Proinflammatory cytokines driving cardiotoxicity in COVID-19

Abstract

Aims: Cardiac involvement is common in patients hospitalized with COVID-19 and correlates with an adverse disease trajectory. While cardiac injury has been attributed to direct viral cytotoxicity, serum-induced cardiotoxicity secondary to serological hyperinflammation constitutes a potentially amenable mechanism that remains largely unexplored.

Methods and results: To investigate serological drivers of cardiotoxicity in COVID-19 we have established a robust bioassay that assessed the effects of serum from COVID-19 confirmed patients on human embryonic stem cell (hESC)-derived cardiomyocytes. We demonstrate that serum from COVID-19 positive patients significantly reduced cardiomyocyte viability independent of viral transduction, an effect that was also seen in non-COVID-19 acute respiratory distress syndrome (ARDS). Serum from patients with greater disease severity led to worse cardiomyocyte viability and this significantly correlated with levels of key inflammatory cytokines, including IL-6, TNF-α, IL1-β, IL-10, CRP, and neutrophil to lymphocyte ratio with a specific reduction of CD4+ and CD8+ cells. Combinatorial blockade of IL-6 and TNF-α partly rescued the phenotype and preserved cardiomyocyte viability and function. Bulk RNA sequencing of serum-treated cardiomyocytes elucidated specific pathways involved in the COVID-19 response impacting cardiomyocyte viability, structure, and function. The observed effects of serum-induced cytotoxicity were cell-type selective as serum exposure did not adversely affect microvascular endothelial cell viability but resulted in endothelial activation and a procoagulant state.

Conclusion: These results provide direct evidence that inflammatory cytokines are at least in part responsible for the cardiovascular damage seen in COVID-19 and characterise the downstream activated pathways in human cardiomyocytes. The serum signature of patients with severe disease indicates possible targets for therapeutic intervention.

Keywords: COVID-19; Cardiotoxicity; Inflammation; Stem cell derived cardiomyocytes.

Graphical Abstract

Figure 1

Serum cardiotoxicity in patients with COVID-19. (A) Schematic of study design. Assessing the effects of serum from patient with confirmed COVID-19 and negative controls on hESC-CM viability and subsequent cytokine profiling. Cardiomyocyte viability was assessed by change in PrestoBlue™ fluorescence and normalized to controls (hESC-CM without serum). (B) Viability of hESC-CM following exposure to serum of COVID-19 positive patients and controls, n = 6 and 12 patients. Viability data was obtained normalizing the fluorescence values to the values obtained in our control culture where cardiomyocytes were cultured in normal medium with no serum set as 1. (C) Results of larger confirmatory study. COVID-19 positive patients and negative controls, n = 8 and 33 patients. (D) Combination of study results from pilot trial and confirmatory study. COVID-19 positive patients and negative controls, n = 12 and 39 patients. The results from the 8 serum samples in common between the first and second study were plotted as the average of the two experiments. (E) Cardiomyocyte viability and sex distribution in COVID-19 positive patients and negative controls. (F–K) Luminex assay of patient serum samples, including IL-6 (F) TNF-α (G), IL1-β (H), IL-10 (I), IFN-γ (J), and high sensitivity CRP (K). Mean values; error bars represent s.d. Two-sided P values were calculated using an unpaired t-test unless otherwise stated. * P < 0.05, *** P < 0.001, **** P < 0.0001. Abbreviations: hESC-CM, human embryonic stem cell-derived cardiomyocytes; n.s., not significant.

Figure 2

Cardiomyocyte viability correlates with COVID-19 disease severity and key pro-inflammatory cytokines. (A) hESC-CM viability data obtained normalizing the PrestoBlue™ fluorescence values to the values obtained in our control culture where cardiomyocytes were cultured in normal medium with no serum set as 1. (B) Age in patients stratified according to disease severity. (C) Correlation of age and hESC-CM viability. Cov-, mild, O₂ NR, O₂ NA, Vent, n = 12, 8, 9, 9, and 13 (A–C). (D1) Gender distribution amongst disease severity groups. Cov-, mild, O₂ NR, O₂ NA, Vent, n = 12, 8, 9, 9, and 13. (D2) hESC-CM viability amongst males and females stratified according to disease severity. (E–J) Luminex cytokine data across disease severity groups. Shown are IL-6 (E), TNF-α (F), IL1-β (G), IL-10 (H), IFN-γ (I), and CRP (J). Mean values; error bars represent s.d. Two-sided P values were calculated using a one-way ANOVA with post-hoc correction for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: Cov-, COVID-19 negative; mild, mild symptoms only; O₂ NR, oxygen not required; O₂ NA, Oxygen-non assisted; vent, assisted ventilation; hESC-CM, human embryonic stem cell-derived cardiomyocytes.

