Abstract
Current models for cardiotoxicity screening are limited to only evaluate hERG inhibition for related prediction of QT prolongation and torsadoegnic potential. More comprehensive in vitro cardiotoxicity assays are being proposed using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), aided with experimental and computational approaches. We tested the potential of CRISPRi modulation in pre-differentiated iPSC-CMs combining all-optical cardiac electrophysiology and molecular analysis to characterize functional phenotypes of key ion channels in the human heart. We designed single guide RNAs (sgRNAs) targeting KCNH2, KCNJ2, and GJA1. The sgRNAs were cloned into a lentiviral vector with eGFP reporter. A dox-inducible dCas9-KRAB was inserted into the AAVS1 safe harbor site of pre-differentiated hiPSC-CMs and transduced with lentivirus carrying the sgRNAs. To deploy all-optical electrophysiology, cells were transduced with an adenoviral vector containing ChR2-eYFP (for optical stimulation) and co-labeled with spectrally-compatible voltage and calcium sensors to obtain functional measurements upon 5 days of dox induction. A sgRNA targeting KCNH2 resulted in about 40% knockdown of KCNH2 mRNA and significant but relatively mild prolongation of action potential duration, e.g. spontaneous APD90 (+12%, p<0.05). A sgRNA targeting KCNJ2 caused about 60% downregulation of mRNA levels and exhibited minimal changes in spontaneous beat frequency in high density cell preparations (+7%, n.s.) but more pronounced effects in reduced density cell preparations (+30%, p<0.01). Additionally, sgRNA targeting GJA1 exhibited about 40% knockdown of mRNA and resulted in significant slowing of conduction velocity (-19%, p<0.01) at 1 Hz pacing conditions. Knockdown of key cardiac ion channels in our system yielded mild but specific functional changes. Using this platform for CRISPRi mediated knockdown of disease-associated genes in pre-differentiated cardiomyocytes can improve the assessment of gene function in cellular cardiac electrophysiology. In combination with sgRNA libraries and/or with CRISPRa mediated gene activation, it can allow a more comprehensive evaluation of the mechanisms controlling cardiac electromechanics.
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