SARS-CoV-2 spike protein-induced inflammation underlies proarrhythmia in COVID-19

Figures

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  Fig. 1. Impact of spike protein on atrial arrhythmia susceptibility. (A) Representative in vivo surface ECG illustrates atrial arrhythmia observed in a spike protein-injected mouse following caffeine + epinephrine challenge. (B) Total atrial arrhythmia burden under caffeine + epinephrine challenge quantified as minutes of arrhythmia per hour of observation (Albumin and vS1 controls n = 6 mice/group, Spike protein n = 18, Spike protein subunits n = 6/group, * p < 0.05 vs. albumin control - Wilcoxon’s rank sum test). (C) Cumulative distribution of atrial arrhythmia duration quantified as seconds of arrhythmia per hour of observation (Albumin control n = 6, Spike-treated (18), † - p < 0.05 vs. albumin control - Kolmogorov-Smirnov test).

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  Fig. 2. Histologic observations following spike protein insult. (A) Representative images of mouse heart samples showing presence of spike protein and elevated inflammatory cytokines, IL-6 and VEGF, in spike protein-injected mice compared to albumin control. (B) Representative H&E images demonstrating normal blood vessels in albumin control (black circle) vs. atypical endothelial cells (enlarged endothelial cells, perivascular edema; red arrows) and evidence of red blood cell extravasation, suggesting microvessel rupture, in high dose spike-injected heart (red circles). (C) High magnification view of dashed box in B) show multiple locations of red blood cell extravasation (circles) and perivascular edema (red arrow). All scale bars are 100 μm.

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  Fig. 3. Ultrastructural effects of spike protein insult. (A) Representative TEM images of IDs from murine atria. White arrows point to GJs, and black arrows point to MJs. (B) Summary plots of median intermembrane distance measured at 50 nm from GJ (perinexal, solid bars) and MJ (MJ-adjacent, striped bars) edges (> 100 measurements/group/location from n = 3 hearts/group, * p < 0.05 vs. albumin control at perinexal sites, † p < 0.05 vs. albumin control at MJ-adjacent sites).

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  Fig. 4. Confocal survey views of murine atria. Representative 2D confocal images showing some instances of Cx43 lateralization as well as elevated detection of NaV1.5 at the lateral membrane and concurrent loss of NaV1.5 at the ID (white arrows) in hearts from mice injected with high dose spike protein (but not low dose; % Cx43 overlap with N-cad: 53 ± 2% in albumin controls vs. 62 ± 5% in vS1 vs. 53 ± 3% in high dose spike, p = ns vs. control; % NaV1.5 overlap with N-cad: 81 ± 6% in albumin controls vs. 90 ± 3% in vS1 vs. 80 ± 4% in high dose spike, p = ns vs. control), n = 3 hearts/group. All scale bars are 25 μm.

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  Fig. 5. 3D Confocal imaging of individual en face IDs. Representative 3D confocal images of en face IDs from murine atria. (A) Albumin control and high dose spike protein (S1 + S2). (B) Cumulative distribution of NaV1.5 distance from Cx43 or Ncad. Dotted lines indicate complete spatial randomness of NaV1.5 relative to Cx43 and Ncad. Solid lines indicate observed NaV1.5 distribution. (C) Summary plot of NaV1.5 attraction to Cx43 or Ncad (n = 3 hearts/group, Albumin control (35 images), S1 + S2 (40), * p < 0.05 vs. albumin, † p < 0.05 vs. complete spatial randomness).