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Link to original content: http://pubmed.ncbi.nlm.nih.gov/27235787/
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. 2016 Sep-Oct:81:183-95.
doi: 10.1016/j.vascn.2016.05.016. Epub 2016 May 25.

Human ex-vivo action potential model for pro-arrhythmia risk assessment

Guy Page  1 , Phachareeya Ratchada  1 , Yannick Miron  1 , Guido Steiner  2 , Andre Ghetti  1 , Paul E Miller  1 , Jack A Reynolds  1 , Ken Wang  2 , Andrea Greiter-Wilke  2 , Liudmila Polonchuk  2 , Martin Traebert  3 , Gary A Gintant  4 , Najah Abi-Gerges  5
Affiliations

Human ex-vivo action potential model for pro-arrhythmia risk assessment

Guy Page et al. J Pharmacol Toxicol Methods. 2016 Sep-Oct.

Abstract

While current S7B/E14 guidelines have succeeded in protecting patients from QT-prolonging drugs, the absence of a predictive paradigm identifying pro-arrhythmic risks has limited the development of valuable drug programs. We investigated if a human ex-vivo action potential (AP)-based model could provide a more predictive approach for assessing pro-arrhythmic risk in man. Human ventricular trabeculae from ethically consented organ donors were used to evaluate the effects of dofetilide, d,l-sotalol, quinidine, paracetamol and verapamil on AP duration (APD) and recognized pro-arrhythmia predictors (short-term variability of APD at 90% repolarization (STV(APD90)), triangulation (ADP90-APD30) and incidence of early afterdepolarizations at 1 and 2Hz to quantitatively identify the pro-arrhythmic risk. Each drug was blinded and tested separately with 3 concentrations in triplicate trabeculae from 5 hearts, with one vehicle time control per heart. Electrophysiological stability of the model was not affected by sequential applications of vehicle (0.1% dimethyl sulfoxide). Paracetamol and verapamil did not significantly alter anyone of the AP parameters and were classified as devoid of pro-arrhythmic risk. Dofetilide, d,l-sotalol and quinidine exhibited an increase in the manifestation of pro-arrhythmia markers. The model provided quantitative and actionable activity flags and the relatively low total variability in tissue response allowed for the identification of pro-arrhythmic signals. Power analysis indicated that a total of 6 trabeculae derived from 2 hearts are sufficient to identify drug-induced pro-arrhythmia. Thus, the human ex-vivo AP-based model provides an integrative translational assay assisting in shaping clinical development plans that could be used in conjunction with the new CiPA-proposed approach.

Keywords: Action potential; Drug discovery and development; Human heart; Pro-arrhythmia assessment; Ventricular trabeculae.

