Jiesheng Kang, Xiao-Liang Chen, Junzhi Ji, Qiubo Lei, and ...

JPET #192609

1

Ca++ channel activators reveal differential L-type Ca++ channel pharmacology between

native and stem cell-derived cardiomyocytes.

Jiesheng Kang, Xiao-Liang Chen, Junzhi Ji, Qiubo Lei, and David Rampe

Disposition, Safety and Animal Research, sanofi, Inc., Bridgewater, NJ, USA

JPET Fast Forward. Published on February 21, 2012 as DOI:10.1124/jpet.112.192609

Copyright 2012 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on February 21, 2012 as DOI: 10.1124/jpet.112.192609

at ASPE

T Journals on M

arch 20, 2022jpet.aspetjournals.org

Dow

nloaded from

http://jpet.aspetjournals.org/

JPET #192609

2

Running Title: Ca++ channel pharmacology in stem cell and native cardiomyocytes

Corresponding Author:

David Rampe, Ph.D.

Sanofi, Inc

Mailstop JR2-203A

Route 202-206

Bridgewater, NJ 08807 USA

Tel: (908) 231-3078

Fax: (908) 231-2520

E0mail: [email protected]

Text Pages: 25

Tables: 1

Figures: 6

References: 33

Abstract: 250 words

Introduction: 376 words

Discussion: 1404 words

Abbreviations: hiPSC-CM, human induced pluripotent stem cell-derived cardiomyocytes; hESC-CM, human embryonic stem cell-derived cardiomyocytes; CL, confidence limits.

Section Assignment: Cardiovascular


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

3

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++ channel in induced pluripotent and embryonic stem

cell-derived cardiomyocytes and compared the results with native guinea pig ventricular cells.

Six structurally distinct antagonist (nifedipine, verapamil, diltiazem, lidoflazine, bepridil, and

MDL 12330) and two structurally distinct activators (Bay K 8644 and FPL 64176) were used.

The IC50 values for the six antagonists showed little variability between the three cell types.

However, while Bay K 8644 produced robust increases in Ca++ channel current in guinea pig

myocytes, it failed to enhance current in the two stem cell lines. Furthermore, Ca++ channel

current kinetics following addition of Bay K 8644 differed in the stem cell-derived

cardiomyocytes compared to native cells. FPL 64176 produced consistently large increases in

Ca++ 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++ 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 cell-

derived cardiomyocytes since these complex proteins contain multiple sites of drug action.


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

4

Introduction

Voltage-dependent L-type Ca++ channels provide the main pathway for Ca++ influx into

the heart and their functioning is crucial for controlling electrical activity and excitation-

contraction coupling. L-type Ca++ channels are complex heteromeric proteins comprised

minimally of α1, α2/δ and β subunits and are modulated by numerous intracellular processes (for

reviews see Bodi et al., 2005 and Benitah et al., 2010). The pharmacology of the L-type Ca++

channel is also complex but has been very well characterized. Numerous structurally distinct

antagonists, working at allosterically coupled sites on the channel, have been discovered and

several of these (e.g. dihydropyridines, benzothiazepines, and phenylalkylamines) are widely

used in the treatment of cardiovascular diseases (Fleckenstein, 1983; Triggle, 1999). Although

not as numerous, activators of L-type Ca++ channels have also been discovered and have

profound effects on both channel activity and cardiac functioning (Rampe and Kane, 1994). L-

type Ca++ channels are also actively studied as anti-targets during drug safety testing since off-

target interactions with these channels can lead to unwanted or dangerous cardiovascular side

effects including altered cardiac contractility and conduction disturbances.

In recent years cardiomyocytes derived from human stem cells have been developed and

provide a new model for the study of the physiology and pharmacology of the human heart. The

cells that have been developed include induced pluripotent stem cell-derived cardiomyocytes and

embryonic stem cell-derived cardiomyocytes (Thomson et al., 1998; He et al., 2003; Zhang et al.,

2009). In addition to their promise in the field of drug discovery and regenerative medicine,

these cells are now also becoming more popular as surrogates for human cardiac tissue for use in

safety and toxicity testing (Anson et al., 2011). Cardiac safety testing is often focused on the

interactions of drugs with various voltage-dependent ion channels since any such interaction has


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

5

the potential to produce arrhythmia. Therefore, the utility of stem cell-derived cardiac myocytes,

either for drug discovery or drug safety testing, will ultimately depend upon whether the detailed

pharmacological profile of their ion channels faithfully recapitulate those found in native

myocytes. To begin an exploration of this area we have surveyed the pharmacology of the L-type

Ca++ channel in both induced pluripotent and embryonic stem cell-derived cardiomyocyte cell

lines and compared the results obtained with native myocytes derived from guinea pig heart.


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

6

Material and Methods

Cell Preparation. Single ventricular myocytes were isolated from guinea pigs as

previously described (Kang et al., 2004). Male Hartley guinea pigs were anesthetized with 5%

isoflurane (Baxter Healthcare Corp., Deerfield IL) in a mixture of nitrous oxide and oxygen

(1:1). A thoracotomy was performed and the heart removed and immediately transferred to

oxygenated (100%) cold saline. The heart was perfused retrogradely at 10 ml/min through the

aorta with oxygenated Ca++ -free saline at 37ºC in three stages: first with standard Ca++-free

saline for 5 min, second with the same solution containing 280U/ml type II collegenase

(Worthington Biochemicals, Lakewood NJ) plus 0.75 U/ml type XIV protease (Sigma Aldrich,

St. Louis, MO) for 8 min, and finally with saline containing 0.2 mM CaCl2 for an additional 7

min. The left ventricle was cut into small pieces and gently shaken at room temperature for about

