key: cord-0974328-m9vo1zoq
authors: Marchiano, Silvia; Hsiang, Tien-Ying; Higashi, Ty; Khanna, Akshita; Reinecke, Hans; Yang, Xiulan; Pabon, Lil; Sniadecki, Nathan J.; Bertero, Alessandro; Gale, Michael; Murry, Charles E.
title: SARS-CoV-2 infects human pluripotent stem cell-derived cardiomyocytes, impairing electrical and mechanical function
date: 2020-08-30
journal: bioRxiv
DOI: 10.1101/2020.08.30.274464
sha: 33fcc44038764a4f8543ecaa1f4bba0d832cc7da
doc_id: 974328
cord_uid: m9vo1zoq

Global health has been threatened by the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2)1. Although considered primarily a respiratory infection, many COVID-19 patients also suffer severe cardiovascular disease2–4. Improving patient care critically relies on understanding if cardiovascular pathology is caused directly by viral infection of cardiac cells or indirectly via systemic inflammation and/or coagulation abnormalities3,5–9. Here we examine the cardiac tropism of SARS-CoV-2 using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and three-dimensional engineered heart tissues (3D-EHTs). We observe that hPSC-CMs express the viral receptor ACE2 and other viral processing factors, and that SARS-CoV-2 readily infects and replicates within hPSC-CMs, resulting in rapid cell death. Moreover, infected hPSC-CMs show a progressive impairment in both electrophysiological and contractile properties. Thus, COVID-19-related cardiac symptoms likely result from a direct cardiotoxic effect of SARS-CoV-2. Long-term cardiac complications might be possible sequelae in patients who recover from this illness.

Global health has been threatened by the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) 1 . Although considered primarily a respiratory infection, many COVID-19 patients also suffer severe cardiovascular disease [2] [3] [4] . Improving patient care critically relies on understanding if cardiovascular pathology is caused directly by viral infection of cardiac cells or indirectly via systemic inflammation and/or coagulation abnormalities 3, [5] [6] [7] [8] [9] . Here we examine the cardiac tropism of SARS-CoV-2 using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and three-dimensional engineered heart tissues (3D-EHTs). We observe that hPSC-CMs express the viral receptor ACE2 and other viral processing factors, and that SARS-CoV-2 readily infects and replicates within hPSC-CMs, resulting in rapid cell death. Moreover, infected hPSC-CMs show a progressive impairment in both electrophysiological and contractile properties. Thus, COVID-19-related cardiac symptoms likely result from a direct cardiotoxic effect of SARS-CoV-2. Long-term cardiac complications might be possible sequelae in patients who recover from this illness.

With over 20 million people affected worldwide, the outbreak of SARS-CoV-2 has already left its permanent mark on human history 1, 10 . Cardiovascular complications, including worsening of preexisting conditions and onset of new disorders, significantly contribute to the increasing mortality of COVID-19 patients, but the underlying mechanisms of cardiopathology are unclear 2, [11] [12] [13] . Upon lung infection, the uncontrolled release of inflammatory cytokines, termed "cytokine storm", could induce multi-organ damage, ultimately leading to organ failure and worsening of pre-existing cardiovascular disorders 5, 6, 8, 14 . Moreover, COVID-19 is associated with coagulopathies, which also can induce heart damage 3, 5, 7 . Lastly, SARS-CoV-2 could directly mediate heart injury by entering cardiomyocytes via binding of the viral spike glycoprotein to its extracellular receptor, angiotensin I converting enzyme 2 (ACE2) 9, 15, 16 . An increasing number of reports have showed presence of SARS-CoV-2 genome in the heart and signs of viral myocarditis in COVID-19 infected individuals, including asymptomatic cases, indicating that SARS-CoV-2 could exhibit cardiac tropism and thus directly impair cardiac function [17] [18] [19] [20] . Myocardial infarction, arrhythmias, and heart failure are the most common cardiovascular complications observed in COVID-19 patients 12, 21, 22 .

In this study we used human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and engineered human heart tissues as platforms to study SARS-CoV-2 cardiac biology 23, 24 , to clarify the functional changes behind these COVID-19-related cardiovascular symptoms.

