Cardiac Pacing

Cardiac Pacing
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Controlling beat rate with electrical stimulation allows you to increase physiological relevance and reduce well-to-well variability. The ability to vary beat rate enables detection of use-dependent drug effects (i.e. beat rate dependent). Optogenetics can further enhance cardiac assays by providing artifact-free, precise, and targeted stimulation.

The Maestro MEA platform with the Lumos optical stimulator offer unprecedented control of cardiac beat rate with electrical or optical stimulation capabilities. Use individually or combine to precisely control cardiomyocyte beating and reduce assay variability.

 

Improve the quality and consistency of your assay

Compound-induced changes to cardiomyocyte field potential duration (FPD) can be used to assess the cardiac safety profile of new drugs. However, repolarization timing is tightly coupled to beat rate, meaning beat rate variability can confound FPD data interpretation. The ability to pace cardiomyocyte activity reduces both FPD well-to-well and inter-assay variability.

Cardiac pacing sets a desired beat by optical or electrical pacing
Cardiac pacing reduced well to well variability
Cardiac pacing reduced variability between plates

Beat period and field potential duration quickly adapt when electrical (or optical) pacing is introduced (A, arrows indicate 1 Hz pace; bottom left). (A bottom left) Key parameters, such as conduction, with real-time visualization. (B) Pacing Pluricyte® Cardiomyocytes with the dedicated E-Stim+ electrode, available in BioCircuit MEA and CytoView MEA plates, significantly reduces FPD variability between wells and plates (p=0.01) as well as variability between plates. (C) Variability was measured as the coefficient of variation (CoV) across wells and across plates.

 

Discover more by controlling cardiac activity

Many compounds exhibit use-dependent effects. Reverse use-dependence, which occurs when a compound produces greater effects at slower beat rates, is an important indicator of proarrhythmic risk. The dedicated E-Stim+ electrode allows the user to systematically vary beat rate to identify such use dependent effects. Thus, pacing with the E-Stim+ electrode significantly increases the information content of your assay.

Reverse use of dependence of E-4031 revealed by pacing Pluricyte Cardiomyocytes
TTX use dependence revealed by pacing Pluricyte Cardiomyocytes with E-stim+ electrode

(D) Reverse use-dependence of E-4031, a hERG channel blocker, revealed by pacing Pluricyte® Cardiomyocytes with the E-Stim+ electrode. (E) Use dependence of TTX, a sodium channel blocker, revealed by pacing Pluricyte Cardiomyocytes with the the E-Stim+ electrode. (D,E) Bars represent mean ± 1 standard error of the mean. Numbers above the bars indicate the number of wells successfully paced.

 

Pacing reveals cell-specific FPD and BP relationship

Cardiac repolarization is intrinsically linked to the beat frequency, both of which are sensitive to pharmacological manipulation. Optogenetic stimulation can be used to control the beat frequency and remove it as a variable, resulting in increased reliability of the repolarization measurement.

A "chirp" assay was used to sequentially increase the beat rate of Plyricyte Cardiomyocytes
The field potential duration adapted with each sequential beat rate increase up to 3 Hz
Typical clinical correction formulas did not accurately predict the FPD

(F, G) The beat rate of Pluricyte® Cardiomyocytes was increased in a step-wise manner (known as a “chirp” assay). The field potential duration (FPD) adapted with each sequential beat rate increase up to 3 Hz. (H) Typical clinical correction formulas, the Fridericia and Bazzett, did not accurately predict the FPD. However, pacing with the Lumos revealed the cell-specific beat rate correction relationship.

 

Quantify arrhythmic risk using cardiac pacing

Optogenetic stimulation in conjunction with LEAP can be used to quantify prolongation independent of beat period and quantify rate-dependent drug effects.

hiPSC-CM transduced with ChR2 and exposed to pulses of blue light paces the cells faster

HiPSC-cardiomyocytes were transduced with channel rhodopsin 2 (ChR2), and pulses of blue light from the Lumos were used to pace cardiomyocytes at successively faster rates. Here, sotalol causes reverse use-dependent prolongation, resulting in longer prolongation at a slower beat rate.

