Abstract 16039: Action Potential Propagation in Monolayers of Cardiac Myocytes Derived from Embryonic Stem Cells
Background: Embryonic stem cell derived cardiomyocytes (ES-CM) have been extensively characterized at the single cell level. However, there has been no systematic study of action potential propagation through ES-CM cells that have formed an electrical syncytium. We generated monolayers of ES-CMs and tested the following hypotheses: 1) ES-CM monolayer electrophysiology will be similar to neonatal rat ventricular myocytes (NRVMs). 2.) ES-CM monolayers can generate sustained reentry.
Methods: ES-CM were purchased from Lonza and used to generate 13 monolayers (18x18 mm2). Optical mapping (Di-8ANEPPS, 40 μmol/L, 200 fps) and confocal Ca2+ imaging (10μM Fluo-4, 150 fps) were performed 3–5 days after plating. Monolayers were immuno-stained for connexin40 (Cx40), Cx43, and α-actinin. Students paired t-test was used to determine statistical significance. All data are presented as mean ± SD
Results: Immunostaining revealed Cx43, but not Cx40. ES-CM monolayers (12/13) had rhythmic activity maintained by a single pacemaker focus. The average rate of spontaneous discharge was 7.87±2 Hz. Overdrive pacing was achieved in 7/13 monolayers. Synchronous calcium release across the field of view suggested sodium dependent propagation. Impulses propagated from the pacemaker focus at uniform conduction velocities (CVs) throughout the monolayer. Interestingly, CV increased from 13.9±0.85 cm/s at 7 Hz to 24.5±2.27 cm/s at 12 Hz, p < 0.05. The monolayers had dynamic action potential duration (APD) restitution for pacing frequencies 7–12 Hz. APD 30, 50, and 80 at 7 vs. 12 Hz was 27.6±0.61 vs. 34.6±2.59, 40.5±2.50 vs. 52.3±4.34, and 58.1±2.74 vs. 84.3±6.64; p <0.05. Tetrodotoxin (TTX, 30 μmol/L) terminated propagation in 3/3 monolayers. Carbachol (10 μM) slowed pacemaker activity by 66% but induced rotors in 2/4 monolayers at frequencies of 11.4 and 8.6 Hz. Rotor frequency in 1/13 without a pacemaker was 16 Hz.
Conclusions: ES-CM monolayers show rhythmic pacemaker activity, uniform CV, and APD restitution, and Ca2+ dynamics similar to NRVM monolayers. Therefore, they are a powerful new model to investigate mechanisms of normal and abnormal cardiac impulse propagation.
- © 2010 by American Heart Association, Inc.