SARS-CoV-2 variants divergently infect and damage cardiomyocytes in vitro and in vivo

Abstract

Background: COVID-19 can cause cardiac complications and the latter are associated with poor prognosis and increased mortality. SARS-CoV-2 variants differ in their infectivity and pathogenicity, but how they affect cardiomyocytes (CMs) is unclear.

Methods: The effects of SARS-CoV-2 variants were investigated using human induced pluripotent stem cell-derived (hiPSC-) CMs in vitro and Golden Syrian hamsters in vivo.

Results: Different variants exhibited distinct tropism, mechanism of viral entry and pathology in the heart. Omicron BA.2 most efficiently infected and injured CMs in vitro and in vivo, and induced expression changes consistent with increased cardiac dysfunction, compared to other variants tested. Bioinformatics and upstream regulator analyses identified transcription factors and network predicted to control the unique transcriptome of Omicron BA.2 infected CMs. Increased infectivity of Omicron BA.2 is attributed to its ability to infect via endocytosis, independently of TMPRSS2, which is absent in CMs.

Conclusions: In this study, we reveal previously unknown differences in how different SARS-CoV-2 variants affect CMs. Omicron BA.2, which is generally thought to cause mild disease, can damage CMs in vitro and in vivo. Our study highlights the need for further investigations to define the pathogenesis of cardiac complications arising from different SARS-CoV-2 variants.

Keywords: COVID-19; Cardiac infection; Cardiomyocytes; Heart; Omicron; SARS-CoV-2.

Fig. 1

Delta, Omicron BA.1, and BA.2 differentially infect hiPSC-CMs. Human iPSC-CMs were infected with Delta (D), Omicron BA.1 (O-BA.1) or BA.2 (O-BA.2). A The cells were infected at a multiplicity of infection (MOI) of 1 and were immunostained for virally-encoded nucleocapsid protein (NP) at 24 h post infection (hpi). Graph shows the percentage of NP⁺ hiPSC-CM, n = 4. B Fluorescence images of infected hiPSC-CMs with MOI of 1 at 24 hpi, NP in Red, DAPI in blue. C Replication of SARS-CoV-2 was determined by infecting iPSC-CMs at an MOI of 0.1. The viral titres of supernatant collected at 24, 48, and 72 hpi were determined by plaque assay, n = 3. Data are presented as mean ± SEM, and n refer to biological replicates. Statistical significance was calculated using A one-way ANOVA or C two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar = 500 μm

Fig. 2

Omicron BA2 induces more severe damage in hiPSC-CMs. Human iPSC-CMs were infected with Delta (D), Omicron BA.1 (O-BA.1) or BA.2 (O-BA.2) for 48 h at MOI of 1. A Mitochondrial redox activity was measured using the PrestoBlue assay, n = 4. B The percentage of hiPSC-CMs with mitochondrial fragmentation was measured, n = 3. C Fluorescence images of hiPSC-CMs showing MLC2V-eGFP in green, NP in red, DAPI nuclear staining in blue. Representative images of 3 batches of cells are shown. D The percentage of hiPSC-CMs with more than one nuclei, n = 4. E The percentage of hiPSC-CMs with condensed nuclei, n = 3. F Cell number was normalised to that of mock infection control, n = 4. Data are presented as mean ± SEM, and n refer to biological replicates. Statistical significance was calculated using the one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar = 25 μm

Fig. 3

SARS-CoV-2 variants Delta and Omicron BA.2 infection induce cardiac damage in hamsters. Hamsters were inoculated intranasally with 10⁴ PFU SARS-CoV-2 Delta or Omicron BA.2 virus. At 2 days post infection (dpi), the hamsters were sacrificed. A Cryosections of the heart were stained with antibodies against the SARS-CoV-2 nucleocapsid protein (NP) in red, cardiac ventricular marker MLC2V in green, and DAPI nuclear staining in blue. Small clusters of NP⁺/MLC2V⁺ cells could be detected in Omicron BA.2 infected heart (arrow). NP⁺ cells could be detected in Delta-infected heart, but they are rarely positive for MLC2v (asterisk). B–E Paraffin sections of the heart were stained with H&E. Representative images of B myocardial blood vessel congestion and interstitial edema (green arrows), C interstitial immune cell infiltration (yellow arrows), D CM degeneration (white arrows). E CM necrosis (blue arrows). F Histological scores of pathological features scaled 0–3, where 0 indicates the absence of pathological changes. Data are presented as mean ± SEM, n = 3 for A, n = 6 for B-F . Statistical significance was calculated using Student’s t-test *p < 0.05, **p < 0.01, ****p < 0.0001. Scale bar A = 25 µM; B–E = 50 µM

Fig. 4

Gene expression analysis of hamster heart. RNA was extracted from hearts of hamsters infected with Delta or Omicron BA.2 at 2 or 7 dpi. The expression of genes important for cardiac function were measured by RT-qPCR, normalised to B2m expression. Data are presented as mean ± SEM, n = 3 biological replicates. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test, relative to mock; *p < 0.05, **p < 0.01

Fig. 5

Transcriptomic profiling of hiPSC-CMs infected by SARS-CoV-2 variants. Human iPSC-CMs were infected with Delta (D), Omicron BA.1 (BA1) or BA.2 (BA2) for 48 h at MOI of 1, n = 3. Venn diagram showing the number of DEGs and their enrichment in different gene pathways commonly and specifically A up-, B down-regulated by the variants. C Heat map of selected genes associated with oxidative phosphorylation (OXPHOS) and contraction

Fig. 6

Delta, Omicron BA.1, and BA.2 induce distinct expression changes in hiPSC-CMs. Human iPSC-CMs were infected with Delta (D), Omicron BA.1 (O-BA.1) or BA.2 (O-BA.2) at MOI of 1. The expression of A RdRp, B genes important for cardiac function was measured by RT-qPCR, normalised to B2M expression. Data are mean ± SEM, n = 4 biological replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Differentially expressed genes in BA.2 group were examined by Ingenuity pathway analysis to predict upstream regulators. C Activation z-score indicates the activation (+) and inhibition (−) of the regulator, significance is indicated by the p-value of overlap. Expression is shown as log of fold change (FC). Regulators with consistent direction of activation score and expression change are shown in bold; D predicted downstream genes of selected regulators and E diagram of regulatory network, Fx: cardiac-related diseases and functions, Lines: direct (solid) and indirect (dotted) interactions

Fig. 7

Omicron infection is mediated via endocytic pathway. Human iPSC-CMs were infected with Delta (D), Omicron BA.1 (O-BA.1) or BA.2 (O-BA.2) for 48 h at MOI of 1. A The level of ACE2 was measured by RT-qPCR, n = 4. B The level of TMPRSS2 mRNA in hiPSC-CMs (n = 3) and Calu-3 cells (positive control, n = 2) were measured by RT-qPCR; C TMPRSS2 protein was evaluated by western blot. D Human iPSC-CMs were infected with the three variants at an MOI of 1, treated with bafilomycin A1 or camostat at the indicated concentrations. Viral RNA levels of SARS-CoV-2 variants were determined by qPCR analysis. Data are presented as mean ± SEM, n refers to biological replicates. Statistical analysis was performed using A one-way ANOVA followed by Tukey’s multiple comparisons test, or D two-way ANOVA. D. Significance is shown relative to the control group (0 µM); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001