5 min to disperse single myocytes. The isolated myocytes were then maintained at 10ºC for

electrophysiological recordings.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) were

purchased from Cellular Dynamics International (iCell Cardiomyocytes; Cellular Dynamics

International, Madison, WI) and cultured for single-cell electrophysiology recordings as

instructed by the manufacturer. Briefly, frozen vials of hiPSC-CM were thawed in a 37ºC water

bath. The thawed cells were mixed with 10 ml of ice-cold plating medium (iCell Cardiomyocyte

Plating Medium, Cellular Dynamics International). The cells were diluted to approximately

20,000-40,000 in 2ml cold plating medium and this cell suspension was transferred into 12-well

culture plates containing 0.1% gelatin-coated glass coverslips. Cells were maintained in a tissue


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

7

culture incubator at 37ºC in an atmosphere of 93% air and 7% CO2. After 2 days of culture, the

Plating Medium was replaced with a cell culture medium (iCell Cardiomyocyte Maintenance

Medium, Cellular Dynamics International). This medium was changed every 48 h. Cells were

maintained on the coverslips for 4-14 days before electrophysiological recordings.

Human embryonic stem cell-derived cardiomyocytes (hESC-CM) were purchased from

Geron Corporation (Menlo Park, CA) and were cultured for single cell electrophysiology

recordings as instructed by the manufacturer. Briefly, frozen vials of hESC-CM were thawed in a

37ºC water bath. The thawed cells were mixed with 10ml pre-warmed RPMI1640/B27 and

centrifuged at 400g for 4 min. The cells were then re-suspended in RPMI1640/B27 at a

concentration of approximately 20,000-40,000 cells/ml placed into 6-well plates containing

Matrigel- (BD Biosciences, San Jose, CA) coated glass coverslips. Cells were maintained in a

tissue culture incubator at 37ºC in an atmosphere of 95% air and 5% CO2. After 2 days and every

other day thereafter, the medium was changed with RPMI1640/B27. Cells were maintained on

the coverslips for 4-14 days before electrophysiological recordings.

Electrophysiological Recordings. All Ca++ channel currents were recorded at room temperature

using the whole-cell configuration of the patch-clamp techniques (Hamill et al., 1981).

Electrodes (1-3 MΏ resistance) were made from TW-150F glass capillary tubes (WPI, Sarasota,

FL). Electrodes were filled with a solution containing 130 mM cesium methanesulfonate, 20 mM

tetraethylammonium chloride, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, 4 mM Tris-ATP,

0.3 mM Tris-GTP, 14 mM phosphocreatine, 50 U/ml creatine phosphokinase, pH 7.2 with

CsOH. The external solution for Ca++ channel recordings contained 137 mM NaCl, 5.4 mM

CsCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4 with NaOH. Ca++

channel currents were recorded using an Axopatch 200B amplifier (Danaher, Inc. Sunnyvale,


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

8

CA). Currents were analyzed using the pCLAMP suite of software (Danaher, Inc). IC50 values

were obtained by nonlinear least-squares fit of the data (GraphPad software, Inc., San Diego,

CA).

Chemicals. Lidoflazine and FPL 64176 were obtained from Sigma Aldrich. Bepridil, diltiazem,

and S-(-)-Bay K 8644 were obtained from Tocris (Ellisville, MD). Nifedipine was obtained from

Acros (Geel, Belgium). Verapamil was obtained from Enzo Life Sciences (Farmingdale, NY).

MDL 12330 was synthesized at sanofi pharmaceuticals (Bridgewater, NJ). All other chemical

were obtained from Sigma Aldrich.


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

9

Results

Figure 1 illustrates the chemical structures of the Ca++ channel antagonists and activators

that were used in this study. The effects of the dihydropyridine Ca++ channel antagonist

nifedipine on Ca++ channel currents recorded from native guinea pig ventricular myocytes,

hiPSC-CM, and hESC-CM are shown in Fig. 2. All cells were held at -40 mV and depolarized

for 200 ms to a test potential of 0 mV to elicit L-type Ca++ channel currents. Consistent with its

high affinity block of this channel, nifedipine inhibited Ca++ channel currents in guinea pig

myocytes with an IC50 value of 9 nM (8-10 nM, 95% confidence limits (CL); Fig. 2A and 2D).

Likewise, nifedipine produced a similar high affinity block of the L-type Ca++ channels recorded

in both the hiPSC-CM and hESC-CM cell lines. In the hiPSC-CM cell line nifedipine inhibited

Ca++ channel currents with an IC50 value of 3 nM (2-4 nM, 95% CL; Fig. 2B and 2D) while in

the hESC-CM cell line this value measured 6 nM (5-7 nM, 95% CL; Fig 2C and 2D).

A wide variety of structurally distinct molecules are known to act as antagonists of the L-

type Ca++ channel. Six of these molecules, were tested for their ability to block L-type Ca++

channel currents in guinea pig myocytes as well as in hiPSC-CM and hESC-CM cell lines. For

these studies, cells were held at -40 mV and depolarized to 0 mV for 200 ms and peak inward

currents in the absence and presence of ascending concentrations of drugs were used to generate

dose-response relationships and corresponding IC50 values. As shown in Table 1, the IC50 values

for any particular antagonist differed little between the three different cell types tested with

variations of approximately three-fold or less.