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Figures

Fig. 1
Fig. 1
Experimental protocol. The protocol consisted of measuring APs from individual human ventricular trabeculae over a period of 2 hrs using sharp-electrode recordings of the membrane potential. Baseline AP stability was assessed by continuous recording for 31 min in control vehicle (Tyrode's, 0.1% DMSO) at pacing rates of 1 Hz (25 min), 2 Hz (3 min) and 1 Hz (3 min). Subsequently, 3 cumulative concentrations of the test article were applied while repeating the same 31-minute pacing protocol for each subsequent drug concentration. For each heart, 3 tissue samples were tested with vehicle control followed by test article and one tissue was used as a vehicle time control. Each one of the 5 drugs was assessed in 5 hearts.
Fig. 2
Fig. 2
Distribution histograms of APD90, triangulation and STV in human ventricular trabeculae at a pacing rate of 1 Hz.
Fig. 3
Fig. 3
Stability of AP recordings over time in human ventricular trabeculae. (A) Typical APs recorded from a human ventricular trabecula at a pacing rate of 1 Hz in the presence of vehicle control and after exposure to 0.1 μM dofetilide, the positive control. V1, V2 and V3 correspond to the 1st, 2nd and 3rd application of vehicle (n=21). (B) Mean % changes in APD30, APD50 and APD90 induced by 3 sequential additions of vehicle and after exposure to dofetilide at 1 and 2 Hz. (C) Mean changes in APD90, triangulation, STV and EAD incidence when trabeculae were incubated with vehicle and dofetilide at 1 and 2 Hz. Note that the effects of vehicle and dofetilide on APD90 / triangulation / EAD activity and STV are plotted on a separate y-axis. (D) Effects of 3 sequential additions of vehicle on STV and triangulation as a function of change in APD90 in human ventricular trabeculae at pacing rates of 1 and 2 Hz. Note that the effects of vehicle on STV and triangulation are plotted on a separate y-axis. 1(in red)/1 (in blue), 2 (in red/2 (in blue) and 3 (in red)/3 (in blue) correspond to V1, V2 and V3 of vehicle, respectively. ٭,*,*P<0.05 versus values from vehicle.
Fig. 4
Fig. 4
Effects of dofetilide on AP in human ventricular trabeculae. (A) Typical APs recorded from a human ventricular trabecula at a pacing rate of 1 Hz in the presence of vehicle control and after exposure to dofetilide at 0.001, 0.01 and 0.1 μM. (B) Mean % changes in APD30, APD50 and APD90 induced by addition of dofetilide at 1 and 2 Hz (n=15). (C) Mean changes in APD90, triangulation, STV and EAD incidence when trabeculae were incubated with dofetilide at 1 and 2 Hz. Note that the effects of dofetilide on APD90 / triangulation / EAD activity and STV are plotted on a separate y-axis. (D) Shows a representative EAD recorded from the same trabecula as in (A) in the presence of 0.1 μM dofetilide and stimulated at 1 Hz. (E) Effects of dofetilide addition on STV and triangulation as a function of change in APD90 in human ventricular trabeculae at pacing rates of 1 and 2 Hz. Note that the effects of dofetilide on STV and triangulation are plotted on a separate y-axis. 1(in red)/1 (in blue), 2 (in red/2 (in blue) and 3 (in red)/3 (in blue) correspond to 0.001, 0.01 and 0.1 μM of dofetilide, respectively. #Decrease in excitabity was observed after exposure to 0.01 and 0.1 μM dofetilide at 1 and 2 Hz. ETPC, Effective therapeutic plasma concentration. ٭,*,*P<0.05 versus values from vehicle.
Fig. 5
Fig. 5
Effects of d,l-sotalol on AP in human ventricular trabeculae. (A) Typical APs recorded from a human ventricular trabecula at a pacing rate of 1 Hz in the presence of vehicle control and after exposure to d,l-sotalol at 1, 10 and 100 μM. (B) Mean % changes in APD30, APD50 and APD90 induced by addition of d,l-sotalol at 1 and 2 Hz (n=15). (C) Mean changes in APD90, triangulation, STV and EAD incidence when trabeculae were incubated with d,l-sotalol at 1 and 2 Hz. Note that the effects of d,l-sotalol on APD90 / triangulation / EAD activity and STV are plotted on a separate y-axis. (D) Effects of d,l-sotalol addition on STV and triangulation as a function of change in APD90 in human ventricular trabeculae at pacing rates of 1 and 2 Hz. Note that the effects of d,l-sotalol on STV and triangulation are plotted on a separate y-axis. 1(in red)/1 (in blue), 2 (in red/2 (in blue) and 3 (in red)/3 (in blue) correspond to 1, 10 and 100 μM of d,l-sotalol, respectively. # Decrease in excitability was observed after exposure to 10 and 100 μM d,l-sotalol at 2 Hz. ETPC, Effective therapeutic plasma concentration. ٭,*,*P<0.05 versus values from vehicle.
