Arrhythmic effects of Epac‐mediated ryanodine receptor activation in Langendorff‐perfused murine hearts are associated with reduced conduction velocity

Summary Recent papers have attributed arrhythmic substrate in murine RyR2‐P2328S hearts to reduced action potential (AP) conduction velocities (CV), reflecting acute functional inhibition and/or reduced expression of sodium channels. We explored for acute effects of direct exchange protein directly activated by cAMP (Epac)‐mediated ryanodine receptor‐2 (RyR2) activation on arrhythmic substrate and CV. Monophasic action potential (MAP) recordings demonstrated that initial steady (8 Hz) extrinsic pacing elicited ventricular tachycardia (VT) in 0 of 18 Langendorff‐perfused wild‐type mouse ventricles before pharmacological intervention. The Epac activator 8‐CPT (8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine‐3′,5′‐cyclic monophosphate) (VT in 1 of 7 hearts), and the RyR2 blocker dantrolene, either alone (0 of 11) or with 8‐CPT (0 of 9) did not then increase VT incidence (P>.05). Both progressively increased pacing rates and programmed extrasystolic (S2) stimuli similarly produced no VT in untreated hearts (n=20 and n=9 respectively). 8‐CPT challenge then increased VT incidences (5 of 7 and 4 of 8 hearts respectively; P<.05). However, dantrolene, whether alone (0 of 10 and 1 of 13) or combined with 8‐CPT (0 of 10 and 0 of 13) did not increase VT incidence relative to those observed in untreated hearts (P>.05). 8‐CPT but not dantrolene, whether alone or combined with 8‐CPT, correspondingly increased AP latencies (1.14±0.04 (n=7), 1.04±0.03 (n=10), 1.09±0.05 (n=8) relative to respective control values). In contrast, AP durations, conditions for 2:1 conduction block and ventricular effective refractory periods remained unchanged throughout. We thus demonstrate for the first time that acute RyR2 activation reversibly induces VT in specific association with reduced CV.

substrate, typically resulting from altered action potential (AP) conduction or recovery properties, this could trigger cardiac arrhythmias. 6 Recent studies have reported reduced atrial 7 and ventricular conduction velocities (CV) 8,9 that could potentially cause re-entrant arrhythmic substrate in murine models of catecholaminergic polymorphic ventricular tachycardia (CPVT) carrying the RyR2-P2328S mutation.
This was attributed to acute effects of altered intracellular Ca 2+ on Na + channel (Na v 1.5) function [7][8][9] and/or sustained downregulation of Na v 1.5 membrane expression. [9][10][11][12][13][14][15][16][17][18] The present experiments assessed whether acute manipulations of Ca 2+ homeostasis could influence CV and introduce arrhythmic substrate in wild-type hearts expressing normal RyR2, as opposed to RyR2-P2328S hearts. They complement recent studies that had modified Ca 2+ homeostasis through exchange protein directly activated by cAMP (Epac) activation of RyR2-Ca 2+ release channels 19 through a phosphokinase A (PKA)-independent pathway. 20,21 These studies had demonstrated that Epac activation increased Ca 2+ spark frequencies in adult rat cardiac myocytes, 22 amplitudes of Ca 2+ -dependent Ca 2+ release after isoproterenol treatment, 23 and amplitudes and frequencies of spontaneous Ca 2+ release in mouse ventricular cardiomyocytes. 24 They also confirmed that Epac activation acutely increased the incidence of both VT and triggered activity, 24  -2′-O-methyladenosine-3′,5′-cyclic monophosphate), 19 and of RyR2antagonism by dantrolene. 25 The latter was both applied alone, and in combination with Epac activation by 8-CPT. Dantrolene is known to inhibit diastolic Ca 2+ release, decrease the frequency and duration of aberrant Ca 2+ sparks in cultures of myocytes modelling CPVT, 26 and heart failure. 27,28 It also increases the threshold for Ca 2+ -induced-Ca 2+release from the sarcoplasmic reticulum in myocytes from failing but not control rabbit hearts. 28 The present study thus additionally throws light on the use of dantrolene in a potential therapeutic approach for the management of Ca 2+ -mediated ventricular arrhythmias.  This simulated the effect of progressively increasing heart rates. The incremental pacing protocols culminated in either a 2:1 loss of capture (where one AP is triggered for every two consecutive stimuli: Figure 2B and C(i)), which provided a measure of refractory period, or VT ( Figure 2C Figure 2C(i)), or in VT ( Figure 2C(ii)). Waveforms of consecutive action potentials observed under such conditions of 2:1 block often showed an alternans pattern. The latter is known to presage an onset of major cardiac arrhythmias in both murine and human hearts. 29 Figure 2C(ii) illustrates a genesis of VT: regular MAPs were recorded at a stimulus BCL of 44 ms. At the arrow, stimulus interval was reduced to 39 ms, and this was followed by an episode of irregular AP firing that eventually developed into VT (marked by square bracket). Table 1 Table 1). Figure 3 illustrates results obtained from programmed applications of extrasystolic (S2) stimuli using the S1S2 programmed electrical stimulation (S1S2 PES) protocol, simulating effects of extrasystolic events that might provoke arrhythmia in the presence of re-entrant substrate. This consisted of runs of repeated stimulus trains, each composed of eight stimuli paced at 8 Hz followed by an S2 stimulus. The stimulus interval between the S2 and preceding S1 stimulus (known as the 'S1S2 stimulus interval') was shortened by 1 ms each time the stimulus train repeated, beginning at 124 ms ( Figure 3A).

