SARS-CoV-2 Infects Human Engineered Heart Tissues and Models COVID-19 Myocarditis

Abstract

There is ongoing debate as to whether cardiac complications of coronavirus disease-2019 (COVID-19) result from myocardial viral infection or are secondary to systemic inflammation and/or thrombosis. We provide evidence that cardiomyocytes are infected in patients with COVID-19 myocarditis and are susceptible to severe acute respiratory syndrome coronavirus 2. We establish an engineered heart tissue model of COVID-19 myocardial pathology, define mechanisms of viral pathogenesis, and demonstrate that cardiomyocyte severe acute respiratory syndrome coronavirus 2 infection results in contractile deficits, cytokine production, sarcomere disassembly, and cell death. These findings implicate direct infection of cardiomyocytes in the pathogenesis of COVID-19 myocardial pathology and provides a model system to study this emerging disease.

Keywords: ACE2, angiotensin converting enzyme 2; COVID-19, coronavirus disease-2019; EHT, engineered heart tissues; LV, left ventricle; SARS-CoV-2, severe acute respiratory syndrome-coronavirus-2; cardiomyocyte; coronavirus disease 2019; engineered heart tissue; hPSC, human pluripotent stem cell(s); myocarditis; severe acute respiratory syndrome coronavirus 2.

Graphical abstract

Figure 1

Specimens From Patients With Severe COVID-19 Myocarditis Show Evidence of SARS-CoV-2 Cardiomyocyte Infection (A) Hematoxylin and eosin staining of cardiac autopsy (anterior left ventricular wall) and biopsy samples (right ventricular septum) from subjects without coronavirus disease-2019 (COVID-19) (control case) and patients with severe COVID-19 myocarditis (case 1-4). (B) In situ hybridization for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) spike and nucleocapsid RNA (red). Hematoxylin: blue. The arrows denote viral RNA staining in cells with cardiomyocyte morphology. (C) Immunostaining of control and COVID-19 myocarditis cardiac autopsy tissue for SARS-CoV-2 nucleocapsid (white) and cardiac actin (red). DAPI: blue. The arrows denote nucleocapsid staining in cardiomyocytes. (D) Immunostaining of control and COVID-19 myocarditis specimens for CD68 (green) and CCR2 (red). DAPI: blue. (E) Immunostaining of control and COVID-19 myocarditis tissue for CD3 (brown). Hematoxylin: blue. DAPI = 4′,6-diamidino-2-phenylindole.

Figure 2

SARS-CoV-2 Infects Cardiomyocytes (A) Focus-forming assay measuring production of infectious virus from human pluripotent stem cell (hPSC)–derived cardiomyocytes (CM), fibroblasts (Fb), and macrophages (Mac) inoculated with SARS-CoV-2 (multiplicity of infection [MOI], 0.1). Media only denotes wells that contain no cells. Assays were performed 3 days following inoculation. Dashed line indicates limit of assay detection. (B) Quantitative real-time polymerase chain reaction (RT-PCR) showing viral N-gene copies in cultures containing CM, Fb, and Macs inoculated with SARS-CoV-2 (MOI 0.1). RNA was collected 3 days post-inoculation (n = 5 per group). (C) Focus-forming assay measuring infectious SARS-CoV-2 (black line indicates wild-type; green line indicates NeonGreen) in supernatant of hPSC-derived cardiomyocytes over time following inoculation (MOI 0.1). A dashed line indicates the limit of detection (n = 4 per group). (D) Two-dimensional cultures of hPSC-derived cardiomyocytes were inoculated with SARS-CoV-2 (MOI 0.1) and analyzed for viability (Zombie-Violet) and infection (NeonGreen) as a function of time by flow cytometry. Right plot shows viability of NeonGreen-positive cells (n = 4 per group). (A–D) Mean values are plotted and error bars denote standard error of the mean. (E) Brightfield microscopy showing cytopathic effect in hPSC-derived cardiomyocytes infected with SARS-CoV-2 (MOI 0.1). Representative images from 5 individual samples. (F) Flow cytometry of 2-dimensional tissues containing CM and Fb (left) or CM, Fb, and Mac (right) harvested on day 3 following mock infection or inoculation with SARS-CoV-2 (MOI 0.1). Representative plot from 4 independent samples. Cardiomyocytes (CD90-CD14-) showed prominent NeonGreen fluorescence (green overlay). NeonGreen signal was not detected in fibroblasts (CD90+CD14-) or macrophages (CD90-CD14+). (G) Quantification of NeonGreen-positive cells from 2-dimensional tissues containing hPSC-cardiomyocytes and fibroblasts or hPSC-cardiomyocytes, fibroblasts, and macrophages (n = 4 per group). Bars denotes median value. ∗p < 0.05 compared to mock infection (Mann-Whitney test). (H) Transmission electron microscopy micrographs of cardiomyocytes in 2-dimensional tissues infected with either mock or SARS-CoV-2 (MOI 0.1). Tissues were harvested on day 3 post-inoculation. Viral budding (blue arrow) and endosomal compartments filled with virions (black arrow) are denoted. Scale bars in insets are 100 nm. Representative image from 4 independent samples. FFU = focus forming units; other abbreviation as in Figure 1.

