Assessment of Positive Inotrpic Compounds Using an Impedance-based System with human iPSC-

derived Cardiomyocytes under Controlled Pacing Conditions Xiaoyu Zhang, Yama A. Abassi

ACEA Biosciences Inc. 6779 Mesa Ridge Rd, San Diego, CA 92121

xCELLigence RTCA CardioECR System

iCell2 cardiomyocytes, provided by Cellular Dynamic International’s (CDI), are highly purified

cardiomyocytes derived from human iPS cells. They are a mixture of spontaneously electrically

active atrial, nodal, and ventricular-like myocytes.

Cardiomyocytes used for this study

*

Understanding drug-mediated modulation of cardiac contractility is an important

question from both a therapeutic angle as well as safety/toxicity angle. While one of

the primary applications for which human iPSC-derived cardiomyocytes (hiPSC-

CMs) are currently being evaluated is for assessment of pro-arrhythmic compounds,

we wanted to understand if these cells can also be used for assessment of inotrpic

compounds which modulate contractility. The impedance-based cardiac contractility

assay has been systematically studied and validated previously (Scott et al., 2014).

While this assay showed comparable sensitivity and specificity using hiPSC-CMs to

the well-validated optical-based contractility assay using adult dog cardiomyocytes

(Harmer et al., 2012) the assay was unable to delineate inotropic effects from

chronotropic effects. In order to address this limitation, we utilized xCELLigence

RTCA CardioECR system, which combines impedance measurement and electrical

pacing, to evaluate the effect of cardio-modulating compounds under pacing and

non-pacing conditions. Our data clearly demonstrate that hiPSC-CMs possess

negative beating rate and amplitude (the amplitude of impedance waveform)

relationship; 2) Modulators of cardiac contractility either increased or decreased

beating amplitude depending on their respective mechanism of actions.

Furthermore, based on our data the positive inotropic effects of cardio-modulating

compounds on beating amplitude could be better assessed under controlled pacing

conditions of hiPSC-CMs. In summary our data shows, that beating amplitude is a

good surrogate for contraction; 2) pacing hiPSC-CMs at a fixed rate would help to

reveal direct effects on contraction; 3) Impedance readout and pacing function

provided by CardioECR system are valuable for assessing inotropic agents.

Result 1. Assessment of Calcium channel inhibitors

Result 1. Relationship of beating amplitude and beating frequency of hiPSC-CMs

Result 3. Positive inotrope tests in isradipine pretreated iCell CM2

Contractility

Cellular toxicity

Ion channels modulation (Ca2+,

Na+, hERG )

Arrhythmic events

Results

Simultaneously record: Impedance (IMP) + Field Potential (FP)

Impedance

Electrodes

Extracellular Recording

(ECR) Electrodes

Introduction

• hiPSC-derived CMs show negative beating amplitude and frequency relationship.

• Beating amplitude is a good surrogate for contraction.

• Pacing hiPSC-CMs at a fixed rate can compensate chronotropic effect and may help

to reveal direct compound effect on contraction.

• Artificial reduction of BAmp by pretreating CMs with L-type Ca2+ channel inhibitor

(isradipine) in conjunction with pacing helps to show positive inotropic effect of

compounds.

• Impedance readout and pacing function provided by CardioECR system are

valuable for assessing inotropic agents.

Results

Beating Amplitude : cell index difference between

positive and negative peak.

Beating Rate: number of beats per min

Beating period: time duration between adjacent

impedance peaks

Result 2. β Adrenergic agonist treatment in paced and spontaneously beating

iCell CM2

Summary

1) BAmp was artificially reduced by partially blocking L-type Ca2+ channel

using isradipine for 30 min;

2) Followed by the 2nd addition of positive inotrope (A. ISO, B. dobutamine and

C. milrinone);

3) The cell responses were measured under pacing and non-pacing conditions

simultaneously on the same plate

Impedance

Contractility

Viability

Field Potential

Electrophysiology

Ion Channel

A

B

C

D

A

B

C

D

Built-in pacing function

y = -0.0015x + 0.4058R² = 0.9745

0

0.1

0.2

0.3

0.4

0 50 100 150 200

Be

atin

g A

mp

litu

de

Beating Rate (beats/min)

