Cardiotoxicity and Safety

Cardiotoxicity traces
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Cardiotoxicity refers to the dysfunction or damage to the heart caused by exposure to natural or synthetic substances. The inability to predict the cardiovascular liability of therapeutic compounds prior to clinical trials has resulted in numerous costly late-stage drug development failures and market withdrawals.

The Maestro Pro and Edge microelectrode array (MEA) systems are uniquely suited to evaluate in vitro cardiotoxicity. With sensitive measurements of the electrical, contractile, and conduction activity of cardiomyocytes in culture, the Maestro MEA platform is a vital tool to inform any cardiac safety testing paradigm.

 

Superior cardiac safety studies

 

 

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative aims to update the existing cardiac safety testing paradigms to better evaluate arrhythmogenic risk of new compounds.  In the CiPA pilot study, six core sites used the Maestro for their MEA studies with two different cell types and eight compounds with known mechanisms. The Maestro and accompanying software tools demonstrated high reliability across replicates, sites, and cell types, while accurately measuring changes in depolarization (spike amplitude), repolarization (field potential duration), and detecting arrhythmias

Moxifloxacin analysis in cardiomyocytes for arrythmias
Moxifloxacin analysis in cardiomyocytes for arrythmias
Nifedipine analysis for arrythmias in cardiomyocytes
Nifedipine analysis for arrythmias in cardiomyocytes
Mexiletine analysis for arrythmias in hiPSC-cardiomyocytes
Mexiletine analysis for arrythmias in hiPSC-cardiomyocytes

(A) CiPA pilot study results for three representative compounds across six Maestro sites using Axiogenesis Cor.4U (orange) or CDI iCell (blue) hiPSC-cardiomyocytes (hiPSC-CMs). Example raw field potential waveforms (inset) are presented before (black) and after (orange or blue) dosing with Moxifloxacin (100 µM), Nifedipine (0.3 µM), and Mexiletine (10 µM). The Maestro reliably detected field potential duration (FPD) prolongation for hERG K+ blockers, FPD reduction for Ca2+ blockers, and spike amplitude reduction for Na+ blockers across sites and cell types.

 

CiPA Pilot Study Results showed that the Maestro MEA was the most reliable and most accurate with the lowest variability

 

In the multi-site, blinded CiPA Pilot Study, the CiPA team demonstrated the utility of hiPSC-cardiomyocytes to detect cardioactive compounds. We are proud that the Maestro MEA system played a pivotal role in the study. Nice work Maestro MEA users.

 

Download CiPA Pilot Study Paper

 

 

 

The follow-up CiPA Myocyte Phase II validation study expanded its predecessor's scope to test 28 drugs from low, intermediate, and high torsades de pointes (TdP) risk categories at 11 sites around the globe. In this study the CiPA team demonstrated the utility of hiPSC-CMs to detect drug-induced arrhythmogenic effects. We are proud that the Maestro MEA system played a pivotal role in the study. More importantly, we are thankful for all the Maestro MEA users at big pharma for making us look great.

 

Download CiPA Phase II Study Paper

 

LEAP assay pharmacology with hiPSC-cardiomyoctyes

The Maestro Pro and Edge’s local extracellular action potential (LEAP) assay noninvasively measures cardiac action potential morphology and easily detects early afterdepolarizations (EADs). Now more subtle alterations to action potential morphology like triangulation can be quantified. Action potential morphology changes measured with LEAP are consistent with previous reports of labor-intensive traditional methods like manual patch clamp.

 
E-4031 first prolonged repolarization and then generated repolarization irregularities.
E-4031 first prolonged repolarization and then generated repolarization irregularities.
E-4031 first prolonged repolarization and then generated repolarization irregularities.
Nifedipine shortened the duration of the LEAP signal in a dose-dependent manner.
Nifedipine shortened the duration of the LEAP signal in a dose-dependent manner.
Nifedipine shortened the duration of the LEAP signal in a dose-dependent manner.
Terodiline induced triangulation at higher concentrations, consistent with patch clamp results.
Terodiline induced triangulation at higher concentrations, consistent with patch clamp results.
 

(B) E-4031 first prolonged repolarization and then generated repolarization irregularities. (C) Nifedipine shortened the duration of the LEAP signal in a dose-dependent manner. (D) Terodiline induced triangulation at higher concentrations, consistent with patch clamp results

 

Measure changes in propagation patterns and conduction velocity in response to pharmacological compounds

In an hiPSC-CM syncytium, beating is generally initiated in one portion of the culture (pacer region) and propagates like a wave through the tissue. Pharmacological agents and cardiac disorders can affect conduction by 1) modifying the excitability of the cells, or 2) altering the gap junction coupling between cells.