Cardiac electrical pacing
Cardiac optical pacing protocol

Getting started with Maestro Pro and Edge couldn't be easier. Culture your cardiomyocytes in an Axion multiwell MEA plate (Day 0). For optogenetic experiments, using adeno-associated virus-based vectors, add the virus to the cell suspension at the time of plating.  Allow 7-14 days for opsin expression.  When transfecting cardiomyocytes with mRNA-based opsins, transfect at peak cardiomyocyte activity and allow 2 days for opsin expression.  Load the MEA plate into the Maestro MEA system at the desired recording times and begin recording.  Perform optical stimulation experiments using the Lumos optical stimulator, or electrical stimulation studies using any of the microelectrodes in the wells of the MEA plate (Day 7+).  Analyze the cardiomyocyte activity with AxIS Navigator Cardiac Module software. 

 

Using an MEA system

 

The advantage of measuring electrically or optically evoked cardiomyocyte activity on the Maestro Pro and Edge systems:

  • Electrical stimulation – Axion’s multiwell plates bring flexibility to your experimental design. Each MEA electrode is dual-purpose, capable of recording or stimulating. The Cardiac Module software makes stimulation simple yet customizable, while optimized artifact elimination and automated detection of electrophysiological features make analysis easy, efficient, and reproducible.

  • Optical stimulation – The patented Lumos optical stimulation system seamlessly pairs with Maestro Pro and Edge, offering precise control over light intensity and duration of four stimulation wavelengths in each well. The Lumos allows researchers to simultaneously direct and record functional cardiomyocyte activity.

  • 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. But, for example, calcium imaging is unable to capture important but subtle changes to Na+ channel functionality, and expression levels of protein markers often poorly correlate with cell model performance. Maestro tracks cardiac activity in real-time allowing you to answer the questions that matter.

  • Analyze cell activity label-free – 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 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.

 

 

Contractility Technology

Contractility

 

Contractility: Using array-based impedance

Every beat of the heart is characterized by a contraction of the heart that pumps the blood out to the body. When cardiomycoytes are cultured on top of electrodes, they form a spontaneously beating syncytium. With each beat, the cells contract and relax, changing their shape and coverage over the electrodes. These changes can be measured as a change in impedance, or contractility.

Contractility is often used to characterize the mechanical properties of induced pluripotent stem cell-derived cardiomyocytes and to detect the effects of compounds on cardiac contractile function (e.g. inotropes). Measures such as beat amplitude, beat period, and excitation-contraction delay reveal changes in contractile function due to cardiomyocyte maturation or compound addition.

Beat Amplitude
Beat timing
excitation-contraction coupling

The Maestro Pro and Edge provide key parameters of cardiomyocyte contractile function, including beat amplitude, beat period, and excitation-contraction delay. The Maestro systems are the only platforms that can measure the coupling between the electrical (field potential) and contractile signals from the same cells over the same electrode at the same time.

Most contractility platforms measure contractility from one or two large electrodes. The Maestro Pro and Edge use an array of microelectrodes to measure high-resolution contractility from multiple locations across the syncytium. Array-based contractility can be used to track propagation and characterize variability. In addition, by simultaneously monitoring both the field potential and the contraction from the same electrode, the Maestro provides highly precise measurements of beat timing and excitation-contraction delays.

Contraction and relaxation of cells being detected
MEA with cells contracting
MEA with cells contracting

(A) Contraction and relaxation are detected as an increase and decrease in contractility (gray arrows). (B) The microelectrode array detects regions that are contracting while others are being stretched. (C) This pattern is represented as a contractility map, where the relative size of each circle indicates whether local cells are contracting or being stretched.

