Cardiac inflammation and microvascular procoagulant changes are decreased in second wave compared to first wave deceased COVID-19 patients

Abstract

Background: Compelling evidence has shown cardiac involvement in COVID-19 patients. However, the overall majority of these studies use data obtained during the first wave of the pandemic, while recently differences have been reported in disease course and mortality between first- and second wave COVID-19 patients. The aim of this study was to analyze and compare cardiac pathology between first- and second wave COVID-19 patients.

Methods: Autopsied hearts from first- (n = 15) and second wave (n = 10) COVID-19 patients and from 18 non-COVID-19 control patients were (immuno)histochemically analyzed. CD45+ leukocyte, CD68+ macrophage and CD3+ T lymphocyte infiltration, cardiomyocyte necrosis and microvascular thrombosis were quantified. In addition, the procoagulant factors Tissue Factor (TF), Factor VII (FVII), Factor XII (FXII), the anticoagulant protein Dipeptidyl Peptidase 4 (DPP4) and the advanced glycation end-product N(^ε)-Carboxymethyllysine (CML), as markers of microvascular thrombogenicity and dysfunction, were quantified.

Results: Cardiac inflammation was significantly decreased in second wave compared to first wave COVID-19 patients, predominantly related to a decrease in infiltrated lymphocytes and the occurrence of lymphocytic myocarditis. This was accompanied by significant decreases in cardiomyocyte injury and microvascular thrombosis. Moreover, microvascular deposits of FVII and CML were significantly lower in second wave compared to first wave COVID-19 patients.

Conclusions: These results show that in our cohort of fatal COVID-19 cases cardiac inflammation, cardiomyocyte injury and microvascular thrombogenicity were markedly decreased in second wave compared to first wave patients. This may reflect advances in COVID-19 treatment related to an increased use of steroids in the second COVID-19 wave.

Keywords: COVID-19; First and second wave; Heart; Inflammation; Thrombosis microvasculature.

Fig. 1

Examples of inflammation, myocytolysis and microvascular thrombosis in the hearts of COVID-19 patients. Shown are examples of increased diffuse presence of CD68+ macrophages (A) and CD45+ leukocytes (B) in a wave 1 COVID-19 patient with diffuse cardiac inflammation (DCI), as well as immunohistochemical examples of clusters of adherent CD45+ leukocytes (C; arrow) and CD3+ T lymphocytes (D; arrows) in a wave 1 COVID-19 patient with lymphocytic myocarditis (LM). In addition an example of myocytolysis, detected as complement factor C3d + cardiomyocytes (E; arrow), and an example of intravascular aggregated CD31+ platelets and fibrin, indicative of a microvascular thrombus (F; arrow) in wave 1 COVID-19 patients.

Fig. 2

Quantification of cardiac inflammation, myocytolysis and microvascular thrombosis in first and second wave COVID-19 patients. Stacked bars of (A): semi-quantitative analysis of the presence of extravasated CD45+ leukocytes, CD68+ macrophages and CD3+ T lymphocytes in the hearts of first wave (Wave 1) and second wave (Wave 2) COVID-19 patients and (B): subdivided between first wave COVID-19 patients with lymphocytic myocarditis (LM; n = 7) and diffuse cardiac inflammation (DCI; n = 8) and second wave patients with DCI (n = 10). Increases in inflammatory cells were quantified as either: no increase (dark green), focal (light green), mild (orange), moderate (dark red) or strong (black). For comparisons Pearson chi-square tests were used. (C): Graph: the number of extravasated CD3+ T lymphocytes supplemented with a maximum of 4 CD68+ macrophages per mm² in in the ventricular endocardium of first wave LM and DCI and second wave DCI COVID-19 patients. Picture: example of extravasated CD3+ T lymphocytes in the ventricular endocardium (arrows). The bars represent mean ± SD. For comparisons a Kruskal-Wallis test with Dunn's multiple comparison test was used. (D): The percentages in stacked bars of first- and second wave COVID-19 patients with (Yes; black) and without (No; white) cardiomyocyte injury (C3d+; left graph) and microvascular thrombi (right graph). For comparisons Pearson chi-square tests were used. n.s indicates not significant. *p < 0.05, **p < 0.01 (exact p-values are given in the text). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Examples of the presence of coagulation regulating factors in the cardiac microvasculature of COVID-19 patients. Immunohistochemical examples of the presence of the procoagulant factors Tissue Factor (A), Factor VII (B), Factor XII (C) in the endothelium of intramyocardial blood vessels of COVID-19 patients (arrows). Anticoagulant dipeptidyl peptidase 4 was detected via immunohistochemistry in the endothelium of most intramyocardial blood vessels of control patients (D; arrows), but was largely absent in the hearts of COVID-19 patients (E).

Fig. 4

Comparison of coagulation regulating factors and CML in the cardiac microvasculature between first and second wave COVID-19 and control patients. The number of blood vessels positive for the procoagulant factors Tissue Factor (A; TF), Factor VII (B; FVII), Factor XII (C; FXII) and anticoagulant dipeptidyl peptidase 4 (D; DPP4) are shown per cm² of left ventricular heart tissue in control patients (Con; n = 18) and first wave (Wave 1; n = 15) and second wave (Wave 2; n = 10) COVID-19 patients. (E): An immunohistochemical example of the presence N(ε)-Carboxymethyllysine (CML) in the endothelium of intramyocardial blood vessels of a COVID-19 patient (arrow) and the immunohistochemical (IH) score for CML per cm² in control patients (Con) and first- and second wave COVID-19 patients. (F): The number of blood vessels with weak, moderate and strong CML staining (staining intensities 1, 2, 3 respectively) in control (Con) and first- and second wave COVID-19 patients. Each point in the graphs represents the value of one individual patient, the bars represent mean ± SD. The bars represent mean ± SD. For comparisons Kruskal-Wallis tests with Dunn's multiple comparison tests were used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (exact p-values are given in the text).

Fig. 5

Comparison of coagulation regulating factors and CML in the cardiac microvasculature between COVID-19 patients with lymphocytic myocarditis and with diffuse cardiac inflammation. Shown are the number of blood vessels positive for Tissue Factor (A; TF), Factor VII (B; FVII), Factor XII (C; FXII), the CML IH-score (D) and intensity scores (E; number of blood vessels with weak, moderate and strong CML staining (staining intensities 1, 2, 3 respectively)) are shown per cm² in first wave (Wave 1) COVID-19 