Characterization and influence of cardiac background sodium current in the atrioventricular node

Abstract

Background inward sodium current (IB,Na) that influences cardiac pacemaking has been comparatively under-investigated. The aim of this study was to determine for the first time the properties and role of IB,Na in cells from the heart's secondary pacemaker, the atrioventricular node (AVN). Myocytes were isolated from the AVN of adult male rabbits and mice using mechanical and enzymatic dispersion. Background current was measured using whole-cell patch clamp and monovalent ion substitution with major voltage- and time-dependent conductances inhibited. In the absence of a selective pharmacological inhibitor of IB,Na, computer modelling was used to assess the physiological contribution of IB,Na. Net background current during voltage ramps was linear, reversing close to 0mV. Switching between Tris- and Na(+)-containing extracellular solution in rabbit and mouse AVN cells revealed an inward IB,Na, with an increase in slope conductance in rabbit cells at -50mV from 0.54±0.03 to 0.91±0.05nS (mean±SEM; n=61 cells). IB,Na magnitude varied in proportion to [Na(+)]o. Other monovalent cations could substitute for Na(+) (Rb(+)>K(+)>Cs(+)>Na(+)>Li(+)). The single-channel conductance with Na(+) as charge carrier estimated from noise-analysis was 3.2±1.2pS (n=6). Ni(2+) (10mM), Gd(3+) (100μM), ruthenium red (100μM), or amiloride (1mM) produced modest reductions in IB,Na. Flufenamic acid was without significant effect, whilst La(3+) (100μM) or extracellular acidosis (pH6.3) inhibited the current by >60%. Under the conditions of our AVN cell simulations, removal of IB,Na arrested spontaneous activity and, in a simulated 1D-strand, reduced conduction velocity by ~20%. IB,Na is carried by distinct low conductance monovalent non-selective cation channels and can influence AVN spontaneous activity and conduction.

Keywords: AVN; Atrioventricular node; Background current; I(B,Na); Pacemaking.

Fig. 1

Background currents elicited by voltage step (Ai–Aiv) and descending voltage ramp (Bi–Biv) protocols in rabbit AVN cells. Ai: Representative families of currents in Tris Na⁺-free and 150 mM-Na⁺ solutions. For clarity of display, only selected current traces are shown (protocol is shown underneath). Aii: Representative Na⁺-dependent inward background currents obtained by subtracting the currents in Tris Na⁺-free from those in 150 mM-Na⁺ solution (see Ai). Aiii: Mean current-voltage relations for currents (end pulse) in Tris Na⁺-free and 150 mM-Na⁺ solutions (mean ± SEM, n = 8 cells). Aiv: Mean current-voltage relation for the subtracted Na⁺-dependent inward background current, I_B,Na (mean ± SEM, n = 8 cells). Bi: Representative currents respectively in Tris Na⁺-free and 150 mmol/L-Na⁺ solutions (protocol is shown underneath). Bii: Representative Na⁺-dependent inward background current obtained by subtracting the current in Tris Na⁺-free from that in 150 mM-Na⁺ solution (see Bi); grey line denotes a fit to the data with a Goldman-Hodgkin-Katz (GHK) current equation for diffusion of permeant ions. Biii: Mean current-voltage relations for currents in Tris Na⁺-free and 150 mM-Na⁺ solutions (mean ± SEM (dotted lines), n = 61 cells). Biv: Mean current-voltage relation for the subtracted Na⁺-dependent inward background current, I_B,Na (mean ± SEM, n = 61cells).

Fig. 2

Na⁺-dependent inward background current (I_B,Na) in mouse AVN cells. A: Representative net currents in Tris- (a) and Na⁺ (b) -containing solutions elicited by descending voltage ramp (lower panel). B: The resulting Na⁺-sensitive subtraction current I_B,Na (b–a). C: The mean I–V relation for I_B,Na from mouse AVN cells (mean ± SEM (dotted lines), n = 6 cells). D: Overlay of the mean Na⁺-dependent background currents I_B,Na from mouse (black line and grey dotted lines show mean ± SEM; n = 6 cells) and rabbit (red line and pink dotted lines show mean ± SEM; n = 61 cells), indicating the similarity between I_B,Na obtained from the two species.

Fig. 3

Background inward current depends on the concentration of Na⁺ in the extracellular solution ([Na⁺]_o). Ai: Representative currents in various Na⁺ concentrations (recorded from a same cell). Aii: The difference curve b-a is the Na⁺-dependent current in 30 mM Na⁺; c-a, 75 mM Na⁺; d-a, 150 mM Na⁺; and e-a in 200 mM Na⁺ solution. B: the relations between log [Na⁺]_o and the current densities of the Na⁺-dependent currents at − 50 and − 100 mV in various [Na⁺]_o (mean ± SEM, n = 8 cells). C: The relation between log[Na⁺]_o and the slope conductance of the Na⁺-dependent currents at − 50 mV in various [Na⁺]_o (mean ± SEM, n = 8 cells). The straight black and dashed lines in B and C show the linear relations.