 

Contractility in 3D

Using an array of microelectrodes for contractility offers many advantages, including assessing variability across the syncytium and variations in cell culture coverage. Recording from microelectrodes enables advanced applications, such as measuring contractility from multiple 3D cardiac spheroids in the same well. A larger electrode would either smear these signals or fail to detect them at all. The microelectrodes provide high spatial resolution recordings to detect the distinct beating patterns in each area of the well.

Spheroid organoids on MEA plate
Organoid beat pattern detected by electrode

Seven spheroids of human induced pluripotent stem cell-derived cardiomyocytes were deposited in a CytoView MEA 6-well plate. The spheroids beat independently, exhibiting unique contractile patterns that were detected and distinguished using array-based contractility on the Maestro.

Optogenetics Technology

Optogenetics

 

Optogenetics: Using light to control cells

Optogenetics is a technique that involves the use of light to control cell function. Cells are first genetically modified to express light-sensitive ion channels, called opsins. Then, light can be used to activate the opsin. The most well-known opsins are light-gated ion channels that can control the excitability of the cell membrane. When activated by the opsin-specific wavelength of light, the channels open allowing ions to flow across the cell membrane to either excite or inhibit the cell. Optogenetics enables precise control over a targeted cell population.

 

Many opsins, many options

Since the first microbial opsin was introduced into mammalian neurons in 2005, many different opsins have been used to control the excitability of electroactive cells such as neurons and cardiomyocytes. Each opsin is sensitive to a specific wavelength range, or color of light and induces a precise biological event.

Opsins activated channel

Channelrhodopsin (ChR2), for example, is activated by blue light. When ChR2 opens, positive cations (like sodium and calcium), flow into the cell, depolarizing or exciting the cell. In contrast, halorhodopsin and archaerhodopsin both inhibit cell excitability by hyperpolarizing the cell in response to yellow or green light, respectively. With optogenetics, you can turn on and off cells like a light switch.

The timing of these light-activated events is fast, facilitating highly precise control. First generation opsins, such as channelrhodopsin, open and close in milliseconds, ideal for kicking off an action potential. Second generation opsins have fined tuned kinetics for even faster, more precise control or slower, longer-lasting inhibition. For example, step-function opsins stay open until another pulse of light switches them off.

Optogenetic silencing of target cell
Optogentic activation of blue light neuron only

Optogenetics can precisely control which cells are turned on or off by employing different gene promoters for opsin expression. Opsins can be expressed in all neurons or used to control specific subpopulations. Unlike electrical stimulation, which excites all nearby cells, optical stimulation can be finely targeted to the cells expressing the opsins responsive to a narrow band of light wavelengths.

ChR2 Neural excitation
ArchT inhibition
ChR2+ Rat cortical neurons (Div 14)

 

In summary, optogenetics is a powerful toolbox for precise control over targeted cell populations at fast time scales. Superior spatial and temporal control, reversibility, and easy stimulus delivery make exploring complex biology simpler than ever before.

 

More than just ion channels

As the field has advanced, opsins have been used to control more than just ion flow. Light-activated gene expression with light-inducible transcription factors can control the proteins made by cells. The combination of optogenetics with CRISPR provides even greater control over CRISPR/Cas9 gene editing.

Opsins have also been incorporated into many biochemical and intracellular signaling pathways to control key protein functions. MAPK and PI3K pathways, Rho family GTPase activation, apoptosis, and protein trafficking can now all be precisely controlled by light.

Opsins in cell membrane

 

Shining light in vitro

Sophisticated biology like optogenetics demands sophisticated technology to explore it. In vitro technology relied on single wavelength lasers and custom lab-specific tools while many in vivo technologies were quickly developed for optical stimulation. The Lumos Optical Stimulation system is the first-of-its-kind multiwell optical stimulator with the ability to deliver up to four wavelengths of light per well with microsecond precision. From controlling the excitability of your neurons to pacing the beating of your cardiomyocytes, discover how the Lumos and optogenetics can revolutionize your assay.

Lumos system