 
The baseline conduction velocity plot of hiPSC-CM
Conduction velocity comparison with different doses of lidocaine
Conduction velocity of hiPSC-CMs
The propagation consistency was more variable and greatly decreased after the anti-cancer drug dosing compared to baseline

(E) When lidocaine, a Na+ channel blocker, was added to a hiPSC-CM network, a decrease in conduction velocity was observed. (F) Addition of an anti-cancer drug treatment to hiPSC-CMs caused a disruption in propagation pattern, and thus a decrease in propagation consistency from beat to beat.

 

 
 
CiPA Cardiotox assay protocol steps

Getting started with Maestro Pro and Edge couldn't be easier. Culture your cells in an Axion multiwell MEA plate (Day 0).  Load the MEA plate into the Maestro MEA system at the desired recording times and begin recording. Analyze the cardiomyocyte activity in the MEA plate label-free and in real-time with AxIS Navigator Cardiac Module software.  Add test compounds as required (e.g. Day 7).

 

 

 

The advantage of measuring cardiotoxicity on the Maestro Pro and Edge systems:

  • 1 system, 4 assays – Record the four key measures of functional cardiac performance, label-free and in real time, in every well of the multiwell MEA plate: [1] action potential; [2] field potential; [3] propagation; and [4] contractility.

  • Measure what matters – Indirect measures are regularly used to infer cardiac activity. Calcium imaging, for example, is unable to capture important changes to sodium channel functionality. Gene and protein expression do not guarantee biological function. Maestro tracks cardiac activity in real time allowing you to answer the questions that matter.

  • Analyze cell activity label-free – Maestro performs noninvasive electrical measurements from the cultured cardiac population, circumventing the use of dyes/reporters that can perturb your cell model and confound results. Track activity over hours, weeks, and months from the same population of cells.

  • Probe cell models in the same plate they were cultured in – Other higher throughput platforms (e.g. automated patch clamp, flow cytometry) often require cell samples to be transferred into a single-cell suspension before testing. This is far from ideal since [1] the heart exists as a functional network of inter-linked cells, and [2] the cell harvesting process requires numerous handling steps. Maestro captures cardiomyocyte functionality while preserving the morphological complexity of your cardiac cell model.

  • It's easy – You don't have to be an electrophysiologist to use Maestro. Just culture your cardiomyocytes in an MEA plate, load your plate into the Maestro system, and record your cardiac data. Axion's data analysis tools will do the rest, even generating the publication-ready graphs you need.

 

 

Cardiac LEAP technology

Cardiac LEAP

 

LEAP: For cardiac action potential recordings

 

 

The cardiac action potential is an electrical signal characterized by the depolarization across the cell membrane of cardiomyocytes, resulting in contraction of the heart. The shapes of an action potential provide vital information about the cardiomyocyte biology, health, and response to a drug. However, measuring the cardiac action potential traditionally requires invasive techniques, such as manual patch clamp, or labels, such as voltage-sensitive dyes.

LEAP technology enables non-invasive, label-free monitoring of the cardiac action potential in a high-throughput real-time format. LEAP can be used for quantification of action potential morphology, repolarization irregularities such early after depolarizations (EADs), and arrhythmic risk factors such as triangulation. Key metrics, such as rise time, action potential duration (APD), triangulation ratio, and percentage of beats with EADs are all automatically detected by LEAP.

depolarization
repolarization
Triangulation
Arrythmias

 

How does LEAP work?

LEAP stands for local extracellular action potential. The theory behind LEAP is similar to that of patch clamp, where the recorded signal amplitude is proportional to the sealing resistance between the electrode and the cell.

In contrast to a field potential signal,  LEAP induction increases the coupling between the cells and electrode,  enabling the measurement of a much larger action potential signal. The increased cell-electrode coupling is stable for 10-20 minutes or longer, allowing extracellular monitoring of the cardiac action potential without disrupting the underlying biology with dyes or invasive electrodes.

"The LEAP assay addresses an important gap in the field, namely providing a non-invasive solution in recording high quality action potentials from cardiac cells using a high throughput format. The LEAP assay may be a game changer."

Bernard Fermini, PH.D.
Chief Scientific Officer, Coyne Scientific

Bernard Fermini

 

How does LEAP differ from the classic field potential?

The field potential has long been the standard for high-throughput in vitro cardiac electrophysiology. The field potential derives from the underlying cardiac action potential, but more closely resembles the low amplitude clinical electrocardiogram (ECG) signal. The initial depolarization phase is seen as a sharp spike, similar to the QRS complex, followed by the slow repolarization analogous to the T wave. Although the field potential has proven highly effective for high-throughput drug screening and other applications, the field potential shape can obscure complex repolarization irregularities and limit comparisons to gold standard manual patch clamp recordings.

LEAP detecting arrythmias

The LEAP signal accurately reflects the shape and duration of the underlying action potential. The large signal allows for automated detection and classification of arrhythmic events, such as notched EADs, rolling EADs, or ectopic beats. LEAP also provides metrics not available from the field potential, such as rise time and triangulation.