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

10

The dose-response relationships for the L-type Ca++ channel activator Bay K 8644 in

guinea pig myocytes, hiPSC-CM, and hESC-CM are shown in Fig. 3. As expected Bay K 8644

produced a dose-dependent increase in peak Ca++ channel current in guinea pig myocytes. The

increase in current was evident throughout the dose-response relationship reaching a maximum

of 267 ± 54% over the control value (set at 100%) at 100 nM, the highest concentration tested

(Fig. 3A and 3D, n=8). In addition, Bay K 8644 also accelerated Ca++ current inactivation during

the depolarizing step pulses. Compared to the results obtained in guinea pig myocytes, the effects

of Bay K 8644 were dramatically different in both hiPSC-CM and hESC-CM. In 23 hiPSC-CM

cells tested we could only find three cells that gave responses similar to those observed in guinea

pig myocytes. In 22 hESC-CM cells examined, none produced a response similar to that seen in

the guinea pig myocytes. Instead, Bay K 8644 produced either no, or very little, stimulation of

calcium channel current amplitude in these cell lines, or a moderate (10-40 % at 100 nM)

inhibition of the current. Further additional exposure to 1 µM Bay K 8644 in some of these cells

also failed to produce an enhancement of current. In addition, Bay K 8644 slowed Ca++ channel

current inactivation especially during the initial fast component of current decay. Typical current

traces in the presence and absence of Bay K 8644 for hiPSC-CM and hESC-CM are illustrated in

Figs. 3B and 3C, respectively, and the dose-response relationship is shown in Fig. 3D.

Bay K 8644 also had effects on L-type Ca++ channel activation kinetics and this was

easiest to see using short depolarizing pulses. Figure 4 shows the effects of 100 nM Bay K 8644

on guinea pig myocytes, hiPSC-CM, and hESC-CM held at -40 mV and depolarized to 0 mV for

30 ms. Because the large capacity transients made measuring the time constant of channel

activation in the guinea pig myocytes difficult, we instead measured time to peak current. In the

absence of Bay K 8644 this value measure 11.4 ± 2.4 ms while after the addition of Bay K 8644


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

11

this value was slightly, although not significantly shortened and measured 10.5 ± 0.8 (Fig. 4A;

n=8). Time to peak current in the hiPSC-CM (n=11) and hESC-CM (n=9) measured 8.7 ± 0.7

and 9.7 ± 1.2, respectively. After addition of Bay K 8644 these values were significantly

prolonged measuring 14.2 ± 0.9 and 14.9 ± 1.0 ms, respectively (Fig. 4B and 4C; p < 0.05 paired

t-test). The one effect of Bay K 8644 that was consistent between the guinea pig myocytes and

all of the stem cell-derived myocytes tested was its ability to prolong Ca++ channel tail current

upon repolarization of the cells (Fig.4 arrows).

Figure 5 illustrates the effects of Bay K 8644 on the L-type Ca++ channel current-voltage

(I-V) relationship measured in guinea pig myocytes, hiPSC-CM, and hESC-CM. To generate the

I-V relationships, all cells were held at -40 mV and depolarized for 30 ms to potentials ranging

from -40 mV to +40 mV in 5 mV increments and peak currents were recorded. All currents were

normalized to that obtained during the 10 mV depolarizing step. Bay K 8644 produced large

increases in calcium current in the guinea pig myocytes over a wide range of potentials. The

peak of the I-V relationship was shifted from 15 mV in control to 5 mV after the addition of Bay

K 8644 (Fig. 5A). By comparison, Bay K 8644 had little effect on current amplitude at the

various test potentials and produced a 5 mV shift (from 10 mV to 5 mV) in the peak of the I-V

relationships in both the hiPSC-CM (Fig. 5B) and the hESC-CM (Fig. 5C).

Figure 6 examines the effects of the benzoylpyrrole Ca++ channel activator FPL 64176 on

L-type Ca++ channel currents recorded from guinea pig myocytes, hiPSC-CM and hESC-CM.

FPL 64176 (30-1000nM) produced a dose-dependent increase in Ca++ current amplitude in all

guinea pig myocytes tested with a maximum increase of 336 ± 97% over the control value at the

top concentration of 1000 nM (Fig. 6a and 6D). At least a two-fold increase in current amplitude

was seen in every cell and this was accompanied by a dramatic slowing of channel activation,


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

12

inactivation, and tail current decay (Fig. 6A). The effects of FPL 64176 were less predictable in

the hiPSC-CM and hESC-CM cell lines. In hiPSC-CM FPL 64176 produced a two-fold or larger

increase in calcium current amplitude in 6/14 cells tested, an increase in current amplitude that

was less than two-fold (25-82% increase) in 5/14 cells (shown in Fig 6B) and a slight decrease

(15-43%) in current amplitude in 3/14 cells. When pooled together, FPL 64176 produced a

maximal increase in Ca++ channel current amplitude of 105 ± 29% over the control value (Fig.

6D). In hESC-CM the effects of FPL 64176 on Ca++ current amplitude were somewhat less

producing a two-fold or larger increase in current in 2/16 of the cells tested, a 15-55% maximal

increase in current in 4/16 cells (Fig. 6C) and no increase or a slight decrease (up to 40%) in

current amplitude in 10/16 cells tested. When pooled together, FPL 64176 produced a 7 ± 11%

increase in current amplitude over the control value in hESC-CM at the 1 µM concentration (Fig.

6D). Regardless of the effects of FPL 64176 on current amplitude, the drug always produced the

characteristic slowing of current activation, inactivation and tail current decay in all hiPSC-CM

and hESC-CM tested.


