Electrophysiological effects of 5-hydroxytryptamine on isolated human atrial myocytes, and the influence of chronic beta-adrenoceptor blockade

Abstract

5-Hydroxytryptamine (5-HT) has been postulated to play a proarrhythmic role in the human atria via stimulation of 5-HT4 receptors. The aims of this study were to examine the effects of 5-HT on the L-type Ca2+ current (ICaL) action potential duration (APD), the effective refractory period (ERP) and arrhythmic activity in human atrial cells, and to assess the effects of prior treatment with beta-adrenoceptor antagonists. Isolated myocytes, from the right atrial appendage of 27 consenting patients undergoing cardiac surgery who were in sinus rhythm, were studied using the whole-cell perforated patch-clamp technique at 37 degrees C. 5-HT (1 nm-10 microm) caused a concentration-dependent increase in ICaL, which was potentiated in cells from beta-blocked (maximum response to 5-HT, Emax=299+/-12% increase above control) compared to non-beta-blocked patients (Emax=220+/-6%, P<0.05), but with no change in either the potency (log EC50: -7.09+/-0.07 vs -7.26+/-0.06) or Hill coefficient (nH: 1.5+/-0.6 vs 1.5+/-0.3) of the 5-HT concentration-response curve. 5-HT (10 microm) produced a greater increase in the APD at 50% repolarisation (APD50) in cells from beta-blocked patients (of 37+/-10 ms, i.e. 589+/-197%) vs non-beta-blocked patients (of 10+/-4 ms, i.e. 157+/-54%; P<0.05). Both the APD90 and the ERP were unaffected by 5-HT. Arrhythmic activity was observed in response to 5-HT in five of 17 cells (29%) studied from beta-blocked, compared to zero of 16 cells from the non-beta-blocked patients (P<0.05). In summary, the 5-HT-induced increase in calcium current was associated with a prolonged early plateau phase of repolarisation, but not late repolarisation or refractoriness, and the enhancement of these effects by chronic beta-adrenoceptor blockade was associated with arrhythmic potential.

Figure 1

Effect of 5-HT on I_CaL current–voltage relationship in human atrial myocytes. (a) An example of original calcium current (I_CaL) traces obtained from a human atrial cell (in this case from a β-blocked patient), during depolarising voltage-clamp pulses (100 ms, 0.33 Hz) from −40 to +40 mV, in 10 mV incremental steps, from a holding potential of −40 mV, under control conditions (open circle) and in the presence of 5-HT at 10 μM (closed circle). (b) Current–voltage relationships of I_CaL expressed in terms of current density, pA pF⁻¹ (n=7 cells, from six patients, treated and not treated with β-blockers). Values are means, with error bars denoting s.e.m., for control (open circles) 5-HT at 10 μM (closed circles) and after 3 min washout of 5-HT (open squares, n=3 cells, three patients). Asterisks indicate P<0.05 between control and 5-HT values at each voltage step (paired t-test).

Figure 2

Effect of the specific 5-HT₄ antagonist GR-113808 on I_CaL stimulated by 5-HT in human atrial myocytes. An example of the time course of change in peak I_CaL density (measured in this case in a cell from a β-blocked patient) plotted at 5 s resolution, in response to 0.001–0.1 μM 5-HT (open boxes), followed by the application of GR-113808 at 0.1 μM (solid box), and the subsequent washout of the antagonist and then of 5-HT. Inset traces show original currents recorded at the time points labelled.

Figure 3

Concentration-dependent effects of 5-HT on I_CaL in human atrial myocytes. (a) Concentration–response relationship for 5-HT (0.001–10 μM) on peak I_CaL in human atrial myocytes. Values are means±s.e.m. (n=17–33 cells, 9–17 patients). The increase in I_CaL is expressed as a percentage of the control value before the addition of 5-HT. The mean data points were fitted by a variable slope sigmoidal curve using the Hill equation (see Methods). (b) Comparison of concentration–response curves for 5-HT on I_CaL and (inset) of maximal I_CaL response to 5-HT, E_max, between cells from patients not treated with β-blockers (open symbols; n=8–15 cells, 5–7 patients) and those from patients treated with β-blockers (closed symbols; n=9–18 cells, 4–11 patients). Asterisk denotes P<0.05 vs non-β-blocked group.

Figure 4

Effect of 5-HT on action potentials and refractoriness in single human atrial cells from patients not treated with β-blockers. (a) Representative examples of original action potential recordings obtained from a single human atrial myocyte, from a patient not treated with a β-blocker, before (upper panel) and in the presence of 10 μM 5-HT (lower panel). Cells were paced at 75 bpm. Dotted lines in bold show the level of 50% of the action potential amplitude. The majority of cells displayed type 3 action potentials, that is, with pronounced phase 1 and a plateau amplitude below the 50% level. The ERP, indicated by solid bars, was calculated as the longest S₁–S₂ interval failing to elicit an S₂ response of amplitude >80% of the preceding S₁ action potential. The S₂ response used to measure this interval is labelled with an arrow. (b) Mean (±s.e.m.) APD (ms) measured at 50 and 90% repolarisation (APD₅₀ and APD₉₀, respectively; n=16 cells, eight patients) and ERP (n=12 cells, eight patients) in cells from patients not treated with β-blockers, in the absence (open bars), in the presence (closed bars) and after removal of 10 μM 5-HT (striped bars; n=11 cells, eight patients for APD and n=9 cells, seven patients for ERP). Asterisk denotes P<0.05 between control and 5-HT values (paired t-test).

Figure 5

Effect of 5-HT on APD and refractoriness in human atrial cells from patients treated with β-blockers. (a) Representative examples of action potentials recorded from a single human atrial myocyte from a patient treated with a β-blocker, before (upper panel) and in the presence of 5-HT at 10 μM (lower panel). Cells were paced at 75 bpm. Dotted lines in bold show the level of 50% of the action potential amplitude. The ERP, indicated by solid bars, was defined and calculated as in the legend of Figure 4a. (b) Mean (±s.e.m.) APD (APD₅₀ and APD₉₀; n=17 cells, nine patients) and ERP (n=12 cells, nine patients) in cells from patients treated with β-blockers, in the absence (open bars), in the presence (closed bars) and after removal of 10 μM 5-HT (striped bars; n=7 cells, five patients for APD and n=3 cells, three patients for ERP). The asterisk denotes P<0.05 between control and 5-HT values (paired t-test).

Figure 6

Effect of 5-HT to promote abnormal depolarisations in cells from β-blocked patients. An example of original recordings of action potentials obtained from a single human atrial myocyte (75 bpm pacing) from a patient treated with a β-blocker, before (top panel), in the presence of 5-HT at 10 μM (middle panel), and then following washout of 5-HT (bottom panel). The arrow indicates the presence of abnormal depolarisations, which occurred only in the presence of 5-HT.