Pacemaker current I_f in adult canine cardiac ventricular myocytes. J. Physiol. (1995) 485:469–483.[Abstract/Free Full Text]
12. Cerbai E., Barbieri M., Mugelli A. Characterization of the hyperpolarization-activated current, I_f, in ventricular myocytes isolated from hypertensive rats. J. Physiol. (1994) 481:585–591.[Abstract/Free Full Text]
13. DiFrancesco D., Tromba C. Inhibition of the hyperpolarization-activated current (I_f) induced by acetylcholine in rabbit sino-atrial node myocytes. J. Physiol. (Lond.) (1988) 405:477–491.[Abstract/Free Full Text]
14. DiFrancesco D., Tortora P. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. Nature (1991) 351:145–147.[CrossRef][Medline]
15. Santoro B., Liu D.T., Yao H., Bartsch D., Kandel E.R., Siegelbaum S.A., Tibbs G.R. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell (1998) 93:717–729.[CrossRef][Web of Science][Medline]
16. Ludwig A., Zong X., Stieber J., Hullin R., Hofmann F., Biel M. Two pacemaker channels from human heart with profoundly different activation kinetics. Embo J. (1999) 18:2323–2329.[CrossRef][Web of Science][Medline]
17. Ishii T.M., Takano M., Xie L.H., Noma A., Ohmori H. Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node. J. Biol. Chem. (1999) 274:12835–12839.[Abstract/Free Full Text]
18. Seifert R., Scholten A., Gauss R., Mincheva A., Lichter P., Kaupp U.B. Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis. Proc. Natl. Acad. Sci. USA (1999) 96:9391–9396.[Abstract/Free Full Text]
19. Isenberg G., Klockner U. Calcium tolerant ventricular myocytes prepared by preincubation in a ‘KB medium’. Pflügers Arch. (1982) 395:6–18.[CrossRef][Web of Science][Medline]
20. Hamill O.P., Marty A., Neher E., Sakmann B., Sigworth F.J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch. (1981) 391:85–100.[CrossRef][Web of Science][Medline]
21. Yasui K., Liu W., Opthof T., Kada K., Lee J.K., Kamiya K., Kodama I. I_f current and spontaneous activity in mouse embryonic ventricular myocytes. Circ. Res. (2001) 88:536–542.[Abstract/Free Full Text]
22. Honjo H., Boyett M.R., Kodama I., Toyama J. Correlation between electrical activity and the size of rabbit sino-atrial node cells. J. Physiol. (1996) 496:795–808.[Abstract/Free Full Text]
23. Rocchetti M., Malfatto G., Lombardi F., Zaza A. Role of the input/output relation of sinoatrial myocytes in cholinergic modulation of heart rate variability. J. Cardiovasc. Electrophysiol. (2000) 11:522–530.[CrossRef][Web of Science][Medline]
24. Noble D., DiFrancesco D., Denyer J. Neuronal and cellular oscillators. Jacklet J.W., ed. (1989) New York: Dekker. 59–85.
25. Denyer J.C., Brown H.F. Rabbit sino-atrial node cells: isolation and electrophysiological properties. J. Physiol. (1990) 428:405–424.[Abstract/Free Full Text]
26. Guo J., Mitsuiye T., Noma A. The sustained inward current in sino-atrial node cells of guinea-pig heart. Pflügers Arch. (1997) 433:390–396.[CrossRef][Web of Science][Medline]
27. Oosthoek P.W., Viragh S., Mayen A.E., van Kempen M.J., Lamers W.H., Moorman A.F. Immunohistochemical delineation of the conduction system. I. The sinoatrial node. Circ. Res. (1993) 73:473–481.[Abstract/Free Full Text]
28. Hagiwara N., Irisawa H., Kameyama M. Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J. Physiol. (1988) 395:233–253.[Abstract/Free Full Text]
29. Verheijck E.E., van Ginneken A.C., Bourier J., Bouman L.N. Effects of delayed rectifier current blockade by E-4031 on impulse generation in single sinoatrial nodal myocytes of the rabbit. Circ. Res. (1995) 76:607–615.[Abstract/Free Full Text]
30. Wilders R., Verheijck E.E., Kumar R., Goolsby W.N., van Ginneken A.C., Joyner R.W., Jongsma H.J. Model clamp and its application to synchronization of rabbit sinoatrial node cells. Am. J. Physiol. (1996) 271:H2168–2182.[Web of Science][Medline]
31. DiFrancesco D., Mangoni M. Modulation of single hyperpolarization-activated channels (I_f) by cAMP in the rabbit sino-atrial node. J. Physiol. (1994) 474:473–482.[Abstract/Free Full Text]
32. Verheijck E.E., Wessels A., van Ginneken A.C., Bourier J., Markman M.W., Vermeulen J.L., de Bakker J.M., Lamers W.H., Opthof T., Bouman L.N. Distribution of atrial and nodal cells within the rabbit sinoatrial node: models of sinoatrial transition. Circulation (1998) 97:1623–1631.[Abstract/Free Full Text]
33. DiFrancesco D. The cardiac hyperpolarizing-activated current, I_f. Origins and developments. Prog. Biophys. Mol. Biol. (1985) 46:163–183.[CrossRef][Web of Science][Medline]
34. Ludwig A., Zong X., Jeglitsch M., Hofmann F., Biel M. A family of hyperpolarization-activated mammalian cation channels. Nature (1998) 393:587–591.[CrossRef][Medline]
35. Moroni A., Barbuti A., Altomare C., Viscomi C., Morgan J., Baruscotti M., DiFrancesco D. Kinetic and ionic properties of the human HCN2 pacemaker channel. Pflügers Arch. (2000) 439:618–626.[CrossRef][Web of Science][Medline]
36. DiFrancesco D. Dual allosteric modulation of pacemaker (f) channels by cAMP and voltage in rabbit SA node. J. Physiol. (1999) 515:367–376.[Abstract/Free Full Text]
37. Shi W., Wymore R., Yu H., Wu J., Wymore R.T., Pan Z., Robinson R.B., Dixon J.E., McKinnon D., Cohen I.S. Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues. Circ. Res. (1999) 85:e1–6.[Abstract/Free Full Text]
38. Wickman K., Nemec J., Gendler S.J., Clapham D.E. Abnormal heart rate regulation in GIRK4 knockout mice. Neuron (1998) 20:103–114.[CrossRef][Web of Science][Medline]
39. Mitsuiye T., Shinagawa Y., Noma A. Sustained inward current during pacemaker depolarization in mammalian sinoatrial node cells. Circ. Res. (2000) 87:88–91.[Abstract/Free Full Text]
40. Platzer J., Engel J., Schrott-Fischer A., Stephan K., Bova S., Chen H., Zheng H., Striessnig J. Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca²⁺ channels. Cell (2000) 102:89–97.[CrossRef][Web of Science][Medline]

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