key: cord-0900657-ju8be0rm
authors: Choi, Seong Woo; Shin, Jin Soo; Park, Soon-Jung; Jung, Eunhye; Park, Yun-Gwi; Lee, Jiho; Kim, Sung Joon; Park, Hun-Jun; Lee, Jung-Hoon; Park, Sung-Min; Moon, Sung-Hwan; Ban, Kiwon; Go, Yun Young
title: Antiviral activity and safety of remdesivir against SARS-CoV-2 infection in human pluripotent stem cell-derived cardiomyocytes
date: 2020-10-19
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2020.104955
sha: c7732368692b33d7eee5cbaa4f42dddf9bf7f57d
doc_id: 900657
cord_uid: ju8be0rm

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is considered as the most significant global public health crisis of the century. Several drug candidates have been suggested as potential therapeutic options for COVID-19, including remdesivir, currently the only authorized drug for use under an Emergency Use Authorization. However, there is only limited information regarding the safety profiles of the proposed drugs, in particular drug-induced cardiotoxicity. Here, we evaluated the antiviral efficacy and cardiotoxicity of remdesivir using cardiomyocytes-derived from human pluripotent stem cells (hPSC-CMs) as an alternative source of human primary cardiomyocytes (CMs). In this study, remdesivir exhibited up to 60-fold higher antiviral activity in hPSC-CMs compared to Vero E6 cells; however, it also induced moderate cardiotoxicity in these cells. To gain further insight into the drug-induced arrhythmogenic risk, we assessed QT interval prolongation and automaticity of remdesivir-treated hPSC-CMs using a multielectrode array (MEA). As a result, the data indicated a potential risk of QT prolongation when remdesivir is used at concentrations higher than the estimated peak plasma concentration. Therefore, we conclude that close monitoring of the electrocardiographic/QT interval should be advised in SARS-CoV-2-infected patients under remdesivir medication, in particular individuals with pre-existing heart conditions.

The world is currently facing unprecedented challenges from the coronavirus disease-19 53 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome-coronavirus-2 (SARS-7 (Qiagen, CA, USA) according to the manufacturer's instructions. RT-qPCR was performed using 142 a SuperScript III Platinum ® SYBR ® Green One-Step qRT-PCR kit (Invitrogen, CA, USA) with a 143 primer set targeting SARS-CoV-2 receptor-binding domain (RBD) gene (sense 5'-144 CAATGGTTTAACAGGCACAGG-3' and antisense 5'-CTCAAGTGTCTGTGGATCACG-3'). 145 The relative viral RNA expression levels were calculated by the ΔΔC T method, and β-actin was 146 used as an endogenous control. Briefly, 1×10 4 Vero E6 cells/well were infected with SARS-CoV-2 at a multiplicity of infection Table   318 1). Similarly, a complete suppression of infectious viral particle production occurred when 319 hESC-CMs and hiPSC-CMs were treated with the highest tested concentration of chloroquine.

Treatment with 33 μM chloroquine led to a 2.3-log 10 PFU/ml and 0.3-log 10 PFU/ml reduction in 321 SARS-CoV-2 titers in hESC-CMs and hiPSC-CMs, respectively, whereas 11 μM chloroquine 322 did not affect the viral titers ( Figure 4C and D) . Likewise, quantification of the viral genome in 323 chloroquine-treated hPSC-CMs determined by RT-qPCR showed a similar trend as the viral 324 yield assay (Figure 4E and F) . Taken together, the data indicated that remdesivir potently inhibits SARS-CoV-2 infection in human cardiomyocyte cultures by reducing the production of 326 infectious progeny virus as well as the synthesis of viral RNA at a nanomolar concentration. In 327 contrast, the inhibitory effect of chloroquine was significantly reduced in hPSC-CMs compared 328 to Vero E6 cells (Table 1) . TMPRSS2 genes in hPSC-CMs (hESCs: H9 and hiPSCs: CMC-11) were measured by RT-qPCR.

The data represent the mean (± SD) of at least two independent experiments performed in 625 triplicate. Statistical analyses were determined using paired student's t-tests, and P < 0.05 was 

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Preclinical Cancer 519

Drug Cardiotoxicity Testing: A Scientific Statement From the

Chemically Defined Culture and Cardiomyocyte square) or infected with SARS-CoV-2 (black circle). (C to F) Reduction of SARS-CoV-2 653 replication by chloroquine and remdesivir in hPSC-CMs as determined by infectious viral titer 654 and RT-qPCR

CoV-2 at an MOI of 2 in the presence of the indicated concentrations of chloroquine or 656 remdesivir. (C and D) Viral titers from hPSC-CM supernatants were determined with plaque 657 assay in Vero E6 cells. (E and F) Quantification of intracellular SARS-CoV-2 genome RNA by 658

Total RNA was isolated from lysates of infected cells for quantification of 659 intracellular SARS-CoV-2 RNA levels (S gene), and results were normalized to β-actin mRNA

Data represent means (±SD) of at least two independent experiments performed in duplicate

Statistical analyses were determined using paired student's t-tests, and significant differences are 662

Cardiomyocyte toxicity of 665 chloroquine and remdesivir in hPSC-CMs. Dose-response curve analyses of drug-induced 666 cardiotoxicity in hPSC-CMs (hESC-CMs and hiPSC-CMs cell lines) were performed in the Arrhythmogenic effects of chloroquine and remdesivir in hiPSC-CMs. The FP signals were 677 analyzed with FP duration (FPD), corrected FPD (FPDc

Fredericia's formula), beat period, the amplitude of Na + Peak (amplitude), beat period 679 irregularity (coefficient of variation of the beat period), and conduction velocity using Cardiac 680

All data are expressed as mean ± SD (n=5, exclusion 681 of quiescent and flat T). Statistical analyses were determined using paired student's t-tests, and P 682 < 0