Agilent Assessing the Impact of Drug Application Note

Application Note

Cell Analysis

Assessing the Impact of Drug
Treatment on Cardiomyocyte Function

Through combined analysis of contractility, metabolic

flux, and cellular oxygenation

iPS Cardiomyocyte Contractility:

Cells cultured on RTCA E-Plate Cardio 96
Measured on xCELLigence RTCA Cardio
Allows interrogation of Contractility

Authors

Ryan McGarrigle, Conn Carey,
and James Hynes
Agilent Technologies, Inc.

Cell Metabolism:

Cells cultured on E-Plate Cardio 96
Measured on TRF Fluorescence Plate Reader
Agilent assays monitor mitochondrial function
(MitoXpress Xtra), glycolytic flux (pH-Xtra) and
cellular oxygenation (MitoXpress Intra).

Workflow Integration:

Allows measurement on E-Plates such that
metabolism and contractility can be measured

sequentially on the same test plate.

Abstract

In this application note, we demonstrate the feasibility of combining
microelectrode-based iPS cardiomyocyte contractility measurements with a
microplate-based bioenergetics assessment to better characterize cellular
responses to drug treatment. Contractility was assessed on 96-well E-Plate

Cardio96 using the Agilent xCELLigence RTCA Cardio system while cell metabolism
was measured on the same E-plate using a multiplexed fluorometric measurement

of O2 consumption with Agilent MitoXpress Xtra, glycolytic flux with Agilent pH Xtra,

and cellular oxygenation using Agilent MitoXpress Intra.

Introduction

Cardiotoxicity and related cardiac impairment remain one

of the main reasons for both drug withdrawal1 and FDA

black box warning2 and are a significant cause of compound

attrition in preclinical development. In vitro assays are capable
of better characterizing cardiac response to drug treatments
and are therefore of significant importance to better predict
such adverse effects in vivo.

Cardiac tissue requires an uninterrupted supply of respiratory
substrates to meet the very high ATP demand imposed by
continuous beating. Over 95% of this ATP is generated by

oxidative phosphorylation (OXPHOS) with the necessary
mitochondrial network taking up approximately one-third

of cardiomyocyte cell volume. Energy starvation and
mitochondrial dysfunction are therefore significant factors

in the progression of cardiotoxicity and so detection of such
metabolic dysfunction is an important aspect of cardiotoxicity

screening. This detection is best achieved by monitoring the

two main ATP generating processes, OXPHOS andglycolysis.

In vivo, the most important respiratory substrates for
ATP production are pyruvate and fatty acyl CoA, however,
cardiomyocyte metabolism is particularly adaptable
and substrates such as amino acids, lactate, and ketone

bodies can also be used. Examples of this adaptability
include hypoxia inducible factor (HIF) mediated metabolic
responses to hypoxia and ischemia and the shift from fatty
acid oxidation (FAO) to glucose metabolism that occurs

in hypertrophic cardiac tissue. These adaptions highlight
the importance of information on substrate preference

and oxygenation when designing and interpreting in vitro
cardiomyocyteanalyses.

As cardiac contraction is the main ATP consumer, the

coupling of contractility to ATP production, and by extension,

mitochondrial activity, is critically important to normal
cardiomyocyte function, particularly as the mitochondrial
reticulum also regulates intracellular calcium homeostasis
and a multitude of critical signally pathways. The ability to
relate cardiomyocyte beating to alter metabolic activity would
therefore be of significant utility.

Figure 1. A simplified schematic of the inter-relationship between cardiomyocyte metabolism and beating activity. OXPHOS produces most of the ATP needed,

with pyruvate and Acyl CoA being the main respirator y substrates. By measuring beating, OXPHOS (via O2 consumption), glycolytic flux (viaextracellular
acidification), and cellular oxygenation a more complete picture of cardiomyocyte function can be established.

2