Figure 3

RNA Sequencing Identified Viral Transcription and Activation of Innate Immune Response in hPSC-Derived Cardiomyocytes and Tissues (A) MDS plot of RNA sequencing data obtained from mock and SARS-CoV-2–infected (MOI 0.1) hPSC-derived CMs, Fbs, Macs, and 2-dimensional tissues (CM + Fb + Mac). Cells and tissues were harvested on day 3 post-inoculation (n = 5 per group). (B) Heatmap of SARS-CoV-2 viral gene expression. Color scale denotes absolute expression as log2 counts per million reads (CPM) (scale: blue = 0, red = 15). (C) Volcano plots showing differentially expressed genes between mock and SARS-CoV-2–infected conditions. Black dots indicate no significant change, red dots indicate upregulated during infection (log2 fold change>2, FDR p < 0.05), and blue dots indicate downregulated during infection (log2 fold change<2, FDR p < 0.05). Data points correspond to individual transcripts. (D) Venn diagram of genes upregulated and downregulated in each cell type and tissues. Differential expression is based on change relative to corresponding uninfected (mock) samples. (E to H) Gene ontology (GO) pathway analysis of CM (E), CM + Fb + Mac (F), Mac (G) and Fib (H) showing top 5 upregulated (red) and downregulated (blue) pathways in SARS-CoV-2–infected samples compared to mock. Color indicates log2 fold change (log2FC). (I) Heat maps of selected differentially expressed genes implicated in metabolism (left), contractile apparatus (center), and immune response (right). CM and tissues (CM + Mac + Fb) are displayed. Color scale denotes relative gene expression (high red, low blue) across cell types and conditions. ATP = adenosine triphosphate; FDR = false discovery rate; IL = interleukin. other abbreviations as in Figures 1 and 2.

Figure 4

EHTs Recapitulate Aspects of COVID-19 Myocarditis (A) Hematoxylin and eosin (H&E)–stained sections of 3-dimensional engineered heart tissue (EHT) consisting of hPSC-derived CM, Fbs, and Macs 5 days following mock infection or inoculation with SARS-CoV-2 (MOI 0.1). Insets are high-magnification images of the boxed areas. Representative images from 4 independent samples. (B) Immunostaining of mock or SARS-CoV-2–infected 3-dimensional EHTs for sarcomeric actin (cardiomyocytes, red), CD68 (macrophages, green), and nucleocapsid protein (white). EHTs were harvested 5 days post-inoculation. Blue: DAPI. Representative images from 4 independent samples. (C) Quantitative RT-PCR of SARS-CoV-2 N-gene expression in EHTs consisting of hPSC-derived CM, Fb, and/or Macs. EHTs were either mock infected or inoculated with SARS-CoV-2 (MOI 0.1) and harvested 5 days post-inoculation. Error bars denote SE of the mean. Dotted line: limit of detection. ∗p < 0.05 compared to uninfected control (mock, Student t test test). (D) In situ hybridization for SARS-CoV-2 spike RNA sense and antisense strands (red) in EHTs 5 days after mock or SARS-CoV-2 infection (MOI 0.1). Blue: hematoxylin. Representative images from 4 independent specimens. Insets are high magnification images of the boxed areas. (E) Spontaneous beating displacement traces for infected and uninfected EHTs on day 5 post-infection. (F) Displacement (relative to uninfected mock condition) generated by spontaneous beating of EHTs as a function of time following inoculation with SARS-CoV-2 (MOI 0.1). Each data point represents a mean value from 4 to 7 independent samples, error bars denote SE of the mean. (G) Quantification of absolute displacement (left) and contraction speed (right) generated by spontaneous beating of EHTs 5 days following mock or SARS-CoV-2 infection (MOI 0.1). Error bars represent SE of the mean; ∗p < 0.05 compared to mock (Student’s t-test test). Abbreviations as in Figures 1 and 2.