| RESULTS
With shortened S1S2 intervals, hearts perfused with control solution containing no drugs ( Figure 3B(i)), dantrolene ( Figure 3B(iii)), and dantrolene combined with 8-CPT ( Figure 3B(iv)) became refractory to the S2 stimulation, as reflected in the absence of an MAP after the S2 stimulus. This S1S2 stimulus interval therefore corresponds to the VERP.
In contrast, with 8-CPT treatment ( Figure 3B(ii)), instead of a loss of AP firing in response to the S2 stimulus, shortening of the S1S2 stimulus interval frequently resulted in VT. During quantification of such results, the S1S2 PES protocol was always repeated at least once in the same heart under the same condition to confirm observations made during the first run of the protocol. In agreement with results from the incremental pacing protocol, the S1S2 PES procedure demonstrated that challenge by 8-CPT (P<.05) but not by dantrolene alone (P>.05) significantly increased arrhythmic incidence but that dantrolene reversed these arrhythmic effects of 8-CPT (8-CPT vs dantrolene+8-CPT, P<.05; control vs dantrolene+8-CPT, P>.05) ( Table 1) and atrial APs that could potentially create arrhythmic substrates. 8,9 (iii) The altered Ca 2+ homeostasis also appeared to modify the Na + current with consequences for CV. The latter could involve (a) a long term downregulation of cardiac Na + channel (Na v 1.5) expression that was observed in both atrial 9 and ventricular RyR2-P228S myocytes. 10 WT rat cardiomyoctes similarly showed increased and decreased surface membrane expression of functionally active Na v 1.5, Na v 1.5 mRNA and total Na v 1.5 protein following challenge by the Ca 2+ channel blocker verapamil and the Ca 2+ ionophore calcimycin respectively. 11,12 (b) Alternatively, acute effects of altered intracellular Ca 2+ resulting from enhanced RyR2-mediated Ca 2+ release might modify Na + channel function. Nav1.5 thus contains two potentially directly Ca 2+ -sensitive regions: (i) a Ca 2+ -binding EFhand motif 13 and (ii) a calmodulin-binding IQ domain in the Na v 1.5 C-terminal region. 14,15 Both these sites have been suggested to mediate Ca 2+ -mediated modulation of Na + channel function. Thus, acute increases and decreases in Na + current densities followed in-  Hearts paced with the S1S2 programmed electrical stimulation (S1S2 PES) protocol to simulate the occurrence of extrasystolic beats within regular pacing. (A) The S1S2 PES protocol was composed of repeats of stimulus trains. Each stimulus is marked out by a vertical marker. Each train consisted of eight S1 stimuli ((A) narrow marker or (B) filled triangles indicating the first and last S1 stimulus in each train; BCL=125 ms), followed by an extrasystolic (S2) stimulus (wide marker in (A) and open triangle in (B)). The interval between the S2 and the preceding S1 stimulus (S1S2 interval) was shortened by 1 ms with each repetition of the S1 stimulus train. The longest S1S2 interval was 124 ms. The S1S2 PES protocol was used to study the effects of progressively shortening S1S2 stimulus intervals on the generation of arrhythmia in hearts perfused with and without drugs. Typical results compared for hearts perfused with (i) control solution, (ii) 8-CPT, (iii) dantrolene, and (iv) with dantrolene combined with 8-CPT treatment, showing either extrasysolic action potentials (clear arrows), refractoriness, or an episode of VT.