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

13

Discussion

Human stem cell-derived cardiomyocytes offer a new approach for studying the cellular

electrophysiological characteristics of the heart. However, detailed pharmacological

characterization of the main cardiac ion channels in these cells requires further study. In the

present report we explored the pharmacology of the L-type Ca++ channel in two distinct stem

cell-derived cardiomyocyte cell lines and compared it to that of native myocytes isolated from

guinea pig heart. The stem cell-derived myocyte cell lines used in this study were chosen

because they were commercially available, they represent two distinct lineages (induced

pluripotent and embryonic), and their basic phenotypic and electrophysiological characteristics

have already been described (Peng et al., 2010; Ma et al., 2011). We chose to study the L-type

Ca++ channel because it possess a rich and well characterized pharmacology comprised of many

structurally distinct antagonists and activators, and because it is a target for therapeutic

intervention as well as an anti-target for use in drug safety assessment.

The L-type Ca++ channel is known to be inhibited by a variety of compounds working at

distinct allosterically coupled sites and we chose six of these to probe the pharmacology of this

channel in the hiPSC-CM and hESC-CM cell lines and compare the results to those obtained

from native guinea pig myocytes. These drugs included representatives from the clinically

popular dihydropyridine (nifedipine), phenylalkylamine (verapamil), and benzothiazepine

(diltiazem) classes (Triggle and Janis, 1987), along with lidoflazine (Barry et al., 1985), bepridil

(Yatani et al., 1986), and MDL 12330 (Rampe et al., 1987). The IC50 values obtained for all of

these drugs were similar to those reported in the literature and displayed a variance of


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

14

approximately three-fold or less between the three cell types studied. In terms of L-type Ca++

channel antagonist pharmacology, the affinity of a wide variety of structurally diverse molecules

is preserved in both hiPSC-CM and hESC-CM cell lines when compared to native mammalian

cardiomyocytes.

It was not until we examined the effects of L-type Ca++ channel activators that we found

pharmacological divergence between stem cell-derived myocytes and native ones. This was

particularly true for the dihydropyridine Bay K 8644. Bay K 8644 is well known as an activator

of L-type Ca++ channels in a wide variety of cells and its stimulatory activity is present

regardless of whether the cells come from primary cultures or from cell lines grown under a

various culture conditions (for reviews see Schramm and Towart, 1985 and Bechem and

Schramm, 1987). In the current study, Bay K 8644 failed to produce its characteristic increases

in current amplitude in either the hiPSC-CM or hESC-CM and furthermore slowed Ca++ channel

activation and inactivation in the stem cell-derived myocytes. These activities are inconsistent

with the well described properties of Bay K 8644 found in guinea pig myocytes in this and other

studies (Hamilton et al., 1987; Zhong et al., 1997), and most importantly in human cardiac

myocytes from atrial (Le Grand et al., 1991; Skasa et al., 2001) and ventricular preparations

(Chen et al., 1999; Chen et al., 2002; Chen et al., 2008) where Bay K 8644 has effects similar to

those found in the guinea pig and other mammalian cells. The lack of effect of Bay K 8644 in the

stem cell lines cannot therefore be considered a species dependent effect, but rather some

specific alteration in the stem cells themselves. The only typical pharmacological characteristic

of Bay K 8644 that appeared to be preserved in all of the stem cell-derived myocytes tested was

a prolongation of tail current decay. Increases in Ca++ channel current amplitude to the

benzoylpyrrole activator FPL 64176 were somewhat variable in the stem cell-derived myocytes


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

15

with some cells showing large increases in current amplitude while others did not.

Characteristically large (defined here as greater than 2-fold) increases in Ca++ channel current

were only observed in some of the stem cells tested. Despite this inconsistency in current

amplitude, the other classic pharmacological effects of FPL 64176 (Rampe et al., 1993; Fan et

al., 2000) were evident in all cells tested and included a dramatic slowing of Ca++ channel

activation, inactivation, and tail current kinetics. While Bay K 8644 and FPL 64176 did not

reproduce some of their well-established effects in the stem cell-derived cardiomyocytes, both

compounds slowed Ca++ channel inactivation and prolonged tail current decay. These actions

should effectively increase the amount of Ca++ entering the cells during a depolarizing step.

Therefore both compounds may still act as Ca++ channel activators when stem cell derived

cardiomyocytes are used in action potential recordings, microelectrode array recordings,

contractility assays, or other assays that indirectly measure Ca++ channel activity. It is only when

the channel is studied directly and in isolation that the differences in pharmacology may become

apparent.

It is unclear why the response to L-type Ca++ channel activators differed between the

stem cell-derived cardiomyocytes used in this study and what is known to occur in native

myocytes. The binding sites for Bay K 8644 and FPL 64176 are thought to be localized in the

IIIS5-S6 linker of the α1 subunit of the channel (Yamaguchi et al., 2000; Yamaguchi et al.,

2003) and single amino acid substitutions in this area dramatically reduce the ability of these

molecules to enhance Ca++ channel current amplitude. It is possible that some genomic

alterations exist in the stem cells cardiomyocytes that limits their ability to respond to Ca++

channel agonists. However, since both cell lines are of human origin, we would expect the

sequence of the L-type Ca++ channel to accurately reflect the normal genotype. Phenotypic


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

16

differences between stem cell-derived cardiomyocytes and native myocytes have been described

and may underlie the pharmacological profile we have observed. Many stem cell-derived

myocytes, including the ones used in this report, are considered to have an immature or

embryonic-like phenotype (Peng et al., 2010; Ma et al., 2011). Electrophysiologically this

presents itself as a more depolarized resting membrane potential and a slower upstroke velocity

during action potential recordings. It is possible that this immature phenotype in some way alters

sensitivity to Ca++ channel activators. If this is the case, such a phenomenon may be limited to

certain stem cell-derived cardiomyocytes since other embryonic-like native cardiomyocytes

including those from embryonic chick heart (Anderson et al., 1990), neonatal rat heart (Rampe et

al., 1993) and fetal human heart (Chen et al., 1999) are all known to retain sensitivity to Bay K

