Pharmacological characterization of adenosine effects on spontaneously beating human-IPSC-derived cardiomyocytes

J. Imredy, B. Balasubramanian, E. Lis, A. Lagrutta, and F. Sannajust. Circulation Research (2015)


Purified human-Induced Pluripotent Stem Cell-derived cardiomyocytes (hIPSC-CMs: iCells®, Cellular Dynamics) form electrically coupled, spontaneously beating cell monolayers (syncytia) upon plating and continuous culture. In this study, we investigate the pharmacology of the hIPSC-CM adenosine (ADO) response via the use of selective A1 and A2A-receptor antagonists and equilibrative nucleoside transporter type-1 (ENT1) inhibitors. After a 14-day culture in 96-well impedance electrode-plates (ACEA Biosciences), the spontaneous iCell beating rates are highly stable from well to well, with a mean beat period of 1.7 sec at 37°C, and a coefficient of variation (CV) \textless 1\% . Extracellularly applied ADO inhibits the spontaneous beating rate of iCells with an IC50 = 2.2 $\mu$M, and a saturation value of near 50\% inhibition. ADO also destabilizes the beat period (CV EC50 = 1.2 $\mu$M) with a maximum CV of 20\% at saturating ADO concentrations. The ADO IC50 is shifted to 248 $\mu$M upon pre-incubation of the iCells with 1 $\mu$M of an A1-selective antagonist DPCPX (A1 Ki=3.9 nM), but not the A2A-selective antagonist ZM241385 (A2A Ki=1nM). The duration of the depressive effect of ADO on beating rate is transient, even at super-saturating ADO concentrations, and depends on the well ADO concentration. The ENT1 inhibitor Draflazine (ENT1 pKi=9.5) pre-applied at 0.1 $\mu$M increases ADO sensitivity by 4-fold one hour after ADO application. Moreover, at any given ADO concentration, the duration of beating rate depression can be prolonged in a concentration-dependent manner by ENT1 inhibitors, with the prolongation-response curve nearly matching the ENT1 IC50s for tested ENT1 inhibitors (Draflazine, NBTI, Dilazep, and Dipyridamole). These findings suggest that the pool of ADO in the test well is cleared by uptake via endogenous ENT1 and support the role of equilibrative ADO uptake in limiting the duration of action of ADO on A1 receptors in cardiomyocytes. We are extending our investigation to the downstream target of A1 receptor activity, and the putative role of IKACH/ADO as the underlying mechanism of beat rate slowing and irregularity. In conclusion, the hIPSC-CM model provides a useful model for investigation of ADO pharmacology of the heart.

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