Figure 5

Mechanisms of Reduced EHT Contractility (A) Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) stain (red) and cardiac actin (green) immunostaining of EHTs (CM +Fb + Mac) 5 days after mock or SARS-CoV-2 infection (MOI 0.1). Blue: DAPI. Representative images from 4 independent experiments. (B) Quantification of TUNEL-positive cells in areas of viral infection. Error bars represent SE of the mean, ∗p < 0.05 compared to mock (Student t test). (C) Immunostaining of hPSC-derived cardiomyocytes for troponin T (red) 3 days after inoculation with mock control or SARS-CoV-2–NeonGreen (MOI 0.1). Blue: DAPI. Arrows denote areas of sarcomere disassembly. (D) Immunostaining of EHTs for troponin T (red) and SARS-CoV-2 nucleocapsid (green) 5 days after inoculation with mock control or SARS-CoV-2–NeonGreen (MOI 0.1). Blue: DAPI. Arrows denote SARS-CoV-2 nucleocapsid–positive cells with reduced troponin T staining. (E) Quantification of troponin T staining in mock (white) and SARS-CoV-2 infected (red) EHTs. Data is presented as mean florescence intensity (MFI). MFI was measured in infected (nucleocapsid-positive [NP+]) cardiomyocytes and uninfected (NP-) cardiomyocytes located proximal or remote to areas of infection. Error bars represent SE of the mean, ∗p < 0.05 compared to mock (Student t test). (F) Quantitative RT-PCR measuring OAS1, MX1, and tumor necrosis factor (TNF) mRNA expression in hPSC-derived cardiomyocytes 3 days post-inoculation with mock control (white) or SARS-CoV-2 (green, MOI 0.1). Cells were treated with vehicle, angiotensin-converting enzyme 2 antibody (ACE2 Ab) (20 μg/ml), remdesivir (10 μM), or TBK inhibitor (MRT67307, 10μM). Error bars indicate SE of the mean. ∗p < 0.05 compared to mock control (Student t test). (G) Quantitative RT-PCR of SARS-CoV-2 N gene expression in hPSC-derived cardiomyocytes that were either mock infected (white) or inoculated with SARS-CoV-2 (green, MOI 0.1) and harvested 3 days post-inoculation. Error bars denote SE of the mean. Dotted line: limit of detection. ∗p < 0.05 compared to uninfected control (analysis of variance [ANOVA]) or vehicle infected as indicated by the bar (ANOVA). (H,I) Flow cytometry measuring the percent of infected (H) and viable (I) cardiomyocytes following either mock infection (white) or inoculation with SARS-CoV-2 (green, MOI 0.1). Cells were harvested and analyzed 3 days post-inoculation. Error bars denote SE of the mean. ∗p < 0.05 compared to uninfected control (ANOVA). (J) Quantitative RT-PCR measuring TNNT2 mRNA expression 3 days post-inoculation with mock control (white) or SARS-CoV-2 (green, MOI 0.1). Error bars indicate SE of the mean. ∗p < 0.05 compared to mock control (ANOVA). (K) Immunostaining for troponin T (red) 3 days post-inoculation with mock control or SARS-CoV-2–NeonGreen (MOI 0.1). Blue: DAPI. Arrows denote areas of sarcomere disassembly. Abbreviations as in Figures 1 and 2.