key: cord-1003098-fp5uxxmh authors: Bojkova, Denisa; Wagner, Julian U G; Shumliakivska, Mariana; Aslan, Galip S; Saleem, Umber; Hansen, Arne; Luxán, Guillermo; Günther, Stefan; Pham, Minh Duc; Krishnan, Jaya; Harter, Patrick N; Ermel, Utz H; Frangakis, Achilleas S; Milting, Hendrik; Zeiher, Andreas M; Klingel, Karin; Cinatl, Jindrich; Dendorfer, Andreas; Eschenhagen, Thomas; Tschöpe, Carsten; Ciesek, Sandra; Dimmeler, Stefanie title: SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes date: 2020-09-23 journal: Cardiovasc Res DOI: 10.1093/cvr/cvaa267 sha: 2de8a647b748c7c3792bb98d9eb7b7a5b6dcb8ec doc_id: 1003098 cord_uid: fp5uxxmh AIMS: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as a global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality. It is unclear whether cardiac injury is caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here, we investigate whether cardiomyocytes are permissive for SARS-CoV-2 infection. METHODS AND RESULTS: Two strains of SARS-CoV-2 infected human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) as demonstrated by detection of intracellular double-stranded viral RNA and viral spike glycoprotein expression. Increasing concentrations of viral RNA are detected in supernatants of infected cardiomyocytes, which induced infections in Caco-2 cell lines, documenting productive infections. SARS-COV-2 infection and induced cytotoxic and proapoptotic effects associated with it abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signalling, apoptosis, and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a 3D cardiosphere tissue model. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Coronavirus particles were further observed in cardiomyocytes of a patient with COVID-19. Infection of iPS-CMs was dependent on cathepsins and angiotensin-converting enzyme 2 (ACE2), and was blocked by remdesivir. CONCLUSIONS: This study demonstrates that SARS-CoV-2 infects cardiomyocytes in vitro in an ACE2- and cathepsin-dependent manner. SARS-CoV-2 infection of cardiomyocytes is inhibited by the antiviral drug remdesivir. TRANSLATIONAL PERSPECTIVE: Although this study cannot address whether cardiac injury and dysfunction in COVID-19 patients is caused by direct infection of cardiomyocytes, the demonstration of direct cardiotoxicity in cardiomyocytes, organ mimics, human heart slices and human hearts warrants the further monitoring of cardiotoxic effects in COVID-19 patients. The coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as global pandemic. SARS-CoV-2 is an enveloped and single-stranded RNA virus type, which mainly invades alveolar epithelial cells and causes adult respiratory distress syndromes. COVID-19 is associated with myocardial injury, as assessed by increased troponin T and NT-proBNP levels accompanying increased cardiovascular symptoms in a significant number of SARS-CoV-2 infected patients [1] [2] [3] . Recent studies further demonstrate significantly reduced ejection fraction, higher left ventricular mass and raised native T1 and T2 assessed by magnetic resonance imaging in patients recovered from severe COVID-19 4 . Elevated levels of cardiac injury markers were associated with higher risk of in-hospital mortality in COVID-19 patients 5 . In patients showing clinical deterioration during COVID-19, left ventricular systolic dysfunction was noted in approximately 20 % of patients according to a most recent study 6 . In addition, patients with underlying cardiovascular disease represent a significant proportion of patients, who may suffer from severe courses after COVID-19 infections 7 . However, it is unclear whether elevated biomarkers of cardiac injury and long term effects on the cardiovascular system are directly caused by viral infection of cardiac tissue or are secondary to hypoxia and systemic inflammation during complicated COVID-19 courses. Earlier studies with cardiac tissue samples revealed mixed results. While one study did not find evidence for viral particles of the first SARS corona virus SARS-CoV 8 , SARS-CoV RNA was identified in 35 % of autopsied human heart samples obtained from patients who succumbed to the SARS crisis during the Toronto SARS outbreak 9 . Other studies suggest that the Middle East respiratory syndrome-related coronavirus (MERS-CoV), which has similar pathogenicity as SARS-CoV-2, can cause acute myocarditis and heart failure 10 . Moreover, substantial amounts of viral SARS-CoV-2 RNA was detected in human hearts of COVID-19 patients [11] [12] [13] [14] Although virus particles were identified in interstitial cells in myocardium of one patient 15 , direct infection in cardiomyocytes of COVID-19 patients has not been described yet. Single cell RNA sequencing and histological analyses demonstrated that human cardiomyocytes express the putative SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2), particularly in patients with cardiovascular diseases 16, 17 suggesting that cardiomyocytes could be targeted by SARS-CoV-2. Therefore, we investigated whether SARS-CoV-2 infects human induced pluripotent stem cell-derived cardiomyocytes in culture and in two models of human cardiac tissue including human heart slices in vitro. MS # CVR-2020-1261 4 The use of all human cells and tissues was approved by the institutional ethics review boards and complies with the Declaration of Helsinki. All subjects gave informed written consent. Cell Culture hiPS-CM of two donors were obtained with an embryoid body-based protocol as described 18 . Cardiospheres were generated by adapting a previously described protocol 19 using hiPS cells. Living human heart slices (300 µm) were generated and cultured as described 9 . Viral infection SARS-CoV-2-FFM1 and FFM2 were isolated and propagated in Caco-2 cells as described 20, 21 . The viral stock was diluted to desired MOI in medium containing 1% fetal bovine