Rate dependency of beta-adrenergic modulation of repolarizing currents in the guinea-pig ventricle

Abstract

Beta-adrenergic stimulation modulates ventricular currents and sinus cycle length (CL). We investigated how changes in CL affect the current induced by isoprenaline (Iso) during the action potential (AP) of guinea-pig ventricular myocytes. Action-potential clamp was applied at CLs of 250 and 1000 ms to measure: (1) the net current induced by 0.1 microm Iso (I(Iso)); (2) the L-type Ca2+ current I(CaL) and slow delayed rectifier current I(Ks) components of I(Iso) (I(IsoCa) and I(IsoK)), identified as the Iso-induced current sensitive to nifedipine and HMR1556, respectively; and (3) I(Iso) persisting after inhibition of both I(Ca) and I(Ks) (I(isoR)). The pause dependency of I(Ks) and its modulation were evaluated in voltage-clamp experiments. The rate dependency of the duration of the action potential at 90% repolarization (APD90) and its modulation by isoprenaline were tested in current-clamp experiments. At a CL of 250 ms I(Iso) was inward during initial repolarization and reversed at 59% of APD90. At a CL of 1000 ms I(Iso) became mostly inward in all cells. Switching to shorter CL did not change I(IsoCa) and I(IsoK) amplitudes, but moved their peak amplitudes to earlier repolarization; I(IsoR) was independent of CL. Acceleration of I(IsoK) at shorter CL was based on faster pause dependency of I(Ks) activation rate. The 'restitution' of activation rates was modulated by isoprenaline. The APD90-CL relation was rotated anticlockwise by isoprenaline and crossed the control curve at a CL of 150 ms (400 beats min(-1)). We conclude that: (1) isoprenaline induced markedly different current profiles according to pacing rate, involving CL-dependent I(Ca) and I(Ks) modulation; (2) the effect of isoprenaline on APD90 was CL dependent, and negligible during tachycardia; and (3) during sympathetic activation, repolarization stability may involve matched modulation of sinus rate and repolarizing currents.

Figure 1. Cycle-length dependency of total isoprenaline-induced current (I_Iso)

A, sample I_Iso recordings at the two CLs with the corresponding AP waveforms (absolute time scales). B, top panels show average AP waveform and net transmembrane current density (I_net = −dV/dt) required to generate it; bottom panels show I_Iso density. In B the time scale was normalized to APD₉₀. Average recordings (n = 21) and ± s.e.m. interval are shown by continuous and dotted lines, respectively.

Figure 2. Cycle-length dependency of isoprenaline-induced calcium current (I_IsoCa)

Description and symbols as in Fig. 1. For average recordings n = 6.

Figure 3. Cycle-length dependency of isoprenaline-induced I_Ks (I_IsoK)

Description and symbols as in Fig. 1. For average recordings n = 6.

Figure 4. Cycle-length dependency of I_Ks conductance (G_Ks) and its modulation

A, representative example of G_Ks profile at two CLs, in control (ctrl) and during isoprenaline (Iso). B, average G_Ks profiles (continuous lines) and their ± s.e.m. interval (dotted lines; n = 6).

Figure 5. Pause dependency of I_Ks reactivation rate

A, sample recordings of I_Ks (protocol shown in the inset) under control conditions (ctrl) and in the presence of isoprenaline (Iso). B, restitution of activation kinetics. The time constant of I_Ks reactivation (τ_react), i.e. activation during S2, is plotted as a function of the S1–S2 interval. Control (•) and isoprenaline (○) data points were fitted (continuous lines) to estimate the time constant of the restitution process (τ_rest, see textl; n = 5). The overall difference between control and isoprenaline data is highly significant (P < 0.001 with ANOVA for repeated measurements); for individual points *P < 0.05 (Bonferroni's correction).

Figure 6. Cycle-length dependency of I_Iso after I_CaL and I_Ks blockade (I_IsoR)

A, average action potential waveforms. B, average I_IsoR density recorded at the two CLs during the action potentials shown in A (time scales normalized to APD₉₀). C, average dynamic I–V relations of I_IsoR. Average recordings (n = 12) and their ± s.e.m. limits are shown by continuous and dotted lines, respectively.

Figure 7. Cycle-length dependency of APD₉₀ and its modulation

APD₉₀ measured during steady-state pacing at various CLs under control conditions (•) and in the presence of isoprenaline (○; n = 3 at all CLs). The continuous lines are logistic data fits, for which the asymptote was fixed at the 60 s APD₉₀ value. The double-headed arrow indicates the heart rate at which the extrapolated APD₉₀–CL relations cross (i.e. APD₉₀ would be unaffected by isoprenaline). The overall difference between control and isoprenaline data is highly significant (P < 0.001 with ANOVA); for individual data points *P < 0.05 (Bonferroni's correction).

Figure 8. Rate of onset of isoprenaline effects on APD₉₀ and membrane current

The time of occurrence of peak inward I_Iso (at steady-state isoprenaline superfusion) was used in each cell as the reference point for current measurement. A and B show examples of the time course of the APD₉₀ (current clamp) and membrane current (AP clamp) responses to isoprenaline (horizontal bar). To highlight CL-dependent changes in the kinetics of response, APD₉₀ (APD) and current (I) were normalized to the steady-state value achieved at each CL (APD_max and I_max, respectively). Continuous lines represent fitting of the exponential phase of response. C, time constants (τ) of APD₉₀ (□, n ≥ 8 at each CL) and membrane current (▪, n ≥ 7 at each CL) response as a function of CL.