Modulation of canine cardiac sodium current by Apelin

Abstract

Apelin, a ligand of the G protein-coupled putative angiotensin II-like receptor (APJ-R), exerts strong vasodilating, cardiac inotropic and chronotropic actions. Its expression is highly up-regulated during heart failure. Apelin also increases cardiac conduction speed and excitability. While our knowledge of apelin cardiovascular actions is growing, our understanding of the physiological mechanisms behind the cardiac effects remains limited. We tested the effects of apelin on the cardiac sodium current (I(Na)) using patch clamp technique on cardiac myocytes acutely dissociated from dog ventricle. We found that apelin-13 and apelin-17 increased peak I(Na) by 39% and 61% and shifted its mid-activation potential by -6.8+/-0.6 mV and -17+/-1 mV respectively thus increasing channel opening at negative voltage. Apelin also slowed I(Na) recovery from inactivation. The effects of apelin on I(Na) amplitude were linked to activation of protein kinase C. Apelin also increased I(Na) "window" current by up to 600% suggesting that changes in intracellular sodium may contribute to the apelin inotropic effects. Our results reveal for the first time the effects of apelin on I(Na). These effects are likely to modulate cardiac conduction and excitability and may have beneficial antiarrhythmic action in sodium chanelopathies such as Brugada Syndrome where I(Na) amplitude is reduced.

Keywords: Arrhythmia; Contraction; Dog; INa; Myocyte; Sodium channels; Ventricle.

Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Figures

Fig. 1

Expression of APJ receptors in dog cardiac left ventricle (LV). (A) Identification of endogenously expressed APJ-R by Western blot in both dog cardiac left ventricle (LV) and rat brain (RB) used as control. Pre-absorbed antibodies (Pa) did not reveal the specific bands observed in LV. (B, C). Cellular distribution of APJ-R in isolated LV myocytes. Staining with rat APJ-R antibodies in green and cellular nucleus by propidium iodide in red. (C) Magnified image of the inset highlighted in B. (D) Control photomicrograph of the APJ-R antibody pre-absorbed against its antigen. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Apelin increases INa amplitude in cardiomyocytes isolated from the canine left ventricle. Protocol used for characterization of the current-voltage relationship (I/V) of INa. Representative recordings of INa in control (Ctrl.), and following perfusion (20 min) with apelin-13 or apelin-17 (100 nM).

Fig. 3

Effect of apelin on the I/V and activation curves of INa in isolated myocytes. (A) Peak INa was normalized to the capacitance of their respective cells. Current densities (pA/pF) were plotted against their respective test potential in control (■ n = 14) and following application of 100 nM apelin-13 (○ n = 7) or apelin-17 (△ n = 7). Peak currents were significantly increased by both active fragments compared to control (p < 0.01). (B) Conductance (G) at each potential was calculated as the ratio INa/(Vm − ENa) where Vm represents the membrane test potential and ENa the reversal potential for INa. Each G values were normalized to the maximum conductance (GMax). Data were plotted against the test potentials to obtain INa activation curve for each condition presented in A. A Boltzmann equation fit to data yielded mid-activation potential (V1/2) values of −31.9 ± 0.3 mV, −38.7 ± 0.5 mV and −40.5 ± 0.3 mV for control, Apelin-13 and Apelin-17, respectively. There was no significant difference between the two active fragments of apelin. Slope factors (k) were similar for control (5.6 ± 0.3 mV), apelin-13 (5.1 ± 0.4 mV) and apelin-17 = (4.3 ± 0.3 mV). Both apelin-13 and apelin-17 activation curves are significantly shifted to more negative potentials compared to control (p < 0,05, F-test). Data are presented as Mean ± SEM.

