A Novel Optical Dynamic Clamp Platform Using Ipsc-Derived Cardiomyocytes For Drug Screening

Quach, Bonnie

A Novel Optical Dynamic Clamp Platform Using Ipsc-Derived Cardiomyocytes For Drug Screening

Abstract

iPSC-derived cardiomyocytes (iPSC-CMs) are a potentially advantageous platform for drug screening because they provide a renewable source of human cardiomyocytes and can be patient specific. One obstacle to their implementation is their neonatal-like electrophysiology, which reduces relevance to adult arrhythmogenesis. One method to address this problem is to electrically mimic deficient currents in iPSC-CMs using a technique called dynamic clamp. Mimicking the missing inward rectifying potassium current, IK1, in iPSC-CMs via dynamic clamp pushes action potential characteristics to resemble more closely an adult cardiomyocyte. However, this method is technically challenging and low throughput, limiting its practical uses for more high-throughput applications, such as large-scale drug screening. To address this, we aim to create an optically-controlled version of dynamic clamp, which because of its contactless nature, could be high-throughput and not limited to a single-cell format. The ideal platform would use optogenetics to supplement the deficient current and use a fluorescent voltage indicator to measure the membrane potential. Optogenetic tools are commonly used statically and to either stimulate or cease electrical activity. This thesis presents a proof of principle of using optogenetic tools in lieu of an electrode by developing an optical dynamic clamp (ODC) platform that uses an LED to dynamically activate a hyperpolarizing opsin, ArchT, to generate an IK1-like current. This ODC platform was verified with the standard electrode-based dynamic clamp (EDC) and gave a similar output, demonstrating a proof-of-concept that optogenetics are able to mimic an electrode. The ODC platform was challenged with E4031, bayK 8664, terfenadine, and verapamil. The ODC platform was able to detect effects of the drugs on action potential characteristics similar to EDC, but the ODC platform did not consistently yield results identical to EDC. Possible reasons and limitations are discussed. With further development, the ODC platform can possibly be refined to be more precise, but maturation of iPSC-CMs may still be needed to make the platform more relevant to adult electrophysiology. The ODC platform has the potential to expand on the possibilities of dynamic clamp by enabling more relevant formats, such as monolayers, co-cultures or with other engineered platforms.

Date Issued

2019

Keywords

cardiology; dynamic clamp; electrophysiology; iPSC-derived cardiomyocyte; optogenetics

Degree Discipline

Pharmacology

Degree Level

Doctor of Philosophy

Rights

Attribution-NonCommercial-NoDerivatives 4.0 International

Rights URI

https://creativecommons.org/licenses/by-nc-nd/4.0/

Types

dissertation or thesis