: S1 stimuli; : S2 stimuli; : MAP in response to S2 stimulus group substitution on its cAMP ribose moiety disrupts its interaction with PKA whilst permitting binding to Epac. This gives 8-CPT a 300fold selectivity for Epac relative to PKA at the low, 1 μmol/L, concentrations employed here. 19,[31][32][33] Further reported actions of 8-CPT on phosphodiesterase isoforms required significantly higher inhibitory binding concentrations than those used here. 34 Epac is implicated in adrenergically-mediated cardiac arrhythmogenesis. 22 Previous studies reported that experimental Epac activation increased the frequencies and amplitudes of spontaneous Ca 2+ release 22,24 and the amplitudes of Ca 2+ waves resulting from Ca 2+ -induced Ca 2+ release. 23 It was arrhythmogenic in Langendorff-perfused whole hearts, 24 likely through altered Ca 2+ homestasis following its interaction with and opening of the RyR2-Ca 2+ -release channel. 19 The present experiments nevertheless first confirmed that 8-CPT acutely produced arrhythmic substrate. Admittedly, steady extrinsic pacing, whether before or following pharmacological intervention, did not induce arrhythmia, but nevertheless provided the AP parameters of APD and latency.  26,27,35 The present experiments demonstrated that applied by itself, dantrolene altered neither arrhythmic tendency nor AP conduction and recovery parameters.
However, it reversed the actions of 8-CPT. Thus when both drugs were added in combination, there was no arrhythmia, and AP parameters were restored to normal.
Together these findings suggest that reversible arrhythmic effects related to slowed AP propagation but unchanged AP recovery can follow acute modifications of Ca 2+ homeostasis accompanying interventions increasing RyR2-mediated sarcoplasmic reticulum Ca 2+ release.
They complement previous observations of a longer term reduction in T A B L E 1 Incidence of ventricular tachycardia (VT) observed with S1S2 PES and incremental pacing protocols Indicates where there is a significant increase in the ratio of AP conduction latency following treatment with 8-CPT compared prior to treatment in the control condition, as shown by an increase in ratio of latency post-treatment vs pre-treatment (P<.01; n=7).
T A B L E 2 Action potential (AP) parameters following pharmacological treatment normalised to control values in the same heart Nav1.5 expression following chronic alterations in Ca 2+ homeostasis associated with the RyR2-P2328S mutation. 10

| METHODS
All animals used for experiments were of the WT sv129 background.
A total of 20 male and female, between the ages of 5 and 7 months, were used for the experiments. Mice were kept in animal house facilities at 21°C and exposed to 12  Experiments were carried out under the following conditions, defined by the perfusate used for the heart.

1.
The control procedure used KH solution alone without any added pharmacological agents. All data are expressed as mean±SEM; where time is the variable, data is presented in milliseconds (ms). The number of hearts from which data is derived from for each condition is designated n. For categorical data, Fisher's exact test was used to test for statistical significance between two data sets, each with two possible outcomes, the presence or absence of VT. Changes in the continuous parameters of AP latency, AP duration at 90% recovery (APD 90 ), basic cycle length (BCL) at the onset of 2:1 block, and VERPs were tested using Student's paired t-test. Statistical significance obtained from the t-test was defined as P<.05.