The susceptibility to SARS-CoV-2 infection depends on the expression of the viral receptor ACE2 9,15,16 . We found that ACE2 is transcriptionally upregulated during cardiac differentiation of both RUES2 embryonic stem cells-derived cardiomyocytes (hESC-CMs; Fig. 1a ) and WTC11c induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs; Supplementary Fig. 1a ).

Single-cell RNA-sequencing analysis detected ACE2 mRNA in ~9% of hESC-CMs, indicating low and/or transitory transcriptional activation (Fig. 1b) . A larger fraction of cells expressed moderate to high levels of endosomal cysteine proteases CTSB (cathepsin B; ~71.0%) and CTSL (cathepsin L; ~46.0%). Detection of these factors is relevant because they can cleave the spike glycoprotein and lead to endomembrane fusion-mediated release of SARS-CoV-2 genome inside the cytoplasm 15, 25, 26 . Importantly, these viral processing factors were often co-expressed with ACE2 ( Supplementary Fig. 1b) . Although viral entry also can be mediated by TMPRSS2 15 , this transmembrane serine protease was not detectable in hESC-CMs ( Supplementary Fig. 1c) , as also reported for the adult human heart 27 . Thus, the ACE2-endosomal viral entry pathway may be the only route for virus entry in cardiomyocytes. Indeed, PIKFYVE, another endosomal viral processing factor, was also broadly expressed in hESC-CMs ( Supplementary Fig. 1c) 26 . Despite the low levels of mRNA, ACE2 protein was strongly expressed in hPSC-CMs derived from multiple lines (RUES2 female hESCs, H7 female hESCs, and WTC11c male hiPSCs), reaching levels comparable to those of VERO cells, a primate kidney epithelial line with established SARS-CoV-2 tropism (Fig. 1c) 15 . Collectively, we concluded that hPSC-CMs express proteins that render them susceptible to SARS-CoV-2 infection 23, 24 .

Since H7 and WTC11c-derived hPSC-CMs showed the highest levels of ACE2, we tested their functional susceptibility to SARS-CoV-2. For this, we incubated highly-pure hPSC-CMs (> 80%; To investigate if hPSC-CMs are permissive to SARS-CoV-2 replication, we quantified extracellular viral particles and intracellular viral RNA (by plaque assay and RT-qPCR, respectively). The onestep growth curve after 5 MOI infection indicated that significant viral replication occurred steadily from 24-72 HPI, followed by a precipitous decline as the cells died (Fig. 2c) . With a multi-step Overall, abnormalities in the generation and propagation of electrical signals were significant even in the absence of substantial cell death, suggesting that SARS-CoV-2 infection in cardiomyocytes could directly create a substrate for arrhythmias. These properties of SARS-CoV-2 infection in cardiomyocytes could explain the high rate of arrhythmia (~14%) which has been observed in COVID-19 patients 5 .

We then evaluated the contractile properties of hPSC-CMs using three-dimensional engineered heart tissues (3D-EHTs), following their contractile behavior through magnetic field sensing 31 (Figs. 4a-b). For these experiments we focused on WTC11c hiPSCs since 3D-EHTs from H7 hESC-CMs proved to spontaneously beat at too high a frequency (> 2 Hz) to enable accurate measurements of contractile behavior (i.e. the tissue had a tetanic-like contraction with minimal relaxation between beats at this frequency). We infected 3D-EHTs from WTC11c hiPSC-CMs with 10 MOI (to facilitate infection within the non-vascularized, cell-dense tissue), and analyzed their contraction for a week. The maximal twitch force decreased as early as 72 HPI, and the contractions continued to subside to < 25% of original force at 144 HPI (Figs. 4c-d, and

Supplementary Videos 1 and 2). Overall, the significant impairment in the contractile properties of 3D-EHTs demonstrates that the mechanical function of cardiomyocytes is impacted by SARS-CoV-2 infection, and this could contribute to whole-organ cardiac dysfunction in patients 32 .

