Arrhythmogenic actions of the Ca2+ channel agonist FPL-64716 in Langendorff-perfused murine hearts

Abstract

The experiments explored the extent to which alterations in L-type Ca(2+) channel-mediated Ca(2+) entry triggers Ca(2+)-mediated arrhythmogenesis in Langendorff-perfused murine hearts through use of the specific L-type Ca(2+) channel modulator FPL-64716 (FPL). Introduction of FPL (1 microm) resulted in a gradual development (>10 min) of diastolic electrical events and alternans in spontaneously beating hearts from which monophasic action potentials were recorded. In regularly paced hearts, they additionally led to non-sustained and sustained ventricular tachycardia (nsVT and sVT). Programmed electrical stimulation (PES) resulted in nsVT and sVT after 5-10 and >10 min perfusion, respectively. Pretreatments with nifedipine, diltiazem and cyclopiazonic acid abolished arrhythmogenic tendency induced by subsequent introduction of FPL, consistent with its dependence upon both extracellular Ca(2+) entry and the degree of filling of the sarcoplasmic reticular Ca(2+) store. Values for action potential duration at 90% repolarization when any of these agents were applied to FPL-treated hearts became indistinguishable from those shown by untreated control hearts, in contrast to earlier reports of their altering in long QT syndrome type 3 and hypokalaemic murine models for re-entrant arrhythmogenesis. These arrhythmic effects instead correlated with alterations in Ca(2+) homeostasis at the single-cell level found in investigations of the effects of both FPL and the same agents in regularly stimulated fluo-3 loaded myocytes. These findings are compatible with a prolonged extracellular Ca(2+) entry that potentially results in an intracellular Ca(2+) overload and produces the cardiac arrhythmogenecity following addition of FPL.

Figure 1. Examples of monophasic action waveforms

Epicardial monophasic action waveforms from epicardial surfaces of the left ventricles of Langendorff-perfused hearts during both non-paced activity (A) and regular pacing at 8 Hz (B) before (a) and at different times following the introduction of 1 μm FPL-64716 (FPL) into the perfusing KH buffer solution (b–d).

Figure 2. Arrhythmic phenomena in non-paced and paced hearts

Numbers of hearts showing arrhythmic phenomena out of a total of n= 8 studied during both non-paced activity (A) and regular pacing at 8 Hz (B) before (a) and at different times following the introduction of 1 μm FPL-64716 (FPL) into the perfusing KH buffer solution (b–d). The bar graphs show incidences of hearts having one or more episodes of alternans (Ab–Ad and Bb–Bd), early and delayed after-depolarizations (EADs and DADs; Ad, Bc and Bd) or episodes of ventricular tachycardia (VT; Bd). The bar graphs also show results of Fisher's exact tests for these incidences when compared with control values obtained in the absence of FPL (*P < 0.01, **P < 0.001 and ***P < 0.0001; open bars indicate and absence or insignificant incidence of the arrhythmic phenomenon).

Figure 3. Monophasic action potentials recorded from the epicardium of hearts in the absence and presence of FPL

Monophasic action potentials recorded from the epicardium of hearts before (A), 5–10 min (B) and >10 min after the addition of 1 μm FPL (C). All experiments were performed during PES at 8 Hz; the arrows indicate S2 extra-stimuli. The examples illustrated that before addition of FPL (A), isolated perfused hearts showed a persistently regular rhythm with no arrhythmogenic events during the PES procedures. The S2 extra-stimuli initiated an episode of nsVT lasting for less than 30 s in 7 out of 8 hearts perfused with FPL for 5–10 min (B). All 8 hearts showed episodes of sVT lasting for more than 30 s with further perfusion with FPL for more than 10 min (C).

Figure 4. Epicardial monophasic action potential recordings in paced hearts and in hearts undergoing PES in the presence of various drugs

Epicardial MAP recordings obtained from both regularly paced hearts (A) and hearts undergoing PES (B) pretreated with 100 nm nifedipine (a), 100 nm diltiazem (c) and 150 nm CPA (e) before restoring 1 μm FPL to the buffer. None of the hearts showed arrhythmic phenomena following pretreatment (a, c and e). Inclusion of either nifedipine or diltiazem during subsequent FPL treatment totally suppressed FPL-induced VT (b and d, respectively) through either pacing protocol. However, 150 nm CPA in combination with a subsequent addition of 1 μm FPL (f) abolished FPL-induced VT although it did permit EAD episodes following the shortest S2 stimuli in each of n= 4 hearts.

Figure 5. The effect of alternans on APD₉₀ values

The APD₉₀ values in alternans were calculated for alternans that appeared 1 min following introduction of FPL. However, the presence of alternans significantly (*P < 0.001) decreased APD₉₀ values with increased FPL perfusion times after 1 min, 5–10 min and >10 min perfusion with FPL.

Figure 6. Fluo−3 fluorescence measurements in isolated myocytes studied using confocal microscopy

Normalized fluorescence (F/F₀) plotted against time with myocyte exposed to periodic field stimulation in perfusion buffer alone prior to any pharmacological manoeuvre (A) and records obtained following addition of 1 μm FPL to the buffer for approximately 15 s (B) and approximately 120 s (C). D shows overall mean peak F/F₀ values taken from the entire set of myocytes. The overall mean peak F/F₀ values are represented for the entire set of myocytes (Di) and subsidiary events (Dii).

Figure 9. Calcium transients in regularly stimulated isolated myocytes in the presence of CPA and FPL

Calcium transients in regularly stimulated (0.5 Hz) isolated myocytes before (A) and after addition of 150 nm CPA (B) and a further addition of FPL (C). D, CPA significantly (***P < 0.05; one-way ANOVA) reduced peak F/F₀ from 3.20 ± 0.04 (n= 9 cells) to 1.80 ± 0.06 (n= 12 cells), but there were no further changes in peak F/F₀ following introduction of FPL, which left a peak F/F₀ of 1.57 ± 0.05 (n= 6 cells), but an appearance of persistent spontaneous ectopic Ca²⁺ peaks in the intervals between stimuli whose peak F/F₀ was 1.32 ± 0.02 (n= 6 cells; C and D).

Figure 8. Calcium transients in regularly stimulated isolated myocytes in the presence of diltiazem and FPL

Calcium transients in regularly stimulated (0.5 Hz) isolated myocytes before (A) and after addition of 100 nm diltiazem (B) and a further addition of FPL (C). D, diltiazem significantly (***P < 0.05; one-way ANOVA) reduced peak F/F₀ from 1.77 ± 0.02 (n= 3 cells) to 1.30 ± 0.01 (n= 3 cells), but there were no further changes following introduction of FPL, which left a peak F/F₀ of 1.31 ± 0.01 (n= 4 cells).

Figure 7. Calcium transients in regularly stimulated isolated myocytes in the presence of nifedipine and FPL

Calcium transients in regularly stimulated (0.5 Hz) isolated myocytes before (A) and after addition of 100 nm nifedipine (B) and a further addition of FPL (C). D, nifedipine significantly (***P < 0.05; one-way ANOVA) reduced peak F/F₀ from 2.11 ± 0.04 to 1.37 ± 0.02 (n= 9 cells), but there were no further changes following introduction of FPL, which left a peak F/F₀ of 1.28 ± 0.05 (n= 6 cells).