Calmodulinopathy Correction using CRISPR Interference in a Cardiac Microtissue Model

Abstract

Calmodulinopathies, caused by mutations in calmodulin (CaM), a ubiquitous Ca2+ sensor, can lead to life-threatening cardiac arrhythmias, such as long QT syndrome (LQTS). In this study, we created a calmodulinopathy model in 3D cardiac microtissues (CMTs), using cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs) with the D130G-CALM2 mutation, and used CRISPR interference (CRISPRi) to rescue the disease phenotype. Lentiviruses that encode a guide RNA (gRNA) targeting CALM2 (both wildtype and mutant) and a Krüppel associated box (KRAB) transcriptional repressor were transduced to CMTs and their electrophysiological and mechanical properties were analyzed. Results show that D130G-CALM2 CMTs reproduced key features of calmodulinopathy associated LQTS, including prolongation of action potential duration (APD), electrical alternans and longer pacing cycle lengths at which loss of 1:1 capture occurred compared to uncorrected mutant CMTs. After transduction with CALM2 gRNA and KRAB lentivirus, we observed significant knockdown of CALM2 mRNA, shortened APD, increased loading rate during contraction and relaxation, and decreased time to peak and relaxation time over a range of pacing frequencies in CMTs. In conclusion, by demonstrating correction of disease electrophysiological and mechanical phenotype by CRISPRi in 3D tissue models, this study provides a proof of concept for the utility of using viral transduction for CRISPRi in cardiac muscle to correct the tissue phenotype.