key: cord-0310516-mcstvrsv authors: Liu, Juli; Zhang, Yucheng; Wu, Shiyong; Han, Lei; Wang, Cheng; Liu, Sheng; Simpson, Ed; Liu, Ying; Wang, Yue; Shou, Weinian; Liu, Yunlong; Rubart-von der Lohe, Michael; Wan, Jun; Yang, Lei title: SARS-CoV-2 Viral Genes Compromise Survival and Functions of Human Pluripotent Stem Cell-derived Cardiomyocytes via Reducing Cellular ATP Level date: 2022-01-23 journal: bioRxiv DOI: 10.1101/2022.01.20.477147 sha: 6ccf212063a949bd3c3f73c15b0e6c599d190079 doc_id: 310516 cord_uid: mcstvrsv Cardiac manifestations are commonly observed in COVID-19 patients and prominently contributed to overall mortality. Human myocardium could be infected by SARS-CoV-2, and human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are susceptible to SARS-CoV-2 infection. However, molecular mechanisms of SARS-CoV-2 gene-induced injury and dysfunction of human CMs remain elusive. Here, we find overexpression of three SARS-CoV-2 coding genes, Nsp6, Nsp8 and M, could globally compromise transcriptome of hPSC-CMs. Integrated transcriptomic analyses of hPSC-CMs infected by SARS-CoV-2 with hPSC-CMs of Nsp6, Nsp8 or M overexpression identified concordantly activated genes enriched into apoptosis and immune/inflammation responses, whereas reduced genes related to heart contraction and functions. Further, Nsp6, Nsp8 or M overexpression induce prominent apoptosis and electrical dysfunctions of hPSC-CMs. Global interactome analysis find Nsp6, Nsp8 and M all interact with ATPase subunits, leading to significantly reduced cellular ATP level of hPSC-CMs. Finally, we find two FDA-approved drugs, ivermectin and meclizine, could enhance the ATP level, and ameliorate cell death and dysfunctions of hPSC-CMs overexpressing Nsp6, Nsp8 or M. Overall, we uncover the global detrimental impacts of SARS-CoV-2 genes Nsp6, Nsp8 and M on the whole transcriptome and interactome of hPSC-CMs, define the crucial role of ATP level reduced by SARS-CoV-2 genes in CM death and functional abnormalities, and explore the potentially pharmaceutical approaches to ameliorate SARS-CoV-2 genes-induced CM injury and abnormalities. Whole mRNA-seq 114 Total RNA was evaluated for quantity and quality by using Agilent Bioanalyzer 2100. The Data comparison between two groups (gene overexpression versus control) was 131 conducted using an unpaired two-tailed t-test. All data were presented as mean ± S.D. Figure 1B) . Therefore, we 151 decided to pursue the functional studies of those three SARS-CoV-2 genes in hPSC-CMs. 152 Firstly, three human ES and iPS cell lines overexpressing M, Nsp8 and Nsp6, respectively, 153 were established by using lentivirus, followed with differentiation into contracting CMs for 154 further assessments ( Figure 1C ). This strategy allows uniformly high gene expression in 155 hPSC-CMs compared with the AAV9-mediated gene delivery into differentiated hPSC- 156 CMs. Since SARS-CoV-2 virus infects human cells through binding with ACE2, 8 TMPRSS2(10,11) or TMPRSS4(12), we profiled their expression dynamics during CM 158 differentiation from WT hESCs using qRT-PCR ( Figure 1D ). Cardiac troponin T (CTNT) 159 is a marker of CMs ( Figure 1D ). ACE2 was lowly expressed in hESCs, but rapidly 160 increased in hESCs-derived CMs (hESC-CMs). TMPRSS2 expression level dropped 161 during CM differentiation ( Figure 1D ), whereas TMPRSS4 expression exhibited a 162 fluctuant patterning ( Figure 1D ). Our single cell RNA-seq data from hESC-CMs (24) 163 confirmed the relatively higher expression level of ACE2 than those of TMPRSS2 and 164 TMPRSS4 in hESC-CMs ( Figure 1E ). Similar as hESC-CMs, adult human heart tissues Figure 1D ) were differentiated into CMs, followed with a published 174 protocol (25) to enrich CMs till over 90% purity by adding lactate medium. Empty lenti-175 vector infected hPSCs were used as control. Next, whole mRNA-seq was performed to 176 profile differentially expressed genes ( Figure 2B ). We found Nsp6 OE , Nsp8 OE The whole mRNA-seq results indicated that overexpression of SARS-CoV-2 viral genes 210 could increase expressions of cell death/apoptosis associated genes in hESC-CMs. Therefore, we next assessed apoptosis of hESC-CMs. Firstly, Nsp6 OE , Nsp8 OE , M OE and 212 control hESCs were differentiated into CMs using a 2D monolayer differentiation 213 method(21,24) ( Figure 4A ). After 12 days' CM differentiation, flow cytometry was 214 conducted to detect the ratios of TUNEL + cells in the CTNT + hESC-CMs. We found 215 significantly increased ratios of TUNEL + cells in the Nsp6 OE , Nsp8 OE -log ( p value) intracellular transport b iosy nthetic process cellular metab olic process response to interleuk in-4 vesicle-mediated transport viral process response to unfolded protein response to cy tok ine immune effector process neutrophil mediated immunity vesicle-mediated transport immune sy stem process positive regulation of translation cellular b iosy nthetic process immune effector process cellular metab olic process leuk ocy te mediated immunity ATP metab olic process metab olic process cell activation in immune response viral process mitochondrial transmemb rane transport ATP b iosy nthetic process C D The treatment time for apoptosis assay was 2 days. All cytokines were from R&D Systems. All chemicals or drugs were from Sigma Aldrich. The lentiviral vector pLVX- The peptide mixture was resuspended in 0.1% TFA (trifluoroacetic acid) and fractionated on a PierceTM High pH reversed-phase peptide fractionation spin column using vendor methodology (cat. num. 84868). Each fraction was dried by speed vacuum and resuspended in 30 µL 0.1% FA. Nano Approximately 30-40M reads per library is generated. A Phred quality score (Q score) is used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 (99.9% base call accuracy). Quality control for raw mRNA-seq data was generated by FastQC v0.11.5 annotation. Analysis of differential expression genes (DEGs) was performed by edgeR v3.32.1 10 , with read counts normalized by the trimmed mean of M-values (TMM) method after lowly expressed genes filtered out by the filterByExpr function using default settings. DEGs due to overexpression of SARS-CoV-2 viral coding genes were identified if their FDR-adjusted p-values were less than 0.05 based on the comparison between the viral gene overexpression and the control. The functional enrichment analysis of gene ontology (GO) biological process was carried out by using DAVID (https://david.ncifcrf.gov/), THE GENE ONTOLOGY RESOURCE (http://geneontology.org/) and Gorilla (http://cbl-gorilla.cs.technion.ac.il/). Canonical signaling pathway and toxicity analysis were performed on QIAGEN Ingenuity Pathway Analysis (IPA). Protein-Protein Interaction Networks analysis were carried out on STRING software (https://string-db.org/). Data comparisons between two groups (gene overexpression versus control) were conducted using an unpaired two-tailed t-test. All data were presented as mean ± S.D. from at least three independent experiments. Differences with P values less than 0.05 were considered significant. 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