The mathematical model

Our team developed a mathematical model describing the oscillation of the membrane potential in a transfected HEK cell. 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. HEK cells transfected with α1G, hERG and HCN2 oscillated when stimulated with 40-150 pA. We did not observe oscillations in cells without stimulation.

Patch clamp results

1. Ethosuximide

Figure 1: A 30 seconds measurement of a transfected HEK cell with α1G, hERG and HCN2 is shown. The measurement was performed in the physiological Krebs buffer, while stimulating with 100pA.

Figure 2: A 30 seconds measurement of the same cell as in Figure 1 is shown at a different timeframe. The measurement was performed in the physiological Krebs buffer with ethosuximide, while stimulating with 100pA.

Figure 3: Comparison of the peak intervals of control vs. ethosuximide.
Using MATLAB, we analyzed the the amount of peaks per 10 seconds for both control and ethosuximide conditions. The analysis showed a 20% decrease in frequency of rhythm when ethosuximide was added. A paired t-test (p = 0.0004) was performed to prove that there is a change in overall rhythm. This means that the decrease in rhythm is significantly different from the control rhythm. The error bars on the figure visualize the 95% confidence interval.

Conclusion

Ethosuximide slowed down the rhythm of the transfected cells significantly. Moreover, this measurement was shown to be continuously and reversibly (not shown in figures). The drug works by directly blocking α1G, which has a considerable impact on the rhythm.

2. Ivabradine

Figure 4: 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 5: This graph shows a 30 seconds measurement of the same HEK cell transfected with α1G, hERG and HCN2. The measurements are done by adding Ivabradine. By adding 100pA stimulation with the patch clamp method.

Figure 6: Comparison of the peak intervals of control vs. Ivabradine 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 ivabradine. 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 no significant decrease in frequency of the rhythm after the 30 seconds wash-in period when ivabradine was added. The bars on the plot show standard deviations in a 95% confidence interval.

Figure 7: Comparison of the amplitudes of control vs. Ivabradine Using MATLAB, we measured the amplitudes in 10 second intervals of all the measurements of our controls and compared these to the amplitude of ivabradine. A paired t-test was used to statistically verify that these intervals are different, proving that there is a change in overall amplitude. This t-test showed a significant increase in amplitude of the rhythm after the 30 seconds wash-in period when ivabradine was added, a p = 1.1541e-05 value was obtained, meaning that this increase in amplitude is significantly different from the control rhythm. The bars on the plot show standard deviations in a 95% confidence interval.

Conclusion

ADD YOUR SOLUTION ABOUT IVABR

2. cAMP

Figure 8: A 30 seconds measurement of a transfected HEK cell with α1G, hERG and HCN2 is shown. The measurement was performed in the physiological Krebs buffer, while stimulating with 40pA.

Figure 9: A 30 seconds measurement of the same cell as in Figure 4 at a different timeframe. The measurement was performed in the physiological Krebs buffer after addition of cAMP, while stimulating with 40pA.

Figure 10: As previously explained, we analyzed the intervals using Matlab. A paired t-test was performed (p = 0.0023). The analysis showed a 30% increase in frequency when adding cAMP compared to control settings. The error bars on the figure visualize the 95% confidence interval.

Conclusion

cAMP has been shown to stimulate HCN in cardiac cells, which causes a faster depolarization phase and subsequently a faster rhythm. Our statistical analysis demonstrated that this hypothesis also holds true for the HEKciting cells.

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 possible. However additional research is still needed to further improve the viability of these cells. Secondly, the adult human retinal pigment epithelium (ARPE-19) cells have also been tested in capsules. These cells appeared to be hardy, have a long-life and have a good viability within the capsule. However, human myoblasts and ARPE-19 cells are only examples of multiple other options, such as non-stem cells: fibroblasts, chondrocytes, osteoblasts, adipocytes and skeletal muscle progenitors. Also, different stem cells are possible such as human mesenchymal stem cells (hMSCs), human embryonic stem cells (hESCs) and hESC-derived mesoderm progenitors.

References

Li, A., Bourgeois, J., Potter, M. and Chang, P. (2007). Isolation of human foetal myoblasts and its application for microencapsulation. Journal of Cellular and Molecular Medicine, 12(1), pp.271-280.
Tao, W., Rein, D., Dean, B., Stabila, P. and Goddard, M. (2002). ARPE-19 AS A PLATFORM CELL LINE FOR ENCAPSULATED CELL BASED DELIVERY. US 6,361,771B1.
Wen, J., Xu, N., Li, A., Bourgeois, J., Ofosu, F. and Hortelano, G. (2007). Encapsulated human primary myoblasts deliver functional hFIX in hemophilic mice. The Journal of Gene Medicine, 9(11), pp.1002-1010.