Fig. 4

Apelin does not modulate sodium channel availability (steady state inactivation) in isolated dog cardiomyocytes from the left ventricle. (A) Standard voltage clamp protocol to measure steady state inactivation consisting of a series of conditioning pulses in increments of 5 mV from a holding potential of −120 mV followed by a test pulse to −15 mV. (B) INa inactivation curves were obtained by plotting the ratio of INa to its maximum value against the conditioning pulse voltage. Data represent values in control (■ n = 12) and with 100 nM apelin-13 (○ n = 5) or apelin-17 (△ n = 7). Mid-inactivation potentials (V1/2) and slopes (k) from a Boltzmann equation fit to data are not statistically significant (p > 0,05). (V1/2; control: −70.9 ± 0.1 mV, apelin-13: −70.4 ± 0.2 mV, apelin-17: −72.1 ± 0.1 mV; k; control: 5.7 ± 0.1, apelin-13: 5.7 ± 0.2, apelin-17: 13 = 5.2 ± 0.1).

Fig. 5

Overlap of steady state activation (m∞) and steady state inactivation (h∞) curves reveals an increase in sodium window current (IW) by apelin. (A) m∞ and h∞ curves in control (■) and following perfusion with 100 nM apelin-13 (○) or apelin-17 (▲). (B) Expanded view of the overlap between the h∞ and m∞ curves from the Boltzmann equation fit to data in A. Intersection points (dotted line) were at −51 mV, −53 mV, −55 mV for control, apelin-13 and apelin-17, respectively. (C) Theoretical IW current voltage relationship calculated from the Hodgkin-Huxley model (IW = GMaxm3h (Vm − ENa), see Materials and methods) in control (■) and following perfusion with 100 nM of apelin-13 (■) or apelin-17 (▲). Maximum IW amplitude was negatively shifted from −29 mV in control to −35 mV and −37 mV with apelin-13 and apelin-17, respectively. (D) Total electric charge carried by IW as obtained from the integral of its I/V relationship presented in C and expressed as percent of the control values.

Fig. 6

Apelin slows INa recovery from inactivation in isolated dog LV myocytes. (A) Standard electrophysiological double pulse protocol (S1-S2) to −20 mV for 20 ms used for measurements of recovery from inactivation. (B) INa recovery from inactivation expressed as the fraction of the initial current elicited during the second pulse and plotted against the inter-pulse interval duration (△t) in control (■ n = 11) and following application of 100 nM apelin-13 (○ n = 5) or apelin-17 (△ n = 7). Data were fitted to a sum of two exponentials. Statistical comparison between the fit using an F-test shows that both apelin isoforms significantly slow recovery from inactivation

Fig. 7

The effect of apelin on INa amplitude is mediated by PKC. (A) Left: Representative current recordings on cardiomyocytes pre-treated with IBMX (100 mM) + 8Br-cAMP (50 μM) for 20 min (control) to inhibit PKA activation followed by concurrent perfusion with Apelin-13 for 15 min. Right: Representative recordings from same protocol as left panel but this time myocytes were pre-treated with chelerythrine (3 μM) for 30 min to inhibit PKC. (B) Left: PKA inhibition did not prevent the increase in INa by apelin-13, as shown by the I/V relationship under perfusion with PKA inhibitor (■ n = 6) and apelin-13 (100 nM) (○ n = 6). Apelin-13 significantly increased the maximum amplitude of INa from −17,8 ± 2,0 pA/pF to −27,6 ± 2,4 pA/pF as seen in Fig. 2 without pre-treatment. Right: I/V curves following concurrent but sequential application of the PKC inhibitor (■ n = 5) and apelin-13 (○ n = 5). Inhibition of PKC activity by chelerethrine reduced the amplitude of INa compared to untreated cells. Apelin-13 increase in INa amplitude was lost following inhibition of PKC (maximum INa chelerythrine: −11,1 ± 2,4 pA/pF, chelerythrine + Apelin-13: −12.1 ± 1.9 pA/pF).

Fig. 8

Apelin increase excitability in canine ventricular preparations. Representative action potential recordings and corresponding maximum rate of depolarization (Vmax) values (A–B) following stimulations at a cycle length (CL) of 800 ms under control and in the presence of Apelin (100 nM) in canine superfused ventricular preparations. Bar graph summarizes the effect of Apelin on Vmax at concentrations of 30 nM and 100 nM (C). *p < 0.05 versus respective control (C); n = 4.