serum and incubated with cells for 2 h. Then the infectious inoculum was removed and cells were supplemented with the respective culture medium 18 . Cardiospheres were cultured with 25µl of viral stock (1.10 7 TCID50/ml) and living human heart slices were incubated with 200µl of viral stock (1.10 7 TCID50/ml) for three to five days. Quantification of SARS-CoV RNA in cell culture supernatants was performed as previously described 21 . For detection of viral titer, hiPS-CM were infected for 2 h, the infection medium was replaced, and supernatants were collected 48h post infection and used to infect confluent layers of CaCo-2 cells in 96-well plates. Cytopathogenic effects were assessed visually 48 h after infection. The infectious titer was determined as TCID50/ml. For further details, see Online Data Supplemental "Expanded Methods". We first addressed if human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) showed the expression of the SARS-CoV-2 receptors ACE2 and the serine proteases TMPRSS2 and cathepsins, which mediate priming of the viral S-protein 22 . ACE2 was well expressed on mRNA level in hiPS-CM but not in undifferentiated iPS cells (Figure 1a , Online Supplement Figure Ia-d) . The cathepsins CTSB and CTSL were highly expressed, whereas TMPRSS2 was detected only at very low levels by RNA sequencing (Figure 1a) . Quantitative RT-PCR confirmed the expression of ACE2, but TMPRSS2 was below the detection level (Figure 1b Next, we determined if SARS-CoV-2 infects cardiomyocytes in a three dimensional tissue environment using human cardiospheres generated by hiPS-cells, which are generated by a modified previously published protocol 19 (Figure 3a Finally, we addressed whether SARS-CoV-2 infects human heart tissue by using living human cardiac tissue slices, which were obtained from explanted hearts 24 (Figure 4a Having demonstrated that SARS-CoV-2 can infect human cardiomyocytes, we tested strategies to interfere with viral infection. First, we determined if interfering with ACE2, which was shown to block virus infection of organoids 25 , also is effective in cardiomyocytes. Indeed, recombinant ACE2 or neutralizing antibodies blocked spike protein expression (Figure 5a) . Since cardiomyocytes essentially lack TMPRSS2 but express cathepsins, we additionally tested the effect of the protease inhibitor N-Acetyl-L-leucyl-L-leucyl-Lmethional (ALLM), which preferentially blocks cathepsins 26 . Indeed, inhibition of cathepsins reduced spike protein expression (Figure 5b) . Moreover, the viral RNA-dependent RNA polymerase inhibitor remdesivir inhibited spike protein expression (Figure 5c ). The study has been supported by the German Center for Cardiovascular Research (DZHK) and the Excellence Strategy Program of the DFG (Exc 2026). KK was supported by the German Heart Foundation. SC was supported by the Pfizer Foundation and MPP and JK by the European Innovation Council (822455). The authors have nothing to disclose The data and analytic methods will be made upon request to other researchers for purposes of reproducing the results or replicating the procedure. The study materials will only be made available if sufficient material can be provided to other researchers for purposes of reproducing the results or replicating the procedure. RNA copies / mL (log 10 ) SARS-CoV-2: a potential novel etiology of fulminant myocarditis COVID-19 and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options Outcomes of Cardiovascular Magnetic Resonance Imaging in Patients Recently Recovered From Coronavirus Disease 2019 (COVID-19) Association of Cardiac Injury With Mortality in Hospitalized Patients With COVID-19 in Wuhan The Spectrum of Cardiac Manifestations in Coronavirus Disease 2019 (COVID-19) -a Systematic Echocardiographic Study Characteristics and Outcomes of 21 Critically Ill Patients With COVID-19 in Washington State Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in-situ hybridization study of fatal cases SARScoronavirus modulation of myocardial ACE2 expression and inflammation in patients with SARS Acute myocarditis associated with novel Middle east respiratory syndrome coronavirus Multiorgan and Renal Tropism of SARS-CoV-2 Evidence of SARS-CoV-2 mRNA in endomyocardial biopsies of patients with clinically suspected myocarditis tested negative for COVID-19 in nasopharyngeal swab Association of Cardiac Infection With SARS-CoV-2 in Confirmed COVID-19 Autopsy Cases Detection of viral SARS-CoV-2 genomes and histopathological changes in endomyocardial biopsies Myocardial localization of coronavirus in COVID-19 cardiogenic shock Cell type-specific expression of the putative SARS-CoV-2 receptor ACE2 in human hearts The ACE2 expression in human heart indicates new potential mechanism of heart injury among patients infected with SARS-CoV-2 Differentiation of cardiomyocytes and generation of human engineered heart tissue Dissection of heterocellular cross-talk in vascularized cardiac tissue mimetics Evidence of SARS-CoV-2 Infection in Returning Travelers from Wuhan SARS-CoV-2 infected host cell proteomics reveal potential therapy targets SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Treatment of SARS with human interferons Long-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2 Inhibitory effect of di-and tripeptidyl aldehydes on calpains and cathepsins Human iPSC-Derived Cardiomyocytes are Susceptible to SARS-CoV-2 Infection What is the role of ER stress in the heart? Introduction and series overview Endothelial cell infection and endotheliitis in COVID-19 GO:0009615: response to virus GO:0034341: response to IFNγ R-HSA-1169410_ antiviral mechanism by IFN-stimulated genes GO: 0035455: response to IFNa hsa04623: cytosolic DNA-sensing pathway GO: 0035356: response to IFNb GO: 0045088: regulation of innate immune pathway hsa04622: RIG-I-like receptor signaling pathway GO