Human iPSC-Derived Cardiomyocytes Are Susceptible to SARS-CoV-2 Infection

Abstract

Coronavirus disease 2019 (COVID-19) is a pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is defined by respiratory symptoms, but cardiac complications including viral myocarditis are also prevalent. Although ischemic and inflammatory responses caused by COVID-19 can detrimentally affect cardiac function, the direct impact of SARS-CoV-2 infection on human cardiomyocytes is not well understood. Here, we utilize human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model to examine the mechanisms of cardiomyocyte-specific infection by SARS-CoV-2. Microscopy and RNA sequencing demonstrate that SARS-CoV-2 can enter hiPSC-CMs via ACE2. Viral replication and cytopathic effect induce hiPSC-CM apoptosis and cessation of beating after 72 h of infection. SARS-CoV-2 infection activates innate immune response and antiviral clearance gene pathways, while inhibiting metabolic pathways and suppressing ACE2 expression. These studies show that SARS-CoV-2 can infect hiPSC-CMs in vitro, establishing a model for elucidating infection mechanisms and potentially a cardiac-specific antiviral drug screening platform.

Keywords: COVID-19; SARS-CoV-2; cardiology; cardiomyocytes; cardiovascular biology; coronavirus; heart; induced pluripotent stem cells; stem cell; viral myocarditis.

Graphical abstract

Figure 1

SARS-CoV-2 Internalizes and Replicates within hiPSC-CMs In Vitro, Eliciting Cytopathic Effect and Contractility Alterations (A) Human iPSC-CMs exhibit standard sarcomeric markers including cardiac troponin T (cTnT) and α-actinin with DAPI as nuclear counterstain. (B) Immunofluorescence for cTnT and SARS-CoV-2 “spike” protein demonstrates that hiPSC-CMs can be infected by SARS-CoV-2. SARS-CoV-2 spike protein is not present in mock infected cultures. (C) HiPSC-CMs after SARS-CoV-2 infection, but not mock infection, exhibit signs of cellular apoptosis, indicated by morphological changes seen in brightfield (BF) and cleaved caspase-3 (CC3) production. A second SARS-CoV-2 antibody marks a viral-specific double-stranded intermediate RNA (dsRNA). (D) Magnified inset from (B) shows a merged immunofluorescence image for SARS-CoV-2 spike protein, cTnT, and DAPI. Arrows indicate perinuclear accumulation of viral particles and suggest active viral protein translation at perinuclear ribosomes. (E) Magnified inset from (C) shows a merged immunofluorescence image for SARS-CoV-2 dsRNA and DAPI. Arrows indicate dsRNA stain. (F) Quantification of immunofluorescence indicates percentage of total DAPI-positive cells that are positive for spike protein, viral dsRNA, CC3, and dsRNA+CC3 in hiPSC-CMs infected with SARS-CoV-2, compared to mock infection. n = 5–7 images (technical replicates) quantified for each stain for mock and infected conditions. ∗p < 0.05. (G) Quantification of beats per minute in wells containing hiPSC-CMs with mock infection versus wells containing hiPSC-CMs infected with SARS-CoV-2. n = 6 videos (technical replicates) recorded for each condition. See Video S1 for representative video clips. ∗p < 0.05.

Figure 2

SARS-CoV-2 Alters hiPSC-CM Transcriptomic Profiles after Infection (A) Graph of mapped reads during RNA sequencing. In virus-infected hiPSC-CMs, more than 50% of genomic reads mapped to the SARS-CoV-2 genome, suggesting viral genome presence within infected hiPSC-CMs. (B) Principal component analysis (PCA) for 3 mock hiPSC-CM samples and 3 SARS-CoV-2-infected hiPSC-CM samples illustrates transcriptional clustering by condition (mock versus infected). Samples represent biological replicates of mock and infected conditions, respectively. (C) Heatmap of differentially expressed genes shows that samples cluster based on transcriptomic profile and condition (mock versus infected). (D) Volcano plot of gene expression change in virus-infected versus mock samples. Significantly changed gene are defined with adjusted p < 0.01 and absolute value fold change >2. Genes of interest noted include cardiac markers LDB3 and TNNC1, cytokines CXCL2, IL11, IL1B, and IFNB1, and antiviral response gene OAS1. (E) Expression of genes of interest in mock versus infected conditions. TNNT2 and TNNC1 represent cardiac markers, whereas CKMT2 represents mitochondrial enzymes. CXCL2, IL1B, IL11, and OAS3 represent innate immune response and viral clearance genes. ∗p < 0.05. (F) Top: downregulated transcriptional pathways based on Gene Ontology (GO) analysis, visualized using REViGO. Significantly downregulated pathways include mitochondrial transport, oxidative phosphorylation, oxidation-reduction processes, and muscle contraction. Bottom: top 10 most significant GO terms associated with downregulated pathways are related to mitochondrial function. (G) Top: upregulated transcriptional pathways based on GO analysis, visualized using REViGO. Significantly upregulated pathways include response to organic substance, immune system process, and apoptotic process. Bottom: top 10 most significant GO terms associated with upregulated pathways are related to response to organic stimulus.