Major adverse cardiovascular events are associated with necroptosis during severe COVID-19

Abstract

Background: The mechanisms used by SARS-CoV-2 to induce major adverse cardiac events (MACE) are unknown. Thus, we aimed to determine if SARS-CoV-2 can induce necrotic cell death to promote MACE in patients with severe COVID-19.

Methods: This observational prospective cohort study includes experiments with hamsters and human samples from patients with severe COVID-19. Cytokines and serum biomarkers were analysed in human serum. Cardiac transcriptome analyses were performed in hamsters' hearts.

Results: From a cohort of 70 patients, MACE was documented in 26% (18/70). Those who developed MACE had higher Log copies/mL of SARS-CoV-2, troponin-I, and pro-BNP in serum. Also, the elevation of IP-10 and a major decrease in levels of IL-17ɑ, IL-6, and IL-1rɑ were observed. No differences were found in the ability of serum antibodies to neutralise viral spike proteins in pseudoviruses from variants of concern. In hamster models, we found a stark increase in viral titters in the hearts 4 days post-infection. The cardiac transcriptome evaluation resulted in the differential expression of ~ 9% of the total transcripts. Analysis of transcriptional changes in the effectors of necroptosis (mixed lineage kinase domain-like, MLKL) and pyroptosis (gasdermin D) showed necroptosis, but not pyroptosis, to be elevated. An active form of MLKL (phosphorylated MLKL, pMLKL) was elevated in hamster hearts and, most importantly, in the serum of MACE patients.

Conclusion: SARS-CoV-2 identification in the systemic circulation is associated with MACE and necroptosis activity. The increased pMLKL and Troponin-I indicated the occurrence of necroptosis in the heart and suggested necroptosis effectors could serve as biomarkers and/or therapeutic targets. Trial registration Not applicable.

Keywords: COVID-19; Heart injury; Necroptosis.

Fig. 1

Flow chart and diagnoses. Flow chart of patient included in the study

Fig. 2

MACE patients increase markers of cardiac injury and SARS-CoV-2 presence. A Log copies per mL of SARS-CoV-2 tested via quantitative RT-PCR. Changes in B Troponin I, C Pro-BNP, D D dimer, and E Presepsin (ng per L of serum). As measured by multiplex analysis, F Cytokine, and chemokine changes in serum (pg per mL). Mean ± SEM analysed by Kruskal–Wallis test with Dunn's multiple-comparison post-test (A–E), t-tests used in F. Asterisks denote the level of significance observed: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001

Fig. 3

Plasma-neutralising titters in patients with MACE show no significant changes. pNT50 values against the four SARS-CoV-2 pseudo-viral variants Beta, Gamma, Delta, and Omicron BA.1 and BA.2 and the control early 2020 strain with the D614 mutation. They were measured in samples collected from patients experiencing MACE and those with no signs of MACE (No-MACE)

Fig. 4

Cardiac transcriptional changes in a hamster model of severe SARS-CoV-2. A Male 5-to-6-week-old golden Syrian hamsters were IT infected with WA1, or mock challenged with vehicle (PBS), and hearts excised at 4 days post-infection for transcriptomics. B Log PFU per gram tissue of SARS-CoV-2 in hearts of hamsters (student t-test, p-value < 0.001 ****). C–F Log fold change (log FC) of transcriptional changes in infected hearts vs uninfected hamsters. Representative gene ontology terms selected were C programmed cell death, D regulation of ROS, E defence response to the virus, and F carbohydrate metabolic processes

Fig. 5

Hamster hearts show increased necroptosis in the model of severe SARS-CoV-2. A–C Male 5-to-6-week-old golden Syrian hamsters were IT infected with WA1 or mock challenged with vehicle (PBS) and hearts excised at 4 days post-infection for transcriptomics. A Normalised Transcript levels of MLKL and gasdermin-D (GSDMD) in uninfected vs SARS-CoV-2 infected hamsters. B Immunoblot for pMLKL in homogenates of hamsters' hearts. C Immunoblot for pMLKL in serum of hospitalised patients experiencing MACE or no-MACE. Mean ± SEM analysed by B student T test or C Kruskal–Wallis test with Dunn's multiple-comparison post-test. Asterisks denote the level of significance observed: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; *** = p ≤ 0.0001

Fig. 6

Markers of cardiac injury, inflammation, necroptosis, and SARS-CoV-2 correlate with MACE. A Correlation plot of serum cytokines, chemokines, biomarkers of cardiac injury, cell death effector MLKL, and SARS-CoV-2 (Log copies/mL). Linear regression plots for pMLKL—Troponin-I, SARS-CoV-2 (Log copies/mL)—Troponin-I and SARS-CoV-2 (Log copies/mL)—pMLKL (densitometry) in patients experiencing B–D MACE or E–G No-MACE