Abstract Body (Do not enter title and authors here): Introduction. Designer receptors exclusively activated by designer drugs (DREADDs) are a robust chemogenetic tool for the interrogation of cellular electrical activity. Particularly, hM4Di activates the Gi signaling pathway, and can induce cellular hyperpolarization, leading to decreased electrical activity. Our aim is to engineer human induced pluripotent stem cell-derived cardiomyocytes (hiSPC-CMs) with controllable electromechanical properties to elucidate the role of transplanted hiPSC-CMs in cardiac regeneration. We hypothesize that activating hM4Di in transgenic hiSPC-CMs will inhibit their electrical activity.
Methods. We developed a new hiPSC line expressing DREADD using the PiggyBac system (PB). The PB transposon included the hM4Di gene under the cTnT promoter, with mCherry and puromycin resistance genes as transfection markers (Fig 1A). After establishing the line, we differentiated these hiPSCs into DREADD-expressing cardiomyocytes (hiPSC-CM^M4Di). We activated hM4Di using clozapine N-oxide (CNO) and determined the optimal concentration through a concentration swipe. Optical mapping was used to analyze the electrical activity of hiPSC-CM^M4Di.
Results. After transfection, we observed the expression of mCherry in hiPSCs (Fig 1B). Additionally, we confirmed that these cells retained their pluripotency by evaluating the stemness markers Sox2 and Nanog (Fig 1C). Then, we performed directed cardiac differentiation. The cells showed spontaneous beating after day 8 of differentiation. We determined that the optimal concentration for hiPSC-CM^M4Di inhibition was 200µM CNO, based on concentration swipe. Furthermore, our optical mapping data showed that spontaneous beating rate was decreased 30 minutes after adding CNO and completely stopped at 60 minutes. The spontaneous beating recovered after CNO was removed. CNO did not affect the electrical behavior of wild type cells (Fig 1D).
Conclusions. We developed a transgenic hiPSC line expressing DREADD. The produced hiPSC-CM^M4Di responded positively to the CNO ligand, allowing inhibition of their electromechanical activity. This cell line holds potential for investigating the electrophysiology of grafted hiPSC-CMs, and for application as disease model in vitro.