Volume 13, Issue 1, Pages 251-261 (January 2016) Gap junction modifier rotigaptide decreases the susceptibility to ventricular arrhythmia by enhancing conduction velocity and suppressing discordant alternans during therapeutic hypothermia in isolated rabbit hearts  Yu-Cheng Hsieh, MD, PhD, Jiunn-Cherng Lin, MD, Chen-Ying Hung, MD, Cheng-Hung Li, MD, Shien-Fong Lin, PhD, FHRS, Hung-I Yeh, MD, PhD, Jin-Long Huang, MD, PhD, Chu-Pin Lo, PhD, Ketil Haugan, MD, PhD, Bjarne D. Larsen, PhD, Tsu-Juey Wu, MD, PhD  Heart Rhythm  Volume 13, Issue 1, Pages 251-261 (January 2016) DOI: 10.1016/j.hrthm.2015.07.023 Copyright © 2016 Heart Rhythm Society Terms and Conditions

Figure 1 A, B: Effects of rotigaptide (Roti) on action potential duration (APD) during 33°C (A) and 30°C (B). C, D: Effects of rotigaptide on ventricular conduction velocity (CV) during 33°C (C) and 30°C (D). PCL = pacing cycle length. Heart Rhythm 2016 13, 251-261DOI: (10.1016/j.hrthm.2015.07.023) Copyright © 2016 Heart Rhythm Society Terms and Conditions

Figure 2 Effect of rotigaptide (Roti) on transverse (CVT) and longitudinal (CVL) ventricular conduction velocities (CVs) during 30°C. A: The pacing site is at the left ventricular free wall. B, top panels: Consecutive phase maps of ventricular conduction at baseline, during 30°C, and after rotigaptide infusion. B, bottom panels: Local optical recordings at sites a–d. Conduction time between 2 epicardial points was evaluated by 50% crossover of the action potential amplitude (AP50) in activation maps (red points). CVT and CVL were obtained by dividing the distances with the conduction time between 2 points. C, top panels: Isochronal maps at 3 stages. C, bottom panels: CVT and CVL with statistical comparison. LAD = left anterior descending coronary artery; PCL = pacing cycle length. Heart Rhythm 2016 13, 251-261DOI: (10.1016/j.hrthm.2015.07.023) Copyright © 2016 Heart Rhythm Society Terms and Conditions

Figure 3 A, B: Effects of rotigaptide (Roti) on the maximal slope of action potential duration (APD) restitutions during 33°C (A) and 30°C (B). C, D: Representative APD restitution curves obtained from anterior surface of left ventricle during 33°C (C) and 30°C (D). BL = baseline; DI = diastolic interval. Heart Rhythm 2016 13, 251-261DOI: (10.1016/j.hrthm.2015.07.023) Copyright © 2016 Heart Rhythm Society Terms and Conditions

Figure 4 Effects of rotigaptide (Roti) on action potential duration (APD) alternans during 33°C (A, B) and 30°C (C, D). A: APD difference maps (from heart 6) showed that alternans properties were not changed by rotigaptide during 33°C. Dashed lines indicate nodal lines. B: Effects of rotigaptide on APD alternans (left) and spatially discordant alternans (SDA) thresholds (right) during 33°C. C: APD difference maps (from heart 4) showed that at pacing cycle length (PCL) 260 ms, rotigaptide converted SDA to no alternans during 30°C. At PCL 240 ms, rotigaptide converted SDA to spatially concordant alternans (SCA). D: Effects of rotigaptide on APD alternans (left) and SDA thresholds (right) during 30°C. Heart Rhythm 2016 13, 251-261DOI: (10.1016/j.hrthm.2015.07.023) Copyright © 2016 Heart Rhythm Society Terms and Conditions

Figure 5 Effects of rotigaptide (Roti) on the incidence of premature ventricular complexes (PVCs) during 33°C and 30°C. A: ECG (from heart 3) before and after rotigaptide infusion during 33°C. Red arrows indicate PVCs. B: PVC incidence before and after rotigaptide infusion during 33°C. C: ECG (from heart 1) before and after rotigaptide infusion during 30°C. Note that the PVC initiated spontaneous ventricular fibrillation (SVF). D: PVC incidence before and after rotigaptide during 30°C. Heart Rhythm 2016 13, 251-261DOI: (10.1016/j.hrthm.2015.07.023) Copyright © 2016 Heart Rhythm Society Terms and Conditions

Figure 6 Effects of rotigaptide (Roti) on the initiation of pacing-induced ventricular fibrillation (PIVF) at pacing cycle length (PCL) 190 ms during 30°C (heart 7). A: Without rotigaptide, consecutive phase maps show that the propagating wave is homogeneous at the first beat (panels a–b). The second beat showed areas of delayed activation at the right ventricular free wall (panels c–d), followed by initiation of a wavebreak (small triangle in panel f). The wavebreak subsequently degenerates into a spiral wave (panels g–h), leading to ventricular fibrillation (VF; panel i). B, top panel: Local optical recordings during PIVF. B, bottom panel: Pseudo-ECG during PIVF. Arrows indicate pacing stimulations. Note that VF became sustained after pacing was stopped. C: Action potential duration (APD) difference maps during burst pacing. Discordant alternans was observed immediately before VF. Dashed lines (a-i) in panels B and C are simultaneous with the phase maps of a-i in panel A. D: With rotigaptide, the propagating wave is homogeneous without wavebreak throughout the pacing train (panels a–i). E: Local optical recordings and pseudo-ECG during burst pacing. No VF was observed after pacing stimulations. F: APD difference maps show discordant alternans during pacing. Dashed lines (a-i) in panels e and f are simultaneous with the phase maps of panels a-i in panel D. RV = right ventricle; LV = left ventricle; A = anterior; P=posterior; SR = sinus rhythm Heart Rhythm 2016 13, 251-261DOI: (10.1016/j.hrthm.2015.07.023) Copyright © 2016 Heart Rhythm Society Terms and Conditions

Figure 7 A, B: Effects of rotigaptide (Roti) on total connexin43 (T-Cx43) and nonphosphorylated connexin43 (NP-Cx43) protein expression during 33°C and 30°C. Top panels: Representative western blots from 4 rotigaptide- and 4 vehicle-treated (Veh) hearts. Lower panels: Quantitative comparisons. C, left panels: Representative images of T-Cx43 distributions in rotigaptide- and vehicle-treated hearts during 33°C and 30°C. C, right panels: Quantitative comparisons of lateralization percentage of T-Cx43. D, left panels: Representative images of NP-Cx43 distributions in rotigaptide- and vehicle-treated hearts during 33°C and 30°C. D, right panels: Quantitative comparisons of lateralization percentage of NP-Cx43. Heart Rhythm 2016 13, 251-261DOI: (10.1016/j.hrthm.2015.07.023) Copyright © 2016 Heart Rhythm Society Terms and Conditions