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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 (If) 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 (If) current in pacemaker cells from the mouse sino-atrial node (SAN). Methods: The If 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 If. Results: In mouse SAN cells, the If current density at –120 mV was 18±2 pA/pF (n=23). If was not detected in cells showing atrial-like morphology that were also found in SAN preparations (n=7). If was blocked by 5 mM Cs+, was inhibited by application of 5 µM acetylcholine, and was increased by 10 µM noradrenaline. The If current reversal potential was –31±2 mV under physiological concentration of Na+ and K+ ions. Lowering the extracellular Na+ concentration reduced If amplitude, while increased when the extracellular K+ concentration was augmented. If voltage for half activation was –87±1 mV (n=6). Conclusions: We conclude that the native If current in mouse SAN cells shows functional properties that are similar to If described in rabbit SAN tissue. This study opens the possibility of investigating the involvement of If in the regulation of heart rate in genetically modified mice.

KEYWORDS Ion channels; Impulse formation; Sinus node

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, (If) has been proposed to play an important role in the control of automaticity [4], but the physiological impact of If in the generation of the diastolic depolarisation is a matter of debate [5].

If 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 If upon different physiopathological conditions [11,12]. If 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 If channels [15–18]. Cloned HCN channels open the perspective of investigating the physiopathological role of If 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 If, 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 If 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).


Figure 1
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  		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 If 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 If 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{Omega}, 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{Omega}.

For recording calcium currents (ICaT, ICa,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 (IKr) was recorded in Tyrode, after addition of 10 µM tetrodotoxin (TTX), and 0.2 µM isradipine. The If current was routinely recorded in Tyrode solution containing 1 mM BaCl2, and 2 mM MnCl2 to block the inward rectifier (IKir) and ICa currents [6]. 10 mM 4-AP, and 10 µM TTX were also added when measuring the If 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 (EMDP), the rate of the diastolic depolarisation (DDR), the AP threshold (Eth), and the voltage jump between the maximum diastolic potential and the AP threshold (Vth). 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.


Figure 3
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  		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 If current analysis, we applied the general set of equations described by Noble and co-workers [24]. The If fully-activated current-to-voltage relation (IFormula) was fitted according to the equation

Formula (1)
where V is the membrane voltage, gmax is the current maximal conductance, and Vrev is the current reversal potential. The If activation curve was fitted according to the equation

Formula (2)
where V1/2 is the voltage for current half activation, and v is the slope factor.

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

Formula (3)
where {alpha}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:

Formula (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.


Figure 2
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  		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 EMDP 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 (INa) was evoked in depolarisation from a HP of –80 mV (Fig. 4a). INa 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 ICa,T and ICa,L [28] (Fig. 4c). ICa,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 ICa,L was activated from –50 mV, and peaked at –20, or –10 mV (Fig. 4d). ICa,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 (IKr) [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).


Figure 4
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  		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 If current
From a HP of –35 mV, hyperpolarising steps elicited inward current (Fig. 4g,h). To isolate If 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 IKir. Five mM Cs+ (Fig. 5a, open square) blocked If by 96±2% (n=9) in a reversible way (Fig. 5a, filled square). Upon deactivation at +5 mV If 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 If block (P<0.0001). We have then used the If sensitivity to extracellular Cs+ to construct the If current-to-voltage relation (Fig. 5b). Fig. 5c shows the If sensitivity to application of the β-adrenergic agonist noradrenaline and the muscarinic agonist acetylcholine, as reported for native If in rabbit SAN cells [6,13]. At –90 mV, application of 10 µM noradrenaline stimulated If by 21%, while application of 5 µM acetylcholine, inhibited If by 37%.


Figure 5
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  		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 If 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 If 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 If 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).


Figure 6
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  		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 If
We next examined the If current reversal potential in mouse SAN cells (Fig. 7a). The apparent reversal potential of If tail currents was between –30 and –20 mV (n=7). Precise quantification of If reversal potential was obtained by measuring the fully activated If current-to-voltage relation (Fig. 7b). Best fit of data points (Fig. 7b, dotted lines) yielded parameters: Vrev=–31±2 mV, and gmax=4±1 nS (n=4). The fully activated current-to-voltage relation was linear, indicating that measurements of If tail currents were not contaminated by residual IKir in depolarisation.


Figure 7
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  		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 If throughout its voltage range of activation (Fig. 8A). Increasing the K+ concentration from 5 to 35 mM significantly increased If (Fig. 8B). Note that If 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).


Figure 8
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  		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 If Kinetics in mouse SAN cells
The If 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 V1/2=–87±1, and v=12±1 (n=6).


Figure 9
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  		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 If 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, If 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.


Figure 10
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  		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 If in mouse, to that of rabbit SAN and cloned HCN channels is shown in Fig. 11. The If 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 {alpha}0=0.00196 s–1, β0=13.26129 s–1, V0=19.58724 mV. Consistently with the calculated V1/2, 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 If, and hHCN2.


Figure 11
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  		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 If, other currents that have been previously reported in other SAN preparations [3] have been consistently found here (Fig. 4).

If recorded in mouse SAN cells is similar to rabbit SAN If. Indeed, the insensitivity of If 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 If current was found to be restricted to spontaneously beating cells (Fig. 6). The possibility that If 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 If 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 If in mouse SAN cells has the same kinetic behaviour of If 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 If in mouse SAN. This view is reinforced by the existence of cells having significantly slower activation kinetics than others (Fig. 10b). Heterogeneity of If kinetics was not due to a negative shift of the If 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 If 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 If density was higher than that reported for rabbit SAN. Enhanced expression of If 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 If would be activated in the diastolic range. Indeed, in this voltage interval, the fractional activation of If varies between 0.02 and 0.1 (Fig. 9c). Accordingly, the densities of the net Cs+-sensitive If 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, ICa,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 IKr (Fig. 4e); the activating If and ICa,L and possibly ICa,T, depending on the relative channel availability close to the MDP (Fig. 4). The possibility that the Ist current [39] could also be involved cannot be excluded, since our recording conditions were not adapted for recording Ist 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 IK(ACh) and ICa,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 If 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 If 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 If and that of the rabbit demonstrates that our preparation would be appropriate to study the consequences of If gene invalidation on pacemaking. Furthermore, new information about the physiological role of If in the autonomic regulation of heart rate could be obtained from mouse strains where genes coding for channels such as ICa,L and IK, 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
 

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