Ca²⁺ Channel Activators Reveal Differential L-Type Ca²⁺ Channel Pharmacology between Native and Stem Cell-Derived Cardiomyocytes

Abstract

Human stem cell-derived cardiomyocytes provide new models for studying the ion channel pharmacology of human cardiac cells for both drug discovery and safety pharmacology purposes. However, detailed pharmacological characterization of ion channels in stem cell-derived cardiomyocytes is lacking. Therefore, we used patch-clamp electrophysiology to perform a pharmacological survey of the L-type Ca 2ϩ channel in induced pluripotent and embryonic stem cell-derived cardiomyocytes and compared the results with native guinea pig ventricular cells. Six structurally distinct antagonists [nifedipine, verapamil, diltiazem, lidoflazine, bepridil, and 2-[(cis-2-phenylcyclopentyl)imino]-azacyclotridecane hydrochloride (MDL 12330)] and two structurally distinct activators [methyl 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate (Bay K8644) and 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL 64176)] were used. The IC 50 values for the six antagonists showed little variability between the three cell types. However, whereas Bay K8644 produced robust increases in Ca 2ϩ channel current in guinea pig myocytes, it failed to enhance current in the two stem cell lines. Furthermore, Ca 2ϩ channel current kinetics after addition of Bay K8644 differed in the stem cellderived cardiomyocytes compared with native cells. FPL 64176 produced consistently large increases in Ca 2ϩ channel current in guinea pig myocytes but had a variable effect on current amplitude in the stem cell-derived myocytes. The effects of FPL 64176 on current kinetics were similar in all three cell types. We conclude that, in the stem cell-derived myocytes tested, L-type Ca 2ϩ channel antagonist pharmacology is preserved, but the pharmacology of activators is altered. The results highlight the need for extensive pharmacological characterization of ion channels in stem cellderived cardiomyocytes because these complex proteins contain multiple sites of drug action.

L-Type Ca2+ Channel Responses to Bay K 8644 in Stem Cell-Derived Cardiomyocytes Are Unusually Dependent on Holding Potential and Charge Carrier

“…10 Furthermore, the stimulatory effects of Bay K 8644 are preserved in a variety of embryonic heart preparations, 17,18 including human fetal heart, 19 indicating that the unusual effects of the drug in stem cell-derived cardiomyocytes is unlikely due directly to the embryonic nature of the cells. To further explore this phenomenon, we utilized three separate stem cell-derived cardiomyocyte cell lines combined with a variety of assay conditions in an attempt to reveal a more typical response to S-( -)-Bay K 8644 as well as to compare and contrast results obtained from these various cell lines.…”

“…11,12,15,16 Conversely, in stem cell-derived cardiomyocytes, the predominate effects of S-( -)-Bay K 8644 include a slowing of both activation and inactivation accompanied by little or no effects (or even inhibition) of current amplitude. 10 The only effect of S-( -)-Bay K 8644 that is preserved is a slowing of tail current decay. It is possible that the failure of these cells to respond normally to S-( -)-Bay K 8644 resides in their origin (embryonic vs. fibroblast-derived) or is due to their well-described embryonic phenotype.…”

“…We have previously undertaken a detailed pharmacological study of the L-type Ca 2 + channel in stem cellderived cardiomyocytes. 10 Although the Ca 2 + channel antagonist pharmacology appeared to be intact, the response of the channel to activators, especially Bay K 8644, was uncharacteristic and unusually weak. Specifically, Bay K 8644 not only failed to produce large increase in current amplitude but also slowed both activation and inactivation kinetics of the current, effects that are opposite to what is normally observed for the drug.…”

Pyrimidine-2,4,6-triones are a new class of voltage-gated L-type Ca2+ channel activators

“…Unexpectedly, the modulation of I Ca by Cp8 closely resembled the actions of the LTCC activator FPL64176. This drug also slows activation and inactivation kinetics, increases peak current and slows current deactivation 23,24,32,33 . It is therefore possible that despite its unrelated structure, channel modulation of I Ca by Cp8 occurs through similar mechanisms than by FPL64176.…”

A New Perspective in the Field of Cardiac Safety Testing through the Comprehensive In Vitro Proarrhythmia Assay Paradigm

“…The potency of some hERG blockers shows marked sensitivity to stimulus protocol and/or measurement temperature. [30][31][32] The extent to which measurement conditions/protocol influence half-maximal inhibitory effect concentration (IC 50 ) may vary significantly between drugs: one comparative analysis of cisapride and dofetilide using manual patch reported variability of I hERG IC 50 for dofetilide between 4 and 46 nM and for cisapride between 7 and 240 nM in published literature derived from mammalian cell lines. 32 This further correlated with greater variability for cisapride than for dofetilide IC 50 (7-72 nM versus 4-15 nM) in experiments at a single temperature in a single study, between step, step-ramp, and AP voltage commands.…”