8644 and/or FPL 64176. Calcium handling has also been reported to differ between stem cell-

derived cardiomyocytes and native ones. In particular the force of contraction in response to β-

adrenergic agonists is reduced in stem cell-derived myocytes (Xi et al., 2010; Pillekamp et al.,

2012). Contractile responses to β-adrenergic stimulation require functional L-type Ca++ channels

and sarcoplasmic reticulum and it is believed that the latter is altered in stem cell-derived

cardiomyocytes (Xi et al., 2010; Pillekamp et al., 2012). Based upon our pharmacological results

it is possible that functional alterations in the L-type Ca++ channel are also present, at least to

some degree, in stem cell-derived cardiomyocytes. Finally, the effects of Bay K 8644 on Ca++

channel activity are known to be reduced in myocytes taken from failing human hearts,

presumably as a result of an increase in basal channel activity due to a heightened

phosphorylation status of the channel in these cells (Chen et al., 2008). Enhance phosphorylation

status could conceivably distort the response of the L-type Ca++ channel in stem cell-derived

cardiomyocytes to further stimulation by agonists like Bay K 8644. Further studies examining


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

17

post-translational processing alterations (e.g. phosphorylation status) in the L-type Ca++ channel

of stem cell-derived cardiomyocytes will be useful and could possibly explain the

pharmacological results obtained in this report.

In summary, we report here the first detailed pharmacological survey of the L-type Ca++

channel in stem cell-derived cardiomyocytes and its comparison with a well-studied native

cardiomyocyte system. We find that the affinity for a wide range of structurally distinct

antagonists is virtually identical in the stem cell-derived myocytes compared to the native cells.

Conversely, Ca++ channel activators, especially Bay K 8644, failed to faithfully reproduce their

established pharmacological effects in the stem cell-derived myocytes. The data point out the

importance of detailed pharmacologic characterization of all ion channels in these and other stem

cell-derived cell lines since these complex proteins contain multiple sites of drug action. With

respect to L-type Ca++ channels, Bay K 8644 and FPL 64176 may represent a useful starting

point when characterizing this channel’s pharmacology in other stem cell-derived cell lines.


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

18

Authorship Contributions:

Participated in research design: Rampe

Conducted experiments: Chen, Ji, Lei

Performed data analysis: Kang

Wrote or contributed to the writing of the manuscript: Rampe


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

19

References

Anderson AJ, Duncan GP, Spedding M, and Patmore L (1990) Pertussis toxin-sensitive G-

protein activation does not influence the response to Bay K 8644 in embryonic chick myocytes. J

Cardiovasc Pharmacol 16: 681-683.

Anson BD, Kolaja KL, and Kamp TJ (2011) Opportunities for the use of human iPS cells in

predictive toxicology. Clin Pharmacol Ther 89: 754-758.

Barry WH, Horowitz JD, Smith TW (1985) Comparison of negative inotropic potency,

reversibility, and effects on calcium influx of six calcium channel antagonists in cultured

myocardial cells. Br J Pharmacol 85: 51-59.

Bechem M, and Schramm M (1987) Calcium-agonists. J Mol Cell Cardiol 19 (suppl 2): 63-75.

Benitah J-P, Alvarez JL, and Gomez AM (2010) L-type Ca2+ current in ventricular

cardiomyocytes. J Mol Cell Cardiol 48: 26-36.

Bodi I, Mikala G, Koch SE, Akhter SA, and Schwartz A (2005) The L-type calcium channel in

the heart: the beat goes on. J Clin Inves 115: 3306-3317.

Chen L, El-Sherif N, and Boutjdir M (1999) Unitary current analysis of L-type Ca2+ channels in

human fetal ventricular myocytes. J Cardiovasc Electrphysiol 10: 692-700.

Chen X, Piacentino V, Furukawa S, Goldman B, Margulies KB, and Houser SR (2002) L-type

Ca2+ channel density and regulation are altered in failing human ventricular myocytes and

recover after support with mechanical assist devices. Circ Res 91: 517-524.


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

20

Chen X, Zhang X, Harris DM, Piacentino V, Berretta RM, Margulies KB, and Houser SR (2008)

Reduced effects of Bay K 8644 on L-type Ca2+ current in failing human cardiac myocytes are

related to abnormal adrenergic regulation. Am J Physiol 294: H2257-H2267.

Fan JS, Yuan Y, and Palade P (2000) Kinetic effects of FPL 64176 on L-type Ca2+ channels in

cardiac myocytes. Naunyn Schmiedebergs Arch Pharmacol 361: 465-476.

Fleckenstein A (1983) Calcium Antagonism in Heart and Smooth Muscle. Experimental Facts

and Therapeutic Prospects, Wiley, New York.

Hamill OP, Marty A, Neher E, Sakmann B, and Sigworth FJ (1981) Improved patch clamp

techniques for high resolution current recording from cells and cell free membrane patches.

Pfluegers Arch 391: 85-100.

Hamilton SL, Yatani A, Brush K, Schwartz A, and Brown AM (1987) A comparison between

the binding and electrophysiological effects of dihydropyridines on cardiac membranes. Mol

Pharmacol 31: 221-231.

He JQ, Ma Y, Lee Y, Thomson JA, and Kamp TJ (2003) Human embryonic stem cells develop

into multiple types of cardiac myocytes: action potential characterization. Circ Res 93: 32-39.

