L-type Ca2+ channel responses to bay k 8644 in stem cell-derived cardiomyocytes are unusually dependent on holding potential and charge carrier

Abstract

Human stem cell-derived cardiomyocytes provide a cellular model for the study of electrophysiology in the human heart and are finding a niche in the field of safety pharmacology for predicting proarrhythmia. The cardiac L-type Ca2+ channel is an important target for some of these safety studies. However, the pharmacology of this channel in these cells is altered compared to native cardiac tissue, specifically in its sensitivity to the Ca2+ channel activator S-(-)-Bay K 8644. Using patch clamp electrophysiology, we examined the effects of S-(-)-Bay K 8644 in three separate stem cell-derived cardiomyocyte cell lines under various conditions in an effort to detect more typical responses to the drug. S-(-)-Bay K 8644 failed to produce characteristically large increases in current when cells were held at -40 mV and Ca2+ was used as the charge carrier, although high-affinity binding and the effects of the antagonist isomer, R-(+)-Bay K 8644, were intact. Dephosphorylation of the channel with acetylcholine failed to restore the sensitivity of the channel to the drug. Only when the holding potential was shifted to a more hyperpolarized (-60 mV) level, and external Ca2+ was replaced by Ba2+, could large increases in current amplitude be observed. Even under these conditions, increases in current amplitude varied dramatically between different cell lines and channel kinetics following drug addition were generally atypical. The results indicate that the pharmacology of S-(-)-Bay K 8644 in stem cell-derived cardiomyocytes varies by cell type, is unusually dependent on holding potential and charge carrier, and is different from that observed in primary human heart cells.

Fig. 1.

Effects of S-(−)-Bay K 8644 on guinea pig cardiomyocytes and Cor.4U^® stem cell-derived cardiomyocytes. (A) Ca²⁺ channel current traces from guinea pig cardiomyocytes under control conditions and following the addition of 10 and 100 nM S-(−)-Bay K 8644. Cells were held at −40 mV and depolarized for 200 ms to 0 mV every 5 s using 1.8 mM external Ca²⁺ as the charge carrier. (B) Ca²⁺ channel current traces in a Cor.4U cardiomyocyte under identical conditions described in (A). The effects of 100 and 1000 nM S-(−)-Bay K 8644 are shown. (C) Concentration–response relationships for S-(−)-Bay K 8644 on peak current in both myocyte preparations are shown. Error bars indicate the standard error of the mean (SEM), n=5–6. (D) The time constant for current inactivation (single exponential fit) is plotted against S-(−)-Bay K 8644 concentration for both myocyte preparations. Error bars indicate SEM and asterisks denote statistical significance (P<0.05, paired t-test, n=5–6).

Fig, 2.

Effects of S-(−)-Bay K 8644 on Ca²⁺ channel currents in Cor.4U cardiomyocytes using short pulses. Cells were held at −40 mV and depolarized to 0 mV for 30 ms every 5 s using 1.8 mM Ca²⁺ as the charge carrier. (A) Ca²⁺ channel current traces under control conditions and following the addition of 10 and 100 nM S-(−)-Bay K 8644 are indicated by the letters (A–C), respectively. Concentration-dependent effects of S-(−)-Bay K 8644 on time to peak current and on tail current decay time constants (tau) are shown in (B, C), respectively. Error bars indicate SEM and asterisks denote statistical significance (P<0.05, paired t-test, n=6).

Fig. 3.

Inhibition of Ca²⁺ channel currents in Cor.4U myocytes by R-(+)-Bay K 8644. (A) Ca²⁺ channel current traces under control conditions and following the addition of 10, 100, and 1000 nM R-(+)-Bay K 8644 are shown. Currents were elicited by 200 ms depolarizing pulse to 0 mV from a holding potential of −40 mV every 5 s using 1.8 mM Ca²⁺ as the charge carrier. (B) Concentration–response relationships for R-(+)-Bay K 8644 alone (open circles) or in the presence of 10 nM S-(−)-Bay K 8644 (filled triangles). R-(+)-Bay K inhibited Ca²⁺ channel currents with an IC₅₀ of 58 (42–79 nM, 95% confidence limits [CL]). In the presence of 10 nM S-(−)-Bay K 8644, this value measured 341 nM (309–377 nM, 95% CL).

Fig. 4.

Calcium channel current responses to S-(−)-Bay K 8644 in Cor.4U cardiomyocytes following pretreatment with acetylcholine. (A) Ca²⁺ channel current traces under control conditions, following treatment with 20 mM acetylcholine, and subsequent treatment with 100 nM S-(−)-Bay K 8644 are shown. Currents were elicited by 200 ms depolarizing pulses to 0 mV from a holding potential of −40 mV every 5 s using 1.8 mM Ca²⁺ as the charge carrier. (B) Concentration–response relationships for S-(−)-Bay K 8644 on peak Ca²⁺ channel currents following preincubation of the cells with acetylcholine. Open circles represent data collected with a normal pipette solution, while filled triangles show data collected in the absence of ATP, GTP, phosphocreatine, and creatine phosphokinase in the pipette. Error bars indicate SEM (n=4).

Fig. 5.

Calcium channel current responses to S-(−)-Bay K 8644 in Cor.4U cardiomyocytes under various assay conditions. (A) Effects of 300 nM S-(−)-Bay K 8644 on currents recorded from a holding potential of −60 mV using 1.8 mM Ca²⁺ as the charge carrier. (B) S-(−)-Bay K 8644 (300 nM) response on calcium channel currents held at −40 mV using 10 mM Ba²⁺ as the charge carrier. (C) Effects of 10 and 300 nM S-(−)-Bay K 8644 on calcium channel currents recorded from a holding potential of −60 mV using 10 mM Ba²⁺ as the charge carrier. Note the enhancement of current activation and inactivation following addition of the drug. (D) Concentration–response curves for S-(−)-Bay K 8644 on peak Ca²⁺ channel currents under the various conditions. Error bars indicate SEM, n=6–9.

Fig. 6.

Calcium channel current responses to S-(−)-Bay K 8644 in Cytiva^® Plus cardiomyocytes. (A) Effects of 300 nM S-(−)-Bay K 8644 on currents recorded from a holding potential of −60 mV using 1.8 mM Ca²⁺ as the charge carrier. (B) S-(−)-Bay K 8644 (300 nM) response on Ca²⁺ channel currents held at −40 mV using 10 mM Ba²⁺ as the charge carrier. (C) Effects of 10 and 300 nM S-(−)-Bay K 8644 on Ca²⁺ channel currents recorded from a −60 mV holding potential using 10 mM Ba²⁺ as the charge carrier. (D) Concentration–response curves for S-(−)-Bay K 8644 on peak calcium channel currents under the various conditions. Error bars indicate SEM, n=8–12.

Fig. 7.

Calcium channel current responses to S-(−)-Bay K 8644 in iCell^® cardiomyocytes. (A) Effects of 300 nM S-(−)-Bay K 8644 on currents recorded from a holding potential of −60 mV using 1.8 mM Ca²⁺ as the charge carrier. (B) S-(−)-Bay K 8644 (300 nM) response on Ca²⁺ channel currents held at −40 mV using 10 mM Ba²⁺ as the charge carrier. (C) Effects of 10 and 300 nM S-(−)-Bay K 8644 on Ca²⁺ channel currents recorded from a −60 mV holding potential using 10 mM Ba²⁺ as the charge carrier. (D) Concentration–response curves for S-(−)-Bay K 8644 on peak calcium channel currents under the various conditions. Error bars indicate SEM, n=6.