Figure 3

Serological profile of patients with COVID-19 and therapeutic blockage mitigating cardiotoxicity. (A) Heatmap showing the Log₂ fold change in median absolute cell counts (bottom mid legend) in patients ordered according to cardiomyocyte viability (bottom left legend). The right column shows the Spearman’s Rho (bottom right legend) and associated P values. (B) Correlation matrix of key pro-inflammatory cytokines, gender, age, and hESC-CM viability. Top legend shows Spearman’s Rho. Values represent two-sided P values. (C) Hierarchical clustering of patients according to COVID-19 disease severity and pro-inflammatory cytokine response. Top legend represent raw Z score. (D) Combinatorial blockage of key pro-inflammatory cytokines increases hESC-CM viability. Viability data was obtained normalizing the fluorescence values to the values obtained in our control culture where cardiomyocytes were cultured in normal medium with no serum set as 1. Mean values; error bars represent s.d. Two-sided P values were calculated using a one-way ANOVA with post-hoc correction for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: Cov-, COVID-19 negative; mild, mild symptoms only; O₂ NR, oxygen not required; O₂ NA, Oxygen-non assisted; vent, assisted ventilation; hESC-CM, human embryonic stem cell-derived cardiomyocytes.

Figure 4

Specificity of COVID-19 serum toxicity and cell response: (A) viability of hESC-CM following exposure to serum from either healthy probands or patients with severe COVID-19 or patients with ARDS but without COVID-19. Viability data was obtained normalizing the PrestoBlue™ fluorescence values to the values obtained in our control culture where cardiomyocytes were cultured in normal medium with no serum set as 1. (B) Beating rate of hESC-CM (normalized to no serum controls) following exposure to serum from either healthy probands or patients with severe COVID-19 or patients with ARDS but without COVID-19. (C) Schematic illustrating the experimental set up using HMVECs to assess whether the COVID-19 serum response has cell-specific effects. (D) Viability of HMVEC (normalized to no serum control) following exposure to serum of control COVID-19 or ARDS patients and controls, n = 5, 7, and 5, respectively. (E) Representative flow cytometry histogram plots showing the percentage of ICAM-1 expressing cells following serum exposure. (F) ICAM-1 MFI of cells treated with serum of control COVID-19 or ARDS patients and controls, n = 5, 7, and 5, respectively. (G) Representative confocal images of HMVEC stained for vWF (red) and DAPI (white). Scale bar 100 µm. (H) Quantification of vWF deposition. *P < 0.05. Abbreviations: ARDS, acute respiratory distress syndrome.

Figure 5

RNA sequencing of serum treated hESC-CM (A) principal component analysis of RNA sequencing data calculated on the 500 most abundant genes across all samples of hESC-CM treated with control (n = 5, red), mild (n = 5, green), or severe (n = 7, blue) serum. (B) Pairwise JSI, calculated on the 500 most abundant genes across all samples. Ranging from 0 to 1, with high values corresponding to increased similarity. (C) Bar chart summarizing the number of genes with log2 (FC) > 0.5 (‘U’, up) or log2 (FC) < −0.5 (‘D’, down) consistently observed when comparing 4/5/6/7 (out of 7) severe samples against all mild/control samples (non-severe); each individual severe sample was compared independently using edgeR. (D) Heatmaps showing Z-scores for quantile-normalized expression levels for selected (differentially expressed) genes grouped according to relevant GO or KEGG pathway terms. (E) Box plots of distributions of |log2 (FC)| for selected genes across the seven comparisons between all mild/control samples and each severe sample, using edgeR. Genes are grouped according to annotated GO or KEGG pathway terms. The colour reflects upregulation (red) or downregulation (blue) in the severe samples. (F) GENIE3 inferred GRNs focused on selected differentially expressed genes. Blue and red nodes correspond to DE genes; the colours reflect downregulation or upregulation, respectively, in severe samples. The width of connecting edges is proportional to weight from the GENIE3 adjacency matrix. Green nodes indicate GO and KEGG pathway terms linked to the DE genes.