Team:KU Leuven/Demonstrate


Demonstrate

Over the course of three months of hard work we gathered a lot of results. Our main source of data was the patch clamp, an instrument used to measure membrane potentials. The goal of our HEKcite project was to design a device that can detect changes in drug concentration by giving a change in overall rhythm as output. Below you will find a summary of our results. Key achievements that we are especially proud of are:
1) Designing an oscillating mathematical model
2) creating oscillating HEK 293 cells
3) Measurements → influencing the rhythm with different types of medications


Key Achievements

  • -Creating an oscillating mathematical model
  • -Creating oscillating HEK293 cells
  • -Adapting the rhythm with different types of medications


The mathematical model


Our team developed a mathematical model describing the oscillation of the membrane potential in transfected HEK cells. The model is based on the (h)existing model of Kharche (2011), which describes the ionic currents through ion channels in sinoatrial node cells in the heart. We created a model containing three ion channels of interest, which are HCN, hERG and α1G. Furthermore, the model also contains equations for Sodium-Potassium exchangers, Sodium-Calcium exchangers, and several background currents. By adjusting the time kinetics of these channels, we fitted the model to our own experimental values. Other parameters that were adjusted to match experimental values were ion channel conductance, membrane capacitance and cell volume.
This electrophysiological model allowed us to perform experiments in silico to learn more about optimal ion channel ratios, rhythm modulation and specific ionic currents.



Creating oscillating HEK cells


Calcium Imagining

We performed calcium imaging to screen for intracellular changes in calcium in the transfected HEK cells. The calcium imaging setup allowed us to visualize more than 100 cells at the same time, which is ideal for an optimization process. The experiments consisted of several transfection ratios and extracellular potassium concentrations. We found an optimal transfection ratio of 2:1 α1G to hERG at an extracellular potassium level of 2-5 mMol. The transfected cells were already stably expressing HCN2, which is ideal since our mathematical model showed that HCN2 is the most important ion channel responsible for a steady rhythm. A stable HCN2 expression across different experiments is beneficial for replicating results with a similar rhythm. Our method allowed to measure the intracellular calcium every two seconds, which was too slow to measure the expected oscillations, but enough to see a subtle change in intensity across different images.

Patch Clamp

Whole-cell patch clamp is a technique where you can measure oscillation at a greater temporal resolution, with a sampling rate up to 10 000 Hz. It measures the electrical current or voltage difference across the membrane of a single cell. After optimizing the experimental setup with calcium imaging, we started using patch clamp with the parameters derived from the experiment. The HEK cells transfected with α1G, hERG and HCN2 were oscillating when stimulated with 40-150 pA. We did not observe oscillations in cells without stimulation.

Patch clamp results

1. Ethosuximide


Figure 1: This graph shows a 30 seconds measurement of a HEK cell transfected with α1G, hERG and HCN2. The measurements are done using Krebs buffer, by adding 100 pA stimulation with the patch clamp method.


Figure 2: This graph shows a 30 seconds measurement of the same HEK cell transfected with α1G, hERG and HCN2. The measurements are done by adding ethosuximide, an α1G antagonist after a wash-in period of 30 seconds, by adding 100 pA stimulation with the patch clamp method.


Figure 3: Comparison of the peak intervals of control vs. ethosuximide
Using MATLAB, we counted the peaks in 10 second intervals of all the measurements of our controls and compared these to the peaks intervals of ethosuximide. A paired t-test was used to statistically verify that these intervals are different, proving that there is a change in overall rhythm. This t-test showed a 20,27% decrease in frequency of the rhythm when ethosuximide was added, a p = 4.0607e-04 value was obtained, meaning that this decrease in rhythm is significantly different from the control rhythm. The bars on the plot show standard deviations in a 95% confidence interval.


conclusion

When comparing the rhythm of the transfected HEK cells in control settings with Krebs buffer with the ethosuximide setting, has shown a significant difference. Ethosuximide, direct blocker of α1G has an overall significant decreasing impact on the rhythm.


2. Ivabradine


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conclusion

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2. cAMP


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Figure 8: Comparison of the peak intervals of control vs. cAMP
Using MATLAB, we counted the peaks in 10 second intervals of all the measurements of our controls and compared these to the peaks intervals of a second messenger, cAMP. A paired t-test was used to statistically verify that these intervals are different, proving that there is a change in overall rhythm. This t-test showed a 30.99% increase in frequency of the rhythm after the 30 seconds wash-in period when cAMP was added, a p = 0.0023 value was obtained, meaning that this increase in rhythm is significantly different from the control rhythm. The bars on the plot show standard deviations in a 95% confidence interval. CI95%(krebs)=[1701.7 1900.4], CI95%(cAMP)=[1235.7 1514.2]


Figure 9: ADD YOUR TEXT


conclusion

When comparing the rhythm of the transfected HEK cells in control settings with Krebs buffer with the cAMP setting, has shown a significant difference. cAMP an activator of the HCN2 ion channel, has an overall significant increasing impact on the rhythm.


