Abstract

Introduction: Parasympathetic nervous system has been reported to provide an
antiarrhythmic effect during ischemia. On the other hand it might exert a proarrhythmic role
in Early Repolarization Syndrome. The exact cardiac electrophysiological effects of
muscarinic agonists in these conditions are not fully understood.
Methods: Action potentials were registered utilizing the standard microelectrode technique
from human donor cardiac preparations and canine papillary muscles (PM) and free-running
Purkinje fibers (PF). Ionic currents were measured using the whole-cell configuration of the
patch clamp method.
Results: 5 μM acetylcholine alone did not alter the action potential duration (APD) either in
canine Purkinje fibers (233.6 ± 4.7 ms vs 231.7 ± 4.6 ms, n=15) or in papillary muscle (172.6
± 5.7 ms vs 172.8 ± 5.3 ms, n=5) preparations . In contrast, after proadministration of I KATP
activator pinacidil (5 µM) (n=8) which caused a significant and large APD 90 reduction (from
207.7 ± 7 ms to 113.1 ± ms, p < 0.05), the cumulative addition of acetylcholine (5 µM)
resulted in significant APD 90 lengthening as early as 3 minutes (from 113.1 ± ms to 147.3 ±
7.4 ms, p < 0.05) in Purkinje fibers. In papillary muscles, after pinacidil induced APD
shortening (187.9 ± 4.5 ms vs 163.7 ± 6.4 ms, p < 0.05) acetylcholine (5 µM) was able to
oppose incompletely the effect of pinacidil (from 163.7 ± 6.4 ms to 172.1 ± 7.4 ms, n=7, p <
0.05). The dispersion of control APD 90 (9.5%, 20 ms, n=8 PF, n=7 PM) was significantly
increased by the administration of pinacidil (APD 90 dispersion: 44.7%, 51 ms, n=8 PF, n=7
PM). However, the cumulative addition of acetylcholine resulted in significant reduction of
this dispersion (16.9%, 28 ms; n=8 PF, n=7 PM, p < 0.05). Carbachol alone did not change
the control current (0 mV control: 0.20 ± 0.2 pA/pF vs 3 μM carbachol: 0.32 ± 0.2 pA/pF, n=6
and +30 mV – control: 0.55 ± 0.4 pA/pF vs 3 μM carbachol: 0.74 ± 0.3 pA/pF, n=6). In case
of prior 5 μM pinacidil application, carbachol administration resulted a significant attenuation
of the current at both voltages (0 mV – control: 0.24 ± 0.2 pA/pF  5 μM pinacidil: 2.03 ±
0.3 pA/pF  3 μM carbachol: 1.51 ± 0.4 pA/pF, n=8, p < 0.05. +30 mV – control: 0.78 ± 0.6
pA/pF  5μM pinacidil: 3.17 ± 0.3 pA/pF  3 μM carbachol: 2.26 ± 0.3 pA/pF, n=8, p <
0.05). Hypoxic condition resulted in shortening of APD 90 (181.4 ± 5.7 ms to 135.0 ± 8.6 ms)
(n=5, p < 0.05), reduction of amplitude (103.7 ± 2.8 mV vs 92 ± 3.5 mV, n=5) and decrease of
the maximum rate of depolarization (185.8 ± 15.8 V/s vs 156.1 ± 20.6 V/s, n=5). After 5 μM
acetylcholine application significant APD 90 prolongation (from 135.0 ± 8.6 ms to 164.4 ± 4.4
ms) and AMP recovery to a normal range (102.1 ± 1.6 mV, n=5) were seen, while V max
remained at the same value (156.0 ± 16.1 V/s, n=5). Acetylcholine (5 μM) exerted various
electrophysiological effects in human donor cardiac preparations. Cilostazol partially reversed
the effects of ionic channel modulators in Early Repolarization Syndrome models.
Conclusions: 5 μM acetylcholine was able to partially antagonize the APD shortening caused
by the previously used I KATP activator (5 µM pinacidil) and decreased the pinacidil induced
APD dispersion elevation between ventricular papillary muscle and Purkinje fibre. 3 μM
carbachol inhibited the ATP-sensitive K + current. After the generation of hypoxic
environment, the use of acetylcholine was able to offset hypoxia-induced APD 90 shortening,
and decrease in amplitude. Activation of the parasympathetic nervous system might reduce
the arrhythmogenic substrate providing antiarrhythmic mechanisms during ischemic

conditions. Cilostazol might be recommended for the treatment of some forms of Early
Repolarization Syndrome.