Team:KU Leuven/Demonstrate
Demonstrate
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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.