Beating Amplitude and Beating Rate Relationship

N=32

35 beats/min

45 beats/min

60 beats/min

75 beats/min

90 beats/min

105 beats/min

120 beats/min

150 beats/min

+

── Beating Period

+

─Bea

tin

g A

mp

litu

de +

1 2 3

Experimental Workflow

1. Directly seed cell on the E-Plate CardioECR48

2. Real time monitor cell performance (viability, contraction and electrical activity) in incubator

3. Conduct compound addition

N=32

-15%

-10%

-5%

0%

5%

10%

15%

20%

CTRL 0.3 µM 3 µM

% c

han

ge o

f bea

tin

g am

plit

ud

e to

th

e ti

me-

mat

ched

co

ntr

ol

Concentration

BAmp_Dobutamine (Spontaneous)

-10%

-5%

0%

5%

10%

15%

20%

CTRL 0.3 µM 3 µM% c

han

ge o

f bea

tin

g

amp

litu

de

to t

he

tim

e-m

atch

ed c

on

tro

l

Concentration

BAmp_Dobutamine (Paced)

Isopreterenol (ISO): non-selective β Adrenergic agonist

Dobutamine: β1 and α1 Adrenergic agonist

-20%

-10%

0%

10%

20%

30%

40%

CTRL 1 µM

% c

ha

nge

of

be

ati

ng

am

plit

ud

e t

o t

he

ti

me

-ma

tch

ed

co

ntr

ol

Concentration

BAmp_ISO in isradipine pretreated CMs

Paced

spontaneous Pa

ce

d

Pre-ISO

Post-ISO

Sp

on

tan

eo

us

Pre-ISO

Post-ISO

1 µM ISO

**

-20%

-10%

0%

10%

20%

30%

40%

CTRL 10 µM 100 µM

% c

han

ge o

f b

eati

ing

amp

ltid

ue

to t

he

tim

e-

mat

che

d c

on

tro

l

Concentration

BAmp_Milrinone in isradipine

pretreated CMsPaced

Spontaneous

-20%

-10%

0%

10%

20%

30%

40%

CTRL 1 µM

% c

han

ge o

f b

eat

ing

amp

litu

de

to

th

e t

ime

-m

atch

ed

co

ntr

ol

Concentration

BAmp_Dobutamine in isradipine

pretreated CMs

Paced

Spontaneous**

1 µM ISO

Abstract

-20%

-15%

-10%

-5%

0%

5%

10%

15%

20%

CTRL 0.3 µM 3 µM

% c

hang

e of

bea

ting

am

plit

ude

to th

e ti

me

-m

atch

ed c

ontr

ol

Concentration

BAmp_Dobutamine (Paced vs. Spontaneous)

Paced

Spontaneous

N=6

**

N=6 N=6

N=4

-20%

-15%

-10%

-5%

0%

5%

10%

15%

20%

CTRL 0.3 µM 1 µM

% c

hang

e of

bea

ting

am

plit

ude

to t

he t

ime

-m

atch

ed c

ontr

ol

Concentration

BAmp_ISO (Paced vs. Spontaneous)

Paced

Spontaneous

**

N=4

Pre-drugISO addition to the

isradipine pretreated cells

**: P < .001

iCell CM2 were electrically paced at successively increased pacing frequency. Beating

amplitude and cell index were measured during the pacing application.

Averaged beating waveform

50 nM Isradipine

1µM

CTRL

Paced

Sp

on

tan

eo

us

CTRL

1µM

A.

B. C.

The cell responses to β Adrenergic agonists were measured under pacing and non-pacing

conditions simultaneously on the same plate. Stimulus setting: rectangular pulse; intensity:

800 mV; length: 0.15 ms; frequency: 1.3 Hz.