The role of intracellular Ca²⁺ (Ca_i) in triggering early (EADs) or delayers (DADs) afterdepolarizations (EADs), the origins of EADs and the mechanisms underlying Torsade de Pointes (TdP) were investigated in drug-induced models of the long QT syndrome (LQTS). LQTS types 1, 2, or 3 were elicited by perfusing the hearts with Anthopleurin-A (APA= 10-15 µM), HMR 1556 (0.1 or 1.0 µM) or E4031 (0.5 µM) which are know to suppress Na²⁺ channel inactivation (SCN5A), K currents involved in the repolarization of the action potential (AP) I_Ks or I_Kr respectively. EADs are frequently observed in LQTS and may precipitate TdP. Caoverload followed by spontaneous Ca²⁺ release from the sarcoplasmic reticulum has been proposed as a trigger of EADs by increasing Ca²⁺ dependent depolarizing currents. Ca_i overload may be due to enhanced Ca²⁺ influx during the long AP durations (APDs) typical of LQTS. While abnormal Ca_i handling, the firing of EADs and DADs was demonstrated in isolated cardiomyocytes, there is little data in whole hearts or evidence that Ca_i triggers EADs.To address these questions, we mapped APs and Ca_i optically from 256 sites of rabbit hearts that were perfused in a Langendorff apparatus. Hearts were loaded with Rhod-2/AM and RH237 to simultaneously map Ca_i and membrane potential (V_m) with 2 photodiode arrays. In some experiments we used a new potentiometric dye PGH1 because of its greater Stokes’ shift, larger fractional fluorescence change per AP and stable recordings for >4 hours. LQTS 1: HMR 1556 caused a marked increase in APDs in male compared to female hearts, indicating a gender difference in the level of I_Ks and had a more pronounced at the base than the apex. Hence, there was no shift in the direction of repolarization (apexÇ base). The I_Ks blocker failed to trigger EADs and TdP in either sex. The greater effects of HMR1556 on the APD of male than female hearts indicates that male hearts express a greater density of I_Ks than in female hearts. In long APs with HMR1556, Ca_i upstrokes followed the AP upstroke (10-12 ms) then gradually recovered to baseline value while V_m remained elevated during the AP plateau. LQTS 2: The I_Kr blocker, E4031 prolonged APDs in rabbits of both sexes from 230±17 ms to 2,364.4±112.9 ms (n=24 hearts), did not alter activation but reversed the repolarization sequence (apexÇ base became baseÇ apex; n=24). There were marked sex-differences in LQTS2. Before puberty rabbits (>60 days), serum levels of sexual hormones (testosterone and estrogen) are very low. E4031 induced EADs and TdP in pre-pubertal male (n=9/10) but not female hearts (n=18). In adult rabbit hearts, females were highly vulnerable to E4031-induced EADs and TdP (n=10) whereas males were protected (n=10). In EADs induced by E4031, Ca_i rises after the AP upstroke, decays 100 ms later then oscillates during the plateau phase. In most experiments (n=5/6), Cai followed V_m resulting in phase maps with counterclockwise trajectories indicating that Ca_i follows V during salvos of EADs. To test if EADs fire

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preferentially from Purkinje and endocardial cell, the septum, right and left endocardium was cryoablated resulting in a thin layer (~ 1 mm) of surviving myocardium. In cryoablated hearts, EADs originated at 2-3 sites on the anterior surface and at the origins of EADs, Ca_i preceded V_m. EADs fired out-of-phase from several sites, propagated and collided, consistent with the undulating EKGs of TdP. Phase maps of EADs had clockwise trajectories; that is Ca_i preceded V_m.LQTS 3: APA (10 µM) prolonged APDs (260 ± 30 ms to 870 ± 67 ms) in both sexes (n = 4/ each sex), elicited EADs and TdP. At EADs, Ca_i rose and peaked during the plateau before EADs fired. Phase maps had clockwise trajectories indicating that Ca²⁺ preceded V_m.Put together, the data show that in the LQTS, Ca_i elevation most likely due to spontaneous Ca²⁺ release from internal stores elicits salvos of EADs, a greater dispersion of repolarization and sustained TdP.