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Opto-electronic feedback control of membrane potential for real-time (re)shaping of action potentials
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Authors
Balázs Ördög*
Tim De Coster*
Sven O. Dekker
Cindy I. Bart
Juan Zhang
Wilhelmina H. Bax
Shanliang Deng
Bram L. den Ouden
Antoine A.F. de Vries
Daniël A. Pijnappels
* = equal authorship
This article originated from a secret project that Balázs and I started. After the BioICD paper was published, I installed a dynamic patch-clamp set-up in the lab. Since we now had this technology at arms-reach, Balázs approached me one evening with the idea of adjusting membrane behavior real-time using feedback, and asked me whether this would be possible using the aforementioned set-up. After having thought about it for 10 minutes, wee both concluded it should bee feasible. In the next weeks, we spent every evening from 6pm-8pm (when everyone else already had left) in the lab trying out this idea. After a month we were certain that this would lead somewhere and the project was taken out of secrecy and moved forward to result in the paper that is presented here.
What did we do exactly?
Over the past two years, I have been collaborating with dr. Ördög on an interesting new technique to control the action potential of an excitable cardiac cell (cardiomyocyte). Through a clever combination of optogenetics with real-time computing, it is possible to let cells react hyper quickly to unwanted changes in the cell membrane. This new technique provides interesting perspectives for in-depth investigations into the regulatory roles of the cell membrane in healthy and diseased cardiomyocytes.
What do you need for this technique?
This technique makes use of four different components. They are the following:
A cell line (in our case the atrial hiAM cell line)
Optogenetic tools to express light-gated ion channels into this cell line. We specifically use one that reacts to blue light to depolarize the cell membrane, and one that reacts to red/orange light to repolarize the cell membrane.
A dynamical patch-clamp set-up to measure cell membrane potential real-time
A real-time Linux computer that takes cell membrane input signals and instantaneously computes a reaction to it.
LEDs that switch on/off based on the reaction term that the real-time computer sends them
These components work effortlessly together in a feedback loop (Figure 1), where we run through the loop in the order 1/2 -> 3 -> 4 -> 5 -> 1/2.
Figure 1: Overview of the experimental set-up that combines real-time computing with optogenetics and dynamic patch-clamp, all effortlessly working together to establish feedback control over cardiomyocyte membrane potential.
How does this new system actually work?
As was mentioned previously, this system relies on a closed-loop feedback mechanism. That means that once started, there is no human influence any more and the whole system operates on itself. What happens is that we first measure a control signal (in this case a control action potential of the excitable cardiac cell) which we store on the computer. Subsequently we measure the action potential of a cell and compare it to the control action potential. When the measured signal is lower than control, we attempt to push the signal up. When the measured signal is higher than control, we attempt to push the signal down. We do this by tiny adaptive steps, after which we measure again to see whether our correction was good or whether we over-corrected. The corrections can be done both electrically (directly through the patch-clamp set-up) or with the use of optogenetics (a more biological way).
An example of the optogenetic technique can be seen in Figure 2. Here we adjusted the action potential with the use of an extra stimulus that created a big bump (panel A). When our set-up (called APqr) was switched on, you can see that the action potential is corrected back to normal again by pushing the signal down through several spikes of red/orange light. We can make use of advanced statistics to confirm that the correction is indeed happening as planned (panels B, C and D).
Figure 2: Action potential correction with the use of optogenetics in our new closed-loop feedback set-up.
What are the advantages of this new technique?
This technique has two major advantages:
It makes use of optogenetics, a biological way to influence the cell membrane potential. When this technique would eventually lead to therapies, this means that this methods will be painless.
As a result of using light, this also means that we are not limited to a single cell. We can perform this technique to multiple cells in a monolayer at the same time. This method therefore creates a technique to control cell membrane potential at the multicellular level.
These two advantages make that it is possible to control the behavior and contraction of multicellular culture dishes. We tried this by imposing different action potentials of sick cells onto a monolayer (Figure 3). By making use of the blue and red/orange light we were able to let cells have a variety of cardiac action potentials corresponding to certain drugs (panel A). Once again, we showd the significance of our findings through statistics (panels B-E for the drugs carbachol and 4AP). Therefore we created an additional use case for our technique.
Figure 3: This same set-up can also impose or force behavior onto cells, allowing us to study how sick cells operate.
What does this now all mean?
We unlocked new research possibilities by the development of an experimental system capable of full cell membrane potential control, including instantaneous modulation of action potential morphology on a multicellular level. Our system restores action potential morphologies in the presence of perturbations of different origin without any prior knowledge about the disturbance. It also enforces arbitrary action potential morphologies with high accuracy in an immediate and self-regulatory manner.
These results therefore set the stage for in-depth investigations into the regulatory roles of action potentials in healthy and diseased excitable heart cells via the application of such real-time opto-electronic control systems.
Journal info
Article type: Original article
Impact factor: 3.8
ISSN: 2667-2375
Cell Reports Methods is an open access, multidisciplinary journal from Cell Press publishing significant methodological advances of broad interest. The primary criterion for publication in Cell Reports Methods is a robust, reproducible method that will spur scientific progress. Papers presenting a new tool or set of reagents that will be of significant utility and interest to the community are also considered. Areas of interest include innovations in sequencing and imaging technologies, genome editing, single-molecule approaches, computational methods, biotechnology, and synthetic biology.
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