Fig. 4

Background inward current with differing external monovalent cations. Ai: Representative current traces in various monovalent cation external solutions as indicated (recorded from the same cell). The background inward current amplitude increased in the order of Li⁺ < Na⁺ < Cs⁺ < K⁺ < Rb⁺. Aii: The difference curves between Tris and various monovalent cation external solutions. B: Mean background inward current densities at − 50 and − 100 mV in various monovalent cation external solutions (mean ± SEM, n = 7 cells), indicating the channel mediating this background current exhibits poor cation selectivity.

Fig. 5

Effects of gadolinium (Gd^3 +), lanthanum (La^3 +), ruthenium red, nickel (Ni^2 +), amiloride, flufenamic acidic (FFA) and acidic pH on Na⁺-dependent inward background current (I_B,Na). Ai and Aii: in control condition, mean I–V relations for currents in Tris Na⁺-free and 150 mM-Na⁺ solutions (Ai), and mean I_B,Na (Aii) (mean ± SEM (dotted lines), n = 9). Bi and Bii: with application of 100 μM Gd^3 +, mean I–V relations for currents in Tris Na⁺-free and 150 mM-Na⁺ solutions (Bi), and mean I_B,Na (Bii) (mean ± SEM (dotted lines), n = 9). C: A summary of the effects of 100 μM Gd^3 +, 100 μM La^3 +, 100 μM ruthenium red, 10 mM Ni^2 +, 1 mM amiloride, 100 μM flufenamic acid (FFA), and acidosis of pH 6.3 on I_B,Na at − 100 mV. *P < 0.05, **P < 0.01; the numbers of cells for each experiment are given in parentheses.

Fig. 6

The single-channel conductance of I_B,Na estimated from power spectral analysis. (A) Mean whole cell Na-dependent inward current-voltage relations from 6 AVN cells. Currents were recorded during voltage steps ranging from − 110 to + 20 mV. Currents recorded in Tris-based solution were subtracted from currents recorded in Na-based solution. Solid line represents a fit to Eq. (1) (full data range not fitted because the equation becomes indeterminate at 0 mV). Dashed line indicates the asymptotic current-voltage relation for the unidirectional flux converging on an E_rev of 0 mV. (Bi) Example current traces recorded in Na (grey) and Tris (black) -based solutions on stepping to − 100 mV. (Bii) Example DC-subtracted current traces recorded at − 100 mV in Na (grey) and Tris (black)-based solutions. Data correspond to those shown in (i). (C) Example power spectral density. Data are from the cell shown in B. Solid line represents a fit to equation S1 (see online Supplementary information). D Mean unitary background channel Na current-voltage relations at the asymptote. Unitary current amplitudes were calculated from the integral of the power spectral density at each voltage according to Eq. (S2). Data correspond to the 6 cells shown in ‘A’. Solid line was fitted by linear regression constrained to reverse at 0 mV. The slope gives a mean open channel conductance of 3.2 ± 1.2 pS. Dotted lines show the 95% confidence intervals.

Fig. 7

Predicted role of I_B,Na in the AV node action potential. (A) predicted role of I_B,Na in AV node pacemaking. The traces show electrical activity calculated using the N cell model from Inada et al. before and after the elimination of I_B,Na from the N cell model. In the presence of I_B,Na, the model shows robust pacemaking, but after elimination of I_B,Na pacemaking is abolished. (B) Predicted role of I_B,Na in the driven AV node action potential. The 10th action potential during 2.5 Hz stimulation is shown. Action potentials before and after the elimination of I_B,Na are shown. After the elimination of I_B,Na, the resting membrane is hyperpolarized. (C) Current-voltage relationships for I_B,Na. Solid black line, experimental I_B,Na from Fig. 1Biv. Solid grey line, GHK flux equation fitted to experimental data. Dashed black line, current-voltage relationship predicted by the GHK equation under physiological conditions (for all simulations [Na⁺]_i = 8 μM; [Na⁺]_o = 140 mM; intracellular and extracellular [K⁺] were set, respectively to 140 mM and 5.4 mM). As shown in panel C (dashed line), the GHK simulated current under ‘physiological’ conditions was slightly smaller than that recorded experimentally (with 150 mM [Na⁺]_o and 0 [Na⁺]_i). It is the smaller current under ‘physiological’ conditions that was incorporated into action potential simulations.