Preclinical In Vitro Cardiac Toxicity

Contractile Activity + Electrophysiological Activity + Viability -> Real Time Cell Analysis of Toxicity

Cardiomyocytes for the predictive assessments of potential cardiotoxic side effects

Significant numbers of drug development projects are terminated in the late preclinical and early clinical stages due to cardiac liability issues. The last two decades has witnessed increased withdrawals or issued safety warnings due to cardiotoxicity. Approximately 1/3 of all drugs withdrawn during 1990-2006 were associated with Torsades de Pointes (TdP). One of the main challenges in preclinical cardiac safety assessment has been the lack of a predictive and biologically relevant model system available in sufficiently high quantities to be used for screening of cardiotoxic and pro-arrhythmic drugs, especially during the hit to lead or lead optimization stage. Recent advances in differentiating embryonic and induced-pluripotent stems cells have created physiologically relevant and disease relevant model systems for preclinical safety assessment of compounds.

Assessment of Contractile Related Toxicities

The existing methods for assessment of contractile activities, such as Langendorff heart assays and other ex vivo whole organ assays are labor intensive, low throughput, and expensive. Alternatively, animal studies can also be conducted, but the predictivity of these assays and translational potential to humans is always an important consideration in addition to the ethical considerations of using animals. Functional monitoring of cardiomyocyte contractility using impedance readout (see figure below) provides incisive information about compound effects on the contractile machinery.

Figure 1: Impedance-based contraction signal of different types of hiPSC-CM.

 

Case study: Blebbistatin, a myosin heavy chain inhibitor, decouples excitation and contraction coupling.

  • Blebbistatin reduced beating amplitude in a dose-dependent manner, but did not alter the field potential signal even after exposure to 10 µM blebbistatin.

(A) 5 seconds of beating amplitude traces obtained from iCell cardiomyocytes before (black traces)  and after (red traces) blebbistatin treatment at 0.01, 1, and 10 µM. The compound addition was performed in one dose one well fashion. (B) 5 seconds of FP traces obtained before (black trace) and after 10 µM of blebbistatin treatment.

Assessment of Ion Channel Toxicities

Assays that more directly assess possible pro-arrhythmic effects of compounds utilize whole animal hearts or Purkinje fibers, and are designed to assess action potential duration. While these assays are considered to be more predictive of arrhythmia, they have higher negative predictivity rate and can be low throughput and technically demanding. Human iPSC-CMs provide a physiologically relevant model that can be used to assess potential toxicity of pharmaceutical compounds or integrated ion channel activity.

Figure 2: Field potential signal of hiPSC-CM.

Assessment of Acute Toxicities:

1. Na+ channel>Mexiletine, a Na+ channel blocker, induced reduction of field potential (FP) spike, suggesting the blockade of sodium channel.

5 seconds of impedance (IMP) (A) and field potential (FP) (B) signals of iCell CMs before (black traces) and after (red traces) exposure to 10 µM Mexiletine. The raw IMP and FP traces were then further calculated to single averaged waveforms (C and D).

2. Ca2+ channelsIsradipine, a Ca2+channel blocker, induced field potential duration (FPD) shortening and decrease in IMP amplitude/beating amplitude (BAmp).

5 seconds of impedance (IMP) (A) and field potential (FP) (B) signals of iCell cardiomyocytes before (black traces) and after (red traces) exposure to 0.01 µM isradipine. The raw IMP and FP traces were then further calculated to single averaged waveforms (C and D).

3. hERG channelE4031, a hERG channel blocker, induced EAD-like FP signal and arrhythmic beating on beating amplitude traces.

5 seconds of impedance (beating amplitude) (A) and field potential (FP) (B) signals of iCell cardiomyocytes before (black traces) and after (red traces) exposure to 0.03 µM E4031. The overlay of IMP and FP traces (C) 30 min post-drug.

4. Multi-ion channelFlecainide: a multichannel blocker, which inhibits Na+ and hERG channels. Flecainide induced FP spike reduction and EAD-like (notch and ectopic beat) on FP traces and arrhythmic beating on IMP traces.