Because LEAP operates on each electrode independently, field potential and LEAP signals can be recorded from the same well simultaneously, providing a direct mapping between features of the field potential and the action potential. In this way, LEAP enables confirmation and automation of feature detection.

LEAP software trace
LEAP software trace

Simultaneous LEAP and field potential (FP) measurements establish translation between FP and LEAP signals. LEAP was induced on half of the microelectrodes in each well of a MEA 48-well plate of iCell CM2. Example LEAP and FP signals from the same well when dosed with E-4031. Depolarization aligned between the LEAP and FP signals, but EADs could be more reliably detected in the LEAP trace.

 

Download the LEAP paper

 

Advantages of LEAP

Due to the larger features of the signal, the LEAP assay is robust against pharmacological manipulations that can render field potential features difficult to detect.

For example, sodium channel blockers cause a detectable change in spike amplitude. At higher doses though, the spike amplitude can become too small to detect. However, rise time prolongation in the LEAP signal can still be easily measured even at high doses. Similar effects can be seen at high doses of hERG blockers when hERG block begins to impact the resting membrane potential.

astemizole traces
atermizole bar graph
astemizole bar graph

Similarly, compounds, such as terodiline, that induce triangulation can flatten the field potential repolarization feature, referred to as the “T wave”. The resulting broad, small amplitude T wave is difficult to detect and quantify. In contrast, triangulation is readily detectable and quantifiable in the LEAP signal.

terodiline
Terodiline induced triangulation at higher concentrations, consistent with patch clamp results.
Terodiline T-wave

 

LEAP applications

LEAP is ideal for capturing even small differences in action potential morphology in cardiac health and disease and in response to compounds. LEAP is a powerful tool for many applications including:

  • Automated APD and EAD detection for high throughput drug screening
  • Predicting arrhythmic risk for cardiac safety and cardiotoxicity testing
  • Characterization of action potential morphology in human induced pluripotent stem cell-derived (hiPSC) cardiomycoytes
  • Studying the effects of genetic manipulation on cardiac electrophysiology
  • Comparing cardiac biology in healthy and diseased states

 

Cardiac MEA technology

Cardiac MEA

 

What is a microelectrode array (MEA)?

Microelectrode arrays (MEA), also known as multielectrode arrays, contain a grid of tightly spaced electrodes embedded in the culture surface of the well. Electrically active cells, such as cardiomyocytes, are cultured on top of the electrodes. When neurons fire action potentials, the electrodes measure the extracellular voltage on a microsecond timescale. As the cells attach and connect with one another, an MEA can simultaneously sample from many locations across the culture to detect propagation and synchronization of cardiac activity across the syncytium.

That’s it, an electrode and your cells. No dyes, no incubation steps, no perfusion, no positioning things just-so; just your cells in a well. Because the electrodes are extracellular (they do not poke into the cells), the recording is noninvasive and does not alter the behavior of the cells, you can measure the activity of your culture for seconds or even months!

CytoView well bottom

An MEA of 64 electrodes embedded in the substate at the bottom of a well.

Rendering of cells growing over the electrodes at the bottom of the well

Cardiomyocytes attach to the array and form a network. The microelectrodes detect the action potentials fired as well as their propagation across the network.

 

Heartbeats in a dish

When cardiomyocytes are cultured on top of an MEA, they attach and connect to form a spontaneously beating sheet of cells, called a syncytium. When one cardiomyocyte fires an action potential, the electrical activity propagates across the syncytium causing each cell to fire and then contract. The electrodes detect each individual action potential and contraction, as well as the propagation of this activity across the array.

The propagating electrical signal is detected by the electrodes as an extracellular field potential. The field potential derives from the underlying cardiac action potential, but more closely resembles a clinical electrocardiogram (ECG) signal. The initial depolarization phase is seen as a sharp spike, similar to the QRS complex, and the slow repolarization is seen as a small slow spike, like a T-wave. The time from the depolarization to repolarization is termed the field potential duration (FPD) and is a key metric in predictive cardiotoxicity screening assays.

While most record the cardiac field potential, the Maestro Pro and Edge MEA systems can also measure local extracellular action potentials, or LEAP. LEAP induction increases the coupling between the microelectrodes and the cardiomyocytes, transforming the extracellular signal from a field potential to an action potential. LEAP provides additional and complementary metrics such as rise time, action potential duration (APD), triangulation, and automated early after depolarization (EAD) detection.

Cardiac Action potential propogates across the cells in the syncytium.

The cardiac action potential propagates from cell to cell across the syncytium. The MEA detects this activity as an extracellular field potential, which closely resembles the clinical ECG.

 

Do more with multiwell  

 

Axion BioSystems offers multiwell plates at many throughputs, from 6-wells to 96-wells, with an MEA embedded in the bottom of each well. Each well represents its own unique cell culture and conditions, creating up to 96 experiments on one plate. Multiwell MEA allows you to study complex human biology in a dish, from a single cell firing to network activity, across many conditions and cell types at once.