Mechanisms underlying the antiarrhythmic effect of ARumenamide-787 in experimental models of the J wave syndromes and hypothermia

Figures

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  Fig 1. Effect of AR-787 on the transient outward current (Ito), inward calcium current (ICa) and action potential recorded from canine right ventricular epicardial myocytes. (n = 6) A: Representative action potential traces recorded in the absence (Control) and presence of AR-787 (10 μM) at a frequency of 0.5 Hz. B and C: Representative traces of Ito and ICa recorded in Control and following exposure to AR-787 (10 μM). D and E: I-V relationships for peak Ito and ICa recorded under control conditions and following exposure to AR-787. F: Molecular structure of Arumenamide-787 (AR-787). All data were obtained at 37°C. Data are reported as mean ± SEM; *p<0.05, ** p<0.001 vs. Control (One Way Repeated Measures ANOVA).

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  Fig 2. Effects of 10mM AR-787 on hERG currents. A: Control currents recorded prior to addition of AR-787. B: Currents recorded at a test potential of +10mV before (control, black) and 7 min after the addition of AR-787 to the perfusate (grey). C: Currents recorded after AR-787 addition (protocol started 7 min after the initiation of AR-787 addition). D: Voltage dependence of steady currents recorded at last 50 msec of the first voltage step (arrow labeled S in panel C). E: Voltage dependence of peak tail currents recorded at -50mV (arrow labeled P in panel C). F: Voltage dependence of activation determined from tail currents normalized to the peak value for each test potential. G: Inactivation time constant determined by curve fitting a single exponential to the tail current. Grey traces in A and C are recorded at a test potential of +10mV. Vertical line below panel A represents 0.7nA while horizontal line represents 2 sec. Data points and vertical bars in panels D-G represent mean ±1 SEM (n = 8). Symbols above or below data points in panels D-G represent statistically significant differences between control and AR-787 determined by a paired data, single-sided Student’s t-Test at p<0.05…

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  Fig 3. The effect of AR-787 on sodium channel activity. The upper panel shows representative traces of the effect of AR-787 (10 μM) on INa (AR-787 -red and Control—black). A: AR-787 (10 μM) induced a significant increase in the conductance of sodium as clearly seen in the significant increase in INa density (B) at -50 mV and -40 mV. C: AR-787 (10 μM) has no significant effect on the voltage-dependence of conductance. D: AR-787 (10 μM) has no significant effect on the voltage-dependence of steady-state fast inactivation.

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  Fig 4. Effect of 1, 10 and 50 μM of AR-787 to suppress the electrocardiographic manifestations in models of JWS (ERS and BrS). From top to bottom, all columns (Col) in each panel shows simultaneously recorded transmembrane action potentials from the M and Epi regions together with a pseudo-ECG from 3 different wedge preparations (panels A [LV wedge 1], B [LV wedge 2] and C [RV wedge]). BCL = 800 ms. A: Recording obtained under Control (Col 1), after 20 min of perfusion with NS5806 (NS, 10 μM) (Col 2) and 25 min after addition of AR-787 (AR, 1 μM) to the perfusate (Col 3). B: Recordings obtained under Control conditions (Col 1), 20 min after perfusion with NS5806 (NS, 10 μM) (Col 2) and 20 min after addition of AR-787 (AR, 10 μM) (Col 3). C: Recordings obtained under Control conditions (Col 1), after 20 and 40 min of exposure to NS5806 (NS, 10 μM) (Col 2 and Col 3, respectively) and 15 min after addition of AR-787 (AR, 50 μM) to the coronary perfusate (Col 4).

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  Fig 5. Effect AR-787 (10 μM) following NS5806 (5–10 μM) on AP and ECG parameters recorded from 3 LV wedge preparations. A: Epi Ph1 magnitude as a % of Ph0 amplitude. B: J wave amplitude as a % of R wave amplitude. C: QT interval (ms). BCL = 800 ms (LV wedges; n = 3). NS5806 (5–10 μM) significantly increased the Epi Ph1 magnitude and the simultaneously recorded electrocardiographic J wave. AR-787 (10 μM) reversed this effect restoring these parameters to control values. The QT interval was unaffected by either intervention. The symbols are data points, and the crossed symbols indicate the mean values (data derived from Table 2); * p<0.005 vs. Control and AR-787 (One Way Repeated Measures ANOVA).

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  Fig 6. AR-787 suppresses ECG and arrhythmic manifestations in a wedge model of BrS in which the disease phenotype was provoked using ajmaline to mimic the loss of function of sodium channel currents caused by mutations in genes encoding the cardiac sodium channel. From top to bottom, each panel shows action potentials recorded from the M region and 2 epicardial sites (Epi 1 and Epi 2) of a RV wedge preparation together with a pseudo-ECG (ECG). BCL: 2000 ms A: Control. B: Recordings obtained after addition of NS5806 (6 μM; 45 min) to the coronary perfusate. C: Further addition of ajmaline (2.5 μM; 30 min) dramatically increased the J wave leading to development of a short run of pVT triggered by a closely coupled premature beat arising from Epi (Phase2-reentry). D: Addition of AR-787 (10 μM; 20 min) restored the epicardial AP dome, greatly diminished the J wave in the ECG and suppressed all arrhythmic activity. E: Washout of AR-787 (85 min) resulted in reappearance of the BrS phenotype with restoration of a prominent J wave in the ECG due to loss of epicardial AP dome.

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  Fig 7. Effect of AR-787 to suppress the electrocardiographic and arrhythmic manifestations in a model of ERS in which the disease phenotype was provoked by exposure to the Ito agonist, NS5806, and the calcium channel blocker, verapamil. From top to bottom, each panel shows simultaneously recorded transmembrane action potentials from the M and Epi regions together with a pseudo-ECG from a LV wedge preparation. BCL = 800 ms. A: Control recordings. B: Recordings obtained after 40 min of perfusion with a combination of NS5806 (NS, 10 μM) and verapamil (2.5 μM) C: Recordings obtained 22 min after the recordings shown in panel B. A closely coupled premature extrasystole is seen to trigger a short run of pVT. D: Recordings obtained 30 min after the addition of AR-787 (AR10 μM) to the coronary perfusate.

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  Fig 8. AR-787 suppresses the ECG and arrhythmic manifestations in a wedge model of ERS in which the disease phenotype was provoked using NS5806 and ajmaline. From top to bottom, each panel shows M and Epi APs and a pseudo-ECG (ECG) recorded from a LV wedge preparation. BCL: 2000 ms. A: Control. B: Recordings obtained after addition of NS5806 (3.75 μM; 60 min) to the coronary perfusate. C: Further addition of ajmaline (2.5 μM; 40 min) dramatically increased the J wave, leading to development of a closely coupled premature beat (CCPB) arising from Epi, consistent with a phase 2-reentry mechanism. D: Further addition of AR-787 (10 μM; 5 min) reduced the manifestation of the epicardial AP notch and J wave in the ECG and suppressed all arrhythmic activity.