Kang J, Chen X-L, Wang H, Ji J, Reynolds W, Lim S, Hendrix J, and Rampe D (2004) Cardiac

ion channel effects of tolterodine. J Pharmacol Exp Ther 308: 935-940.

Le Grand B, Hatem S, Deroubaix E, Couetil JP, and Coraboeuf E (1991) Calcium current

depression in isolated human atrial myocytes after cessation of chronic treatment with calcium

antagonists. Circ Res 69: 292-300.


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

21

Ma J, Guo L, Fiene SJ, Anson BD, Thomson JA, Kamp TJ, Kolaja KL, Swanson BJ, and

January CT (2011) High purity human-induced pluripotent stem cell-derived cardiomyocytes:

electrophysiological properties of action potentials and ionic currents. Am J Physiol 301: H2006-

H2017.

Peng S, Lacerda AE, Kirsch GE, Brown AM, and Bruening-Wright A (2010) The action

potential and comparative pharmacology of stem cell-derived human cardiomyocytes. J

Pharmacol Toxicol Meth 61: 277-286.

Pillekamp F, Haustein M, Khalil M, Emmelheinz M, Nazzal R, Adelmann R, Nguemo F,

Rubenchyk O, Pfannkuche K, Matzkies M, Reppel M, Bloch W, Brockmeier K, and Hescheler J

(2012) Contractile properties of early human embryonic stem cell-derived cardiomyocytes: Beta-

adrenergic stimulation induces positive chronotropy and lusitropy but not inotropy. Stem Cells

Dev doi:10.1089/scd.2011.0312.

Rampe D, and Kane JM (1994) Activators of voltage-dependent L-type calcium channels. Drug

Dev Res 33: 344-363.

Rampe D, Triggle DJ, and Brown AM (1987) Electrophysiological and biochemical studies on

the putative Ca++ channel blocker MDL 12,330A in an endocrine cell. J Pharmacol Exp Ther

243: 402-407.

Rampe D, Anderson B, Rapien-Pryor V, Li T, and Dage RC (1993) Comparison of the in vitro

and in vivo cardiovascular effects of two structurally distinct Ca++ channel activators, Bay K

8644 and FPL 64176. J Pharmacol Exp Ther 265: 1125-1130.


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

22

Schramm M, and Towart R (1985) Modulation of calcium channel function by drugs. Life Sci

37: 1843-1860.

Skasa M, Jungling E, Picht E, Schondube F, and Luckhoff A (2001) L-type calcium currents in

atrial myocytes from patients with persistent and non-persistent atrial fibrillation. Basic Res

Cardiol 96: 151-159.

Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, and Jones

JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282: 1145-1147.

Triggle DJ, and Janis RA (1987) Calcium channel ligands. Annu Rev Pharmacol Toxicol 27:

347-369.

Triggle DJ (1999) The pharmacology of ion channels: with particular reference to voltage-gated

Ca2+ channels. Eur J Pharmacol 375: 311-325.

Yamaguchi S, Okamura Y, Nagao T, and Adachi-Akahane S (2000) Serine residue in the IIIS5-

S6 linker of the L-type Ca2+ channel α1C subunit is the critical determinant of the action of

dihydropyridine Ca2+ channel agonists. J Biol Chem 275: 41504-41511.

Yamaguchi S, Zhorov BS, Yoshioka K, Nagao T, Ichijo H, and Adachi-Akahane S (2003) Key

roles of Phe1112 and Ser1115 in the pore-forming IIIS5-S6 linker of the L-type Ca2+ channel α1C

subunit (Cav1.2) in binding of dihydropyridines and action of Ca2+ channel agonists. Mol

Pharmacol 64:235-248.

Xi J, Khalil M, Shishechian N, Hannes T, Pfannkuche K, Liang H, Fatima A, Haustein M, Suhr

F, Bloch W, Reppel M, Saric T, Wernig M, Janisch R, Brockmeier K, Hescheler J, and

Pillekamp F (2010) Comparison of contractile behavior of native murine ventricular tissue and


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

23

cardiomyocytes derived from embryonic or induced pluripotent stem cells. FASEB J 24: 2739-

2751.

Yatani A, Brown AM, and Schwartz A (1986) Bepridil block of cardiac calcium and sodium

channels. J Pharmacol Exp Ther 237: 9-17.

Zhang J, Wilson GF, Soerens AG, Koonce CH, Yu J, Palecek SP, Thomson JA, and Kamp TJ

(2009) Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ Res

104: e30-e41.

Zhong J, Hwang T-C, Adams HR, and Rubin LJ (1997) Reduced L-type calcium current in

ventricular myocytes from endotoxemic guinea pigs. Am J Physiol 273: H2312-H2324.


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

24

Figure Legends

Figure 1. Chemical structures of the L-type Ca++ channel ligands used in this study.

Figure 2. Effects of nifedipine on L-type Ca++ channel currents in guinea pig and stem cell-

derived cardiomyocytes. Cells were held at -40 mV and depolarized to 0 mV for 200 ms at 10 s

intervals. The inhibitory effects of 3, 10 and 100 nM nifedipine on L-type Ca++ channels

recorded from guinea pig myocytes, hiPSC-CM , and hESC-CM are shown in panels A, B, and

C, respectively. Dose-response curves are shown in panel D. Error bars denote S.E.M. (n=6 for

guinea pig myocytes, 11 for hiPSC-CM, and 9 for hESC-CM).

