Human Induced Pluripotent Stem Cell-Derived Cardiac Cell Sheets Expressing Genetically Encoded Voltage Indicator for Pharmacological and Arrhythmia Studies

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe introduction of the human induced pluripotent stem cell (hiPSC) technology () coupled with improved methods for cardiomyocyte differentiation (, ) brought a unique value to the fields of cardiac disease modeling (, , , , , ) and drug testing (, , , ). In the field of cardiac electrophysiology, most studies examined the electrical properties of the hiPSC-derived cardiomyocytes (hiPSC-CMs) at the single-cell level, focusing primarily on action potential (AP) and ionic current characteristics. Only a few studies have utilized multicellular hiPSC-based cardiac tissue models to evaluate conduction and arrhythmogenesis (, , href="#bib18" rid="bib18" class=" bibr popnode">Laksman et al., 2017, href="#bib19" rid="bib19" class=" bibr popnode">Lee et al., 2012).Traditional methodologies used for studying conduction in cardiomyocyte cultures include multielectrode extracellular recordings (href="#bib39" rid="bib39" class=" bibr popnode">Yankelson et al., 2008), which may be limited in terms of spatial resolution and degree of complexity of the information gained, or optical mapping (href="#bib9" rid="bib9" class=" bibr popnode">Herron et al., 2012). The latter approach utilizes voltage-sensitive dyes (VSDs) to follow changes in membrane potential (href="#bib23" rid="bib23" class=" bibr popnode">Lopez-Izquierdo et al., 2014, href="#bib24" rid="bib24" class=" bibr popnode">Matiukas et al., 2007). These indicators, although of great utility, can cause phototoxicity and hamper the ability to obtain long-term and repeated recordings.To overcome the aforementioned challenges, we propose to combine the hiPSC technology, two-dimensional (2D) cardiac tissue models, and genetically encoded voltage indicators (GEVIs), such as ArcLight (href="#bib20" rid="bib20" class=" bibr popnode">Leyton-Mange et al., 2014, href="#bib31" rid="bib31" class=" bibr popnode">Shinnawi et al., 2015), VSFP-CR (href="#bib5" rid="bib5" class=" bibr popnode">Chen et al., 2017), CaViar (href="#bib6" rid="bib6" class=" bibr popnode">Dempsey et al., 2016), and VSFP2.3 (href="#bib22" rid="bib22" class=" bibr popnode">Liao et al., 2015), for probing membrane potentials. Specifically, we chose to focus on ArcLight and hypothesized that the generated ArcLight-expressing hiPSC-derived cardiac cell sheets (hiPSC-CCSs) will allow gaining high-resolution information regarding tissue conduction and repolarization in both acute and long-term studies. Following establishment of this unique multicellular model, we aimed to evaluate its potential for drug testing using agents known to alter conduction and/or repolarization with specific emphasis on studying the mechanisms of drug-induced arrhythmias at the tissue level. Finally, we aimed to evaluate the biophysical properties of reentrant arrhythmias (spiral waves) induced in this model through either a drug-related pro-arrhythmia mechanism or following programmed electrical stimulation and to test the efficacy of clinically relevant therapeutic interventions.