Fig. 6
Fig. 6
Effects of quinidine on AP in human ventricular trabeculae. (A) Typical APs recorded from a human ventricular trabecula at a pacing rate of 1 Hz in the presence of vehicle control and after exposure to quinidine at 0.1, 1 and 10 μM. (B) Mean % changes in APD30, APD50 and APD90 induced by addition of quinidine at 1 and 2 Hz (n=15). (C) Mean changes in APD90, triangulation, STV and EAD incidence when trabeculae were incubated with quinidine at 1 and 2 Hz. Note that the effects of quinidine on APD90 / triangulation / EAD activity and STV are plotted on a separate y-axis. (D) Effects of quinidine addition on STV and triangulation as a function of change in APD90 in human ventricular trabeculae at pacing rates of 1 and 2 Hz. Note that the effects of quinidine on STV and triangulation are plotted on a separate y-axis. 1(in red)/1 (in blue), 2 (in red/2 (in blue) and 3 (in red)/3 (in blue) correspond to 0.1, 1 and 10 μM of quinidine, respectively. ETPC, Effective therapeutic plasma concentration. ٭,*P<0.05 versus values from vehicle.
Fig. 7
Fig. 7
Effects of paracetamol on AP in human ventricular trabeculae. (A) Typical APs recorded from a human ventricular trabecula at a pacing rate of 1 Hz in the presence of vehicle control, and after exposure to paracetamol (2, 20 and 200 μM) and 0.1 μM dofetilide (the positive control). (B) Mean % changes in APD30, APD50 and APD90 induced by addition of paracetamol and after exposure to dofetilide at 1 and 2 Hz (n=15). (C) Mean changes in APD90, triangulation, STV and EAD incidence when trabeculae were incubated with paracetamol and dofetilide at 1 and 2 Hz. Note that the effects of paracetamol and dofetilide on APD90 / triangulation / EAD activity and STV are plotted on a separate y-axis. (D) Effects of paracetamol addition on STV and triangulation as a function of change in APD90 in human ventricular trabeculae at pacing rates of 1 and 2 Hz. Note that the effects of paracetamol on STV and triangulation are plotted on a separate y-axis. 1(in red)/1 (in blue), 2 (in red/2 (in blue) and 3 (in red)/3 (in blue) correspond to 2, 20 and 200 μM of paracetamol, respectively. ETPC, Effective therapeutic plasma concentration. ٭,*,*P<0.05 versus values from vehicle.
Fig. 8
Fig. 8
Effects of verapamil on AP in human ventricular trabeculae. (A) Typical APs recorded from a human ventricular trabecula at a pacing rate of 1 Hz in the presence of vehicle control, and after exposure to verapamil (0.01, 0.1 and 1 μM) and 0.1 μM dofetilide (the positive control). (B) Mean % changes in APD30, APD50 and APD90 induced by addition of verapamil at 1 and 2 Hz (n=15). (C) Mean changes in APD90, triangulation, STV and EAD incidence when trabeculae were incubated with verapamil at 1 and 2 Hz. Note that the effects of verapamil on APD90 / triangulation / EAD activity and STV are plotted on a separate y-axis. (D) Effects of verapamil addition on STV and triangulation as a function of change in APD90 in human ventricular trabeculae at pacing rates of 1 and 2 Hz. Note that the effects of verapamil on STV and triangulation are plotted on a separate y-axis. 1(in red)/1 (in blue), 2 (in red/2 (in blue) and 3 (in red)/3 (in blue) correspond to 0.01, 0.1 and 1 μM of verapamil, respectively. ETPC, Effective therapeutic plasma concentration. ٭,*,*P<0.05 versus values from vehicle.
Fig. 9
Fig. 9
Variability in measurements performed from different donor hearts and from different trabeculae from the same heart. (A), (B) and (C) show the “Total” and “intra-heart” variabilities for APD90, triangulation and STV at a pacing rate of 1 Hz, respectively (n=96). V1, V2 and V3 correspond to the 1st, 2nd and 3rd application of vehicle. Conc., concentration; SD, Standard deviation; 10x ETPC, 10-fold Effective Therapeutic Plasma Concentration.
Fig. 10
Fig. 10
Estimation of sample size with 2 hearts. (A) and (B) show the estimation of sample size for APD90 and triangulation at a pacing rate of 1 Hz for the concentration of around 10-fold the ETPC of dofetilide (0.01 μM), d,l-sotalol (100 μM) or quinidine (10 μM). Power calculation was performed on a % change scale. Each tissue sample served as its own control. Treatment effect unequal to zero was detected with Linear Mixed Effect Model. Target power 80% (dashed black line) corresponds to an 80% chance of concluding there was a real effect.
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