Figure 3. Effects of Bay K 8644 on L-type Ca++ channels recorded from guinea pig and stem

cell-derived cardiomyocytes. Cells were held at -40 mV and depolarized for 200 ms to 0 mV at

10 s intervals. The effects of 10 and 100 nM Bay K 8644 on L-type Ca++ channel currents

recorded from guinea pig myocytes, hiPSC-CM, and hESC-CM are shown in panels A, B, and C,

respectively. The dose-response relationship for Bay K 8644 in these cells is shown in panel D.

Bay K 8644 produced a dose-dependent increase in Ca++ channel current in the guinea pig

myocytes while no such stimulation was observed in the hiPSC-CM or hESC-CM cell lines.

Error bars indicated S.E.M. (n=8 for guinea pig myocytes, 23 for hiPSC-CM and 22 for hESC-

CM).

Figure 4. Bay K 8644 effects on L-type Ca++ channel currents in guinea pig myocytes, hiPSC-

CM and hESC-CM during a short depolarization. Cells were held at -40 mV and depolarized for

30 ms to 0 mV in the absence and presence of 100 nM Bay K 8644 at 10 s intervals. Ca++

channel recordings from guinea pig myocytes, hiPSC-CM and hESC-CM are illustrated in panels


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

25

A, B, and C, respectively. Note the slowing of the time to peak current in hiPSC-CM and hESC-

CM cells and the prolongation of tail current decay (arrows) in all cell types.

Figure 5. Effects of Bay K 8644 on L-type calcium channel current-voltage (I-V) relationships.

Cells were held at -40 mV and depolarized for 30 ms to potentials ranging from -40 to +40 mV

in 5 mV increments. Peak Ca++ channel currents were recorded in the absence and presence of

100 nM Bay K 8644. I-V relationships in guinea pig myocytes, hiPSC-CM and hESC-CM are

shown in panels A, B, and C, respectively. Currents were normalized to that measured during the

+10 mV step pulse for each cell type. Error bars indicate S.E.M. (n=9 for guinea pig myocytes,

11 for hiPSC-CM, and 9 for hESC-CM).

Figure 6. Dose-response relationship of FPL 64176 on L-type Ca++ channel currents recorded in

guinea pig and stem cell-derived cardiomyocytes. Cells were held at -40 mV and depolarized to

0 mV for 200 ms in the absence and presence of increasing concentrations of FPL 64176. The

effects of 100 and 1000 nM FPL64176 on Ca++ channel currents recorded from guinea pig

myocytes, hiPSC-CM and hESC-CM are shown in panels A, B, and C, respectively. The dose-

response relationships for these cells is shown in panel D. Error bars indicate S.E.M. (n=8 for

guinea pig myocytes, 14 for hiPSC-CM, and 16 for hESC-CM).


at ASPE

T Journals on M


Dow

nloaded from


JPET #192609

26

Table 1. Inhibitory effects of Ca++ channel antagonists on L-type Ca++ channel currents recorded

in native guinea pig myocyte, hiPSC-CM and hESC-CM. The IC50 value of each compound

along with the 95% confidence limits (95% CL) and number of replicates (n) is given.

IC50 (95% CL, n)

Native Myocyte hiPSC-CM hESC-CM

Lidoflazine 238 nM (140-406 nM, 7) 230 nM (179-294 nM, 9) 101 nM (93-110, 9)

MDL 12330 2133 nM (1824-2494 nM, 6) 1085 nM (818-1440 nM, 11) 910 nM (675-1225 nM, 8)

Verapamil 167 nM (135-205, 6) 70 nM (62-80 nM, 9) 93 nM (73-120 nM, 9)

Diltiazem 276 nM (225-338 nM, 7) 233 nM (212-257 nM, 10) 262 nM (214-320 nM, 9)

Nifedipine 9 nM (8-10 nM, 6) 3 nM (2-4 nM, 11) 6 nM (5-7 nM, 9)

Bepridil 115 nM (93-141 nM, 6) 54 nM (51-58 nM, 9) 71 nM (54-93 nM, 8)


at ASPE

T Journals on M


Dow

nloaded from


Lidoflazine MDL 12330

Verapamil Diltiazem

Nifedipine Bepridil

FPL 64176 S-(-)-Bay K 8644

Figure 1


at ASPE

T Journals on M


Dow

nloaded from


A. Native Myocyte B. hiPSC-CM

C. hESC-CM D. Dose-Response

-9 -8 -70

25

50

75

100

hESC-CM

Native Myocyte

hiPSC-CM

[Nifedipine], logM

Cal

cium

Cur

rent

(%C

ontr

ol)

0 100 200 300Time (ms)

Cal

cium

Cur

rent

(pA

)

-600

-400

-200

0100 nM

10 nM

3 nM

Control

0 100 200 300Time (ms)

Cal

cium

Cur

rent

(pA

)

-600

-400

-200

0100 nM

10 nM

3 nM

Control

0 100 200 300

Time (ms)

Cal

cium

Cur

rent

(pA

)

-200

-100

0100 nM

10 nM

3 nM

Control

0 100 200 300

Time (ms)

Cal

cium

Cur

rent

(pA

)

-200

-100

0100 nM

10 nM

3 nM

Control

0 100 200 300

Time (ms)

Cal

cium

Cur

rent

(pA

)

-300

-200

-100

0

10 nM

3 nM

Control

100 nM

0 100 200 300

Time (ms)

Cal

cium

Cur

rent

(pA

)

-300

-200

-100

0

10 nM

3 nM

Control

100 nM

Figure 2


at ASPE

T Journals on M


Dow

nloaded from


Figure 3



Control -8.5 -8.0 -7.5 -7.0 -6.50

100

200

300

400

hESC-CM

Native Myocyte

hiPSC-CM

[Bay K 8644], logM

Ca

lciu

m C

urre

nt(%

Co

ntro

l)