A rapidly increasing number of reports acknowledge cardiovascular involvement as a prevalent complication observed in COVID-19 patients, but discriminating between direct versus indirect effects is still an open challenge 12, 14, 18, 19 . Our results provide important evidence that SARS-CoV-2 has the ability to directly infect cardiomyocytes, to impair both their electrophysiological and contractile properties, and eventually to induce cell death. These results support the hypothesis that, independent of inflammation or coagulopathy, SARS-CoV-2 can cause direct functional heart damage.

One limitation of this study is our reliance on hPSC-CMs, which are well-known for their functional immaturity 33 . Moreover, while the in vitro systems we used have been successfully leveraged to model electrophysiological and contractile alterations due to drugs or inherited mutations 34, 35 , their application to modeling COVID-19 will require further validation. A recent report by Dolhnikoff et al. identified coronaviral particles in the cytoplasm of cardiomyocytes, endothelial cells, and fibroblasts by electron microscopy in the heart of an 11 year-old child who died from multi-system inflammatory syndrome in children (MIS-C) following COVID-19 infection 20 . This indicates that in vivo cardiomyocytes with substantially greater maturity than used here are susceptible to SARS-CoV-2 infection. COVID-19 patients are commonly treated with steroids to control systemic inflammation. However, our data suggests that treatments aimed to control the direct damage of SARS-CoV-2, e.g. antiviral medications and/or cardioactive drugs, should also be taken into consideration to prevent long-term cardiovascular complications.

Cell culture. Undifferentiated RUES2 hESCs (RUESe002-A; WiCell) and WTC11c hiPSCs (a gift of Dr. Bruce Conklin, Gladstone Institutes, San Francisco) were maintained in mTeSR1 (Stemcell Technologies) on tissue culture dishes coated with Matrigel (Corning) at 0.17 mg/mL, and passaged as small clumps using Versene (Gibco). Cardiomyocytes from RUES2 and WTC11c were differentiated as previously described 36 Following washes with DPBS with 5% FBS, samples were run on a BD FACSCanto II flow cytometer and data from 10,000 valid events were acquired with the BD FACSDIVA software.

Analysis was performed with FlowJo v10.7.

Bulk RNA-seq datasets from differentiating RUES2 hESC-CMs had been previously generated and analyzed 36 Differences versus non infected (mock) control by two-way ANOVA followed by Sidak correction for multiple comparisons (* = p <0.05; ** = p < 0.01). 

Author contributions

MEA and 3D-EHTs assays, and wrote the first draft of the manuscript. T.Y.H. cultured and expanded SARS-CoV-2 and performed immunofluorescence. T.H. designed and fabricated the 3D-EHTs magnetic sensing system, and analyzed the data. A.K. performed and analyzed RT-qPCR. H.R. contributed to experimental design and differentiated hiPSC-CMs. X.Y. contributed to experimental design and analyzed sc-RNA-seq data. L.P. contributed to experimental design and supervised the experiments. A.B. contributed to the study's conception, experimental design and execution, and assisted in writing the manuscript

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Scale bars: 50µm (b) Representative recordings of spontaneous electrical activity of SARS-CoV-2-infected H7 hESC-CMs at 72 HPI. (c) Representative quantifications of electrophysiological properties from MEA analyses in SARS-CoV-2-infected H7 hESC-CMs. Mean ± SEM of 8 wells. Differences versus mock control by two-way ANOVA with Sidak correction for multiple comparisons (*** = p < 0.001). (d) Representative field potential traces in SARS-CoV-2-infected H7 hESC-CMs at 72 HPI, and quantification of field potential duration corrected by beat rate (FPDc) in spontaneous and paced experiments. Mean ± SEM of 8 and 6 wells for spontaneously beating and paced cells, respectively

We thank Drs. Farid Moussavi-Harami, Kenta Nakamura, and Daniel Yang for helpful discussions during the development of this project, and Dr. Aidan Fenix for experimental support. We are grateful to Axion Biosystem for proving material and assistance for the MEA experiments. 

The authors declare that there is no conflict of interest.