SARS-CoV-2 infects human cardiomyocytes promoted by inflammation and oxidative stress

Abstract

Introduction: The respiratory illness triggered by severe acute respiratory syndrome virus-2 (SARS-CoV-2) is often particularly serious or fatal amongst patients with pre-existing heart conditions. Although the mechanisms underlying SARS-CoV-2-related cardiac damage remain elusive, inflammation (i.e. 'cytokine storm') and oxidative stress are likely involved.

Methods and results: Here we sought to determine: 1) if cardiomyocytes are targeted by SARS-CoV-2 and 2) how inflammation and oxidative stress promote the viral entry into cardiac cells. We analysed pro-inflammatory and oxidative stress and its impact on virus entry and virus-associated cardiac damage from SARS-CoV-2 infected patients and compared it to left ventricular myocardial tissues obtained from non-infected transplanted hearts either from end stage heart failure or non-failing hearts (donor group). We found that neuropilin-1 potentiates SARS-CoV-2 entry into human cardiomyocytes, a phenomenon driven by inflammatory and oxidant signals. These changes accounted for increased proteases activity and apoptotic markers thus leading to cell damage and apoptosis.

Conclusion: This study provides new insights into the mechanisms of SARS-CoV-2 entry into the heart and defines promising targets for antiviral interventions for COVID-19 patients with pre-existing heart conditions or patients with co-morbidities.

Keywords: Cardiomyocytes; Heart damage; Inflammation; Oxidative stress; SARS-CoV-2.

Fig. 1

Cardiomyocyte force production and passive stiffness (Fpassive). (A) Representative image of skinned cardiomyocyte and elasticity test protocol with original recordings of force development at sarcomere length (SL) 2.2 μm (B) in activating buffer and response to stepwise cell stretching (SL 1.8–2.3 μm) (C) in relaxing buffer. In (B) at a constant sarcomere length of 2.2 μm calcium dependent force development is presented from donor, HF, and SARS-CoV-2 cardiomyocytes. (C) Passive stiffness (pCa 9.0) is measured between sarcomere lengths of 1.8 and 2.3 μm donor, HF, and SARS-CoV-2 cardiomyocytes. Fit curves are 2-order polynomials to the means. (D) is titin Coomassie blue, (E) Western blot with titin specific antibody which recognizes the elastic region of titin the N2B unique sequence (N2Bus). (F,G) Western blot with cardiac myosin binding protein C (cMyBPC) and cardiac troponin I (cTnI) specific antibodies which recognizes both proteins. Data are shown as mean ± SEM; (n = 16–20/4–5) cardiomyocytes/hearts. ‡P < 0.05 SARS-CoV-2 vs. donor, ϮP < 0.05 HF vs. donor, *P < 0.05 SARS-CoV-2 vs. HF by Student t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

SARS-CoV-2 infection of the heart and proteolytic enzymes. In (A) schematically pathway and effects of SARS-CoV-2 infection on cardiomyocytes are presented with integrated inflammatory, proteolytic and oxidative stress pathways. The enzymatic activities of MMP-2 (B), MMP-9 (C) and cathepsin (D) in donor, HF patients and SARS-CoV-2 infected patients. In cardiac tissue a viral spike/cathepsin interaction is shown using immunfluorescence and duolink (E) DAPI staining and WGA (anti-wheat agglutinine 555 conjugate, red) staining are used for nucleic acids and membranes, respectively. Bax (Bcl-2-associated X protein) (F,G) and NFAT (nuclear factor of activated T-cells) (H,I) activity and expression. In addition, activities of apoptotic enzymes caspase 3 (J) and caspase 9 (L) and their expression level (K) and (M) respectively. Intracellular calpain activity (N) and expression (O). Calcineurin activity (P) and expression (Q). Inserts show characteristic Western blot section. Data are shown as mean ± SEM; n = 8–9 patient/group. *P < 0.05/**P < 0.01/***P < 0.001/****P < 0.0001 HF and SARS-CoV-2 vs. donor, ϮϮϮϮP<0.0001 SARS-CoV-2 vs. HF by one-way ANOVA. P-values were corrected for multiple comparisons by the Tukey method. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Inflammasome and oxidative stress markers in SARS-CoV-2 infected cardiac tissue. (A) Represents the whole inflammatory pathway investigated in this section, enzymatic activities of (B) HMGB1 (high mobility group box 1), (C) calprotectin, (D,E) TLR (toll-like receptor) 2 and 4, (F) RAGE (receptor for advanced glycation endproducts), (G) expression of JNK (C-jun-N-terminal kinase), enzymatic activities of (H) NF-κB (nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells), (I) NLRP3 (NLR family pyrin domain containing 3), (J) IL-1, (K) IL-2, (L) IL-18 (interleukin), (M) ICAM-1 (intracellular adhesion molecule-1) and (N) VCAM1 (vascular cell adhesion molecule-1) and (O) TNFα (tumor necrosis factor α). Further enzymatic activities of (P) H2O2 (hydrogen peroxide), (Q) H2O2 level in cytosol, (R) GSH (reduced glutathione), (S) GSH level in cytosol (T) LPO (lipid peroxidase), (U) LPO level in cytosol in cardiac tissue from healthy donors, heart failure (HF) patients and severely infected SARS-CoV-2 patients. Data are shown as mean ± SEM; n = 8–9 patient/group. *P < 0.05/**P < 0.01/****P < 0.0001 HF and SARS-CoV-2 vs. donor, ϮϮP < 0.01/ ϮϮϮP<0.001/ϮϮϮϮP<0.0001 SARS-CoV-2 vs. HF by one-way ANOVA. P-values were corrected for multiple comparisons by the Tukey method.

Fig. 4

SARS-CoV-2 and Neuropilin-1 cascade. IL-6 activity and expression in SARS-CoV-2 patients compared to donors and HF patients (A,B), myeloperoxidase activity (C) and expression levels (D). Neuropilin-1 (E) and expression (F,G,I) in SARS-CoV-2 infected patients. (H) Neutrophil elastase activity. (J,K) immunofluorescence showing the interaction/colocalization of SARS-CoV-2 spike with IL-6 (J) or Neuropilin-1 (K) single staining. DAPI staining (blue) and WGA (anti-wheat agglutinine 555 conjugate, red) staining are used for nucleic acids and membranes. (L-P) HDAC4 expression level. Histone expression and modifications (Q-T), histone 3 expression level (Q), acetylated (R), Di-methylated (S), phosphorylated (T) in SARS-CoV-2 and donors and compared to HF patients. Data are shown as mean ± SEM; n = 8–9 patient/group. *P < 0.05/**P < 0.01/****P < 0.0001 HF and SARS-CoV-2 vs. donor, ϮP < 0.05/ϮϮP < 0.01/ϮϮϮP<0.001/ϮϮϮϮP<0.0001 SARS-CoV-2 vs. HF by one-way ANOVA. P-values were corrected for multiple comparisons by the Tukey method. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)