Control -8.5 -8.0 -7.5 -7.0 -6.50

100

200

300

400

hESC-CM

Native Myocyte

hiPSC-CM

[Bay K 8644], logM

Ca

lciu

m C

urre

nt(%

Co

ntro

l)

10 nM Bay K 8644

Control

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300

-1500

-1000

-500

0

100 nM Bay K 8644

10 nM Bay K 8644

Control

Time (ms)

Cal

cium

Cur

rent

(pA

)

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300

-1500

-1000

-500

0

100 nM Bay K 8644

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300

-200

-100

0

10 nM Bay K 8644

Control100 nM Bay K 8644

Time (ms)

Cal

cium

Cur

rent

(pA

)

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300

-200

-100

0

10 nM Bay K 8644

Control100 nM Bay K 8644

Control

10 & 100 nM Bay K 8644

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300-300

-200

-100

0

100

Control

10 & 100 nM Bay K 864410 & 100 nM Bay K 8644

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300-300

-200

-100

0

100

Time (ms)

Cal

cium

Cur

rent

(pA

)

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300-300

-200

-100

0

100


at ASPE

T Journals on M


Dow

nloaded from


A. Native Myocyte

B. hiPSC-CM

C. hESC-CM

Figure 4

0 20 40 60Time (ms)

Cal

cium

Cur

rent

(pA

)

-800

-400

0

100 nM Bay K 8644

Control

0 20 40 60Time (ms)

Cal

cium

Cur

rent

(pA

)

-800

-400

0

0 20 40 60Time (ms)

Cal

cium

Cur

rent

(pA

)

-800

-400

0

100 nM Bay K 8644

Control

Time (ms)

Cal

cium

Cur

ren

t (pA

)

100 nMBay K 8644

Control

0 20 40 60

-200

0

Time (ms)

Cal

cium

Cur

ren

t (pA

)

100 nMBay K 8644

Control

0 20 40 60

-200

0

0 20 40 60

-200

0

Time (ms)

Ca

lciu

m C

urre

nt (

pA)

100 nM Bay K 8644

Control

0 20 40 60-2000

-1000

0

Time (ms)

Ca

lciu

m C

urre

nt (

pA)

100 nM Bay K 8644

Control

0 20 40 60-2000

-1000

0

0 20 40 60-2000

-1000

0


at ASPE

T Journals on M


Dow

nloaded from


A. Native Myocyte

B. hiPSC-CM

C. hESC-CM

Figure 5

Test Pulse (mV)

Nor

ma

lize

d C

urr

ent

0-40 -30 -20 -10 10 20 30 40

1.0

2.0

3.0

4.0

100 nM Bay K 8644

Control

Test Pulse (mV)

Nor

ma

lize

d C

urr

ent

0-40 -30 -20 -10 10 20 30 40

1.0

2.0

3.0

4.0

100 nM Bay K 8644

Control

Test Pulse (mV)N

orm

aliz

ed C

urr

ent

0-40 -30 -20 -10 10 20 30 40

0.2

0.4

0.6

0.8

1.0100 nM Bay K 8644

Control

Test Pulse (mV)N

orm

aliz

ed C

urr

ent

0-40 -30 -20 -10 10 20 30 40

0.2

0.4

0.6

0.8

1.0100 nM Bay K 8644

Control

Test Pulse (mV)

Nor

ma

lize

d C

urr

ent

0-40 -30 -20 -10 10 20 30 40

0.2

0.4

0.6

0.8

1.0

1.2100 nM Bay K 8644

Control

Test Pulse (mV)

Nor

ma

lize

d C

urr

ent

0-40 -30 -20 -10 10 20 30 40

0.2

0.4

0.6

0.8

1.0

1.2100 nM Bay K 8644

Control


at ASPE

T Journals on M


Dow

nloaded from




Control -7.5 -7.0 -6.5 -6.0 -5.50

100

200

300

400

500

hESC-CM

Native Myocyte

hiPSC-CM

[FPL 64176], logM

Cal

cium

Cur

ren

t(%

Con

trol

)

Control -7.5 -7.0 -6.5 -6.0 -5.50

100

200

300

400

500

hESC-CM

Native Myocyte

hiPSC-CM

[FPL 64176], logM

Cal

cium

Cur

ren

t(%

Con

trol

)

Figure 6

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300

-2000

-1000

0 Control

100 nM FPL 64176

1000 nM FPL 64176

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300

-2000

-1000

0

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300

-2000

-1000

0 Control

100 nM FPL 64176

1000 nM FPL 64176

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300

-600

-300

0 Control

100 nM FPL 64176

1000 nM FPL 64176

Time (ms)

Cal

cium

Cur

rent

(pA

)

0 100 200 300

-600

-300

0

0 100 200 300

-600

-300

0 Control

100 nM FPL 64176

1000 nM FPL 64176

Time (ms)

Cal

cium

Cur

rent

(pA

)

1000 nM FPL 64176

0 100 200 300

-600

-300

0

100 nM FPL 64176

Control

Time (ms)

Cal

cium

Cur

rent

(pA

)

1000 nM FPL 64176

0 100 200 300

-600

-300

0

100 nM FPL 64176

Control1000 nM FPL 64176

0 100 200 300

-600

-300

0

100 nM FPL 64176

Control


at ASPE

T Journals on M


Dow

nloaded from


Jiesheng Kang, Xiao-Liang Chen, Junzhi Ji, Qiubo Lei, and ...

Documents

Transcript of Jiesheng Kang, Xiao-Liang Chen, Junzhi Ji, Qiubo Lei, and ...