5 seconds of impedance (IMP) (A) and field potential (FP) (B) signals of iCell cardiomyocytes before (black traces) and after (red traces) exposure to 3µM flecainide. (C)The overlay of IMP and FP traces (C) 30 min post-drug. (D) Magnified in FP trace.

Long-term Ion Channel Toxicities:

Pentamidine, a hERG channel trafficking inhibitor, showed delayed FPD and contractility period duration.

(A) 15 seconds of IMP traces obtained from iCell cardiomyocytes before and after pentamidine treatment at the indicated time points. (B) The expanded FP traces recorded before and after 10 µM pentamidine treatment at the indicated time points. (C) Time course of FPD measurement over the course of 10 µM pentamidine treatment.

Assessment of Structural Related Toxicities

Chemotherapeutic drugs, such as anthracyclines and certain kinase inhibitors, are known to compromise the structural integrity of cardiomyocytes. Identifying and delineating these types of toxicities are an important component of overall cardiotoxicity assessment of pharmaceutical compounds. While there may be laborious orthogonal end-point assays that can provide information regarding the impact of pharmaceutical compounds on structural integrity of cardiac myocytes, some effects may be transient and missed by end-point analysis. The xCELLigence CardioECR system is the only assay or platform that provide both structural integrity and viability information, while simultaneously providing data about ion channel activity and contractility. The totality of the three types of toxicities makes the CardioECR a unique platform well positioned for early cardiotoxicity screening.


Assessment of Anthracycline Toxicity:

Doxorubicin, an anthracycline and chemotherapy drug, is known to cause cardiac toxicity

(A) Overall cell index change over the course of 72 hour treatment. (B) 15 seconds of impedance traces obtained from iCell cardiomyocytes before and after doxorubicin treatment at the indicated time points.

  • Doxorubicin induced overall cell index decrease in a dose and time-dependent manner, suggesting a loss of cell viability and/or structural damage to the cells.
  • Doxorubicin also affected the contractility of cardiomyocytes in a dose and time dependent manner, which may be related to the loss of viability.

Assessment of Kinase Inhibitor Toxicity:

Sorafenib is a well-studied cardiotoxic compound that’s known to cause myocardial infarction and ischaemia.

  • Sorafenib induced overall cell index decrease in a dose and time-dependent manner. It caused cell quiescence at 20 µM

(A) Overall cell index change over the course of 48-hour treatment. (B) 15 seconds of impedance traces obtained from iCell cardiomyocytes before and after sorafenib treatment at the indicated time points. 

Overcome hurdles in your research to find your next insight of your therapeutic compound interaction

Ncardia webinar

Webinar: Rethinking Translational Predictivity using HiPSC-Derived Models

A Retrospective Analysis for Cardiac Liabilities, a webinar hosted by Ncardia and ACEA Biosciences.

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Astrazeneca webinar

Webinar: Unraveling Kinase Inhibitor Cardiotoxicity with Cardiomyocyte Impedance Assays

Dr. Sarah Lamore (AstraZeneca) presents her research utilizing cellular impedance measurement of beating cardiomyocytes to deconvolute kinase inhibitor cardiotoxicity. 

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cardiac safety paper

Publication: A Tool for Cardiac Safety Assessment

Multi-parametric assessment of cardiomyocyte excitation-contraction coupling using impedance and field potential recording.

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Easy and Flexible Work Flow: Simply plate the cells, start acquiring data, and perform combination treatments & chronic dosing

Simplify your analysis – powerful software to help you elucidate information from data
Unique software monitors integrated ion channel activity, contractility, and longer term viability to elucidate adverse compound action.

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Are you collecting all the needed data to make a true safety assessment?

The xCELLigence CardioECR® system combines real-time impedance monitoring with both multielectrode array (MEA) technology and a pacing function for the evaluation of cardiomyocyte function along with integrated ion channel activity.

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The xCELLigence® RTCA Cardio system uses non-invasive, label-free impedance monitoring to quantitatively evaluate cardiomyocyte health/function in real-time.

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Are your hiPSC-CMs displaying neonatal gene expression profile? Pacing hiPSC-CM under increasing pacing frequencies leading to functionally mature hiPSC-CM which display more adult-like gene expression profile.

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