Future directions


Stable cell line

The biggest drawback of our project is the variability of gene expression in our manipulated cell line. Since we only transfect in a transient manner, the exact concentration of the DNA containing our ion channels differs slightly with every transfection. In the future, a stable cell line, containing the three ion channels with the DNA of the ion channels in a stable concentration, could reduce this variability. All the cells would oscillate at the same frequency, causing a more coherent rhythm.

Extra ion channel modulation

If in the future, we have our stable cell line, thus a stable and intrinsic oscillating system. A logical next step could be to induce a fourth ion channel, for example the temperature-activated ion channel TRPV1. This fourth ion channel could influence the rhythm too, which would strengthen the overall oscillating device. Introducing a fourth ion channel into our oscillating system, could have numerous applications.
By introducing Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) for example, we can influence the depolarizing rate by a designer drug that has no effect on the human body, such as clozapine-N-oxide (CNO). This sensing system could be used to control drug adherence in patients by giving CNO together with their medication orally, followed by quantification in the urine due to fluctuations in the rhythm of the cells. Temperature-sensitive TRPV4 channels could be introduced to create a temperature sensor with this cell-line. Other channels such as KATP channels could be used to indirectly measure extracellular glucose concentration and report it by a change of pace of the bioluminescence that is coupled to depolarization. These examples provide only a few possibilities that this extra ion channel could provide.

Receptor modulation

We have created a biosensor that enables us to sense in real-time the fluctuations drug concentration in the blood, by measuring the frequency differences in our manipulated HEK cells. It needs no explanation that drugs that directly affect our ion channels, would influence the overall rhythm of the cells. But also drugs that affect the cAMP signal transduction system could influence the rhythm. For example, dopamine antagonists, which are drugs used for patients suffering from Parkinson’s disease. The effect of these drugs vary from person to person and their therapeutic effect decreases over time, resulting in more side effects. If a dopamine receptor is be transfected into our HEKcite cell line, it would enable our cells to sense the fluctuations in the blood concentration of dopamine antagonists. When these dopamine antagonists bind to their receptors, which are now present on the membrane of our HEKcite cells, the cAMP signal transduction system is activated, resulting in an increase of cAMP. This raise in cAMP results in a change in overall rhythm, as we have shown.
Thus, our therapeutic drug monitoring device could be generalized for numerous types of drugs that need a close follow-up.

Genetic link

As the options are endless, the genes of our ion channels could be coupled to an inducible promoter, enabling us to modulate the rhythm through gene expression. For example, if we would couple the HCN channel to an inducible promoter with a short half-life, this will rapidly accelerate the rhythm when gene expression is induced. Moreover, the short half-life will cause faster deactivation. Coupling one, two or all the ion channels to promoters would provide endless option of modulate the frequency of the oscillating cells, providing us with numerous applications in the field of medicine, biotechnology and many more.

Considerations for replicating the experiments

We made an inquiry about the type of cells we would use. HEK 293 cells, like the ones we created a sinus rhythm in, are known to have a high growth rate. This would result in the cells quickly filling the capsule, which would cause them to starve and die. Furthermore, they contain adenoviral DNA. The possible risks of an infection with an adenovirus and its effect on cells with adenoviral DNA present in the body are not yet known. This is why cells containing foreign DNA are not allowed to be used in humans. These two factors reduce the applicability of our HEK cells strictly to this proof of concept study. For this reason, we started looking for other cells.
As noted by professor Cosson, the key decision criteria for choosing new cells are: (1) the ease with which they are engineered, (2) the ability to store them in vitro in large batches, (3) the ease to be cultured in cheap media, and finally (4), the cells need to be in biosafety category 1 to comply with regulation. Two cell types that meet these requirements, human myoblasts and ARPE-19 cells, were investigated to be used in the finalized capsule. Different studies already confirm that using human myoblasts in capsules is a possibility. However further research is still needed for improvement of the viability of these cells. Secondly, the ARPE-19 cells which are retinal pigment epithelium cells have also been tested in capsules. This cells appeared to be hardy, have a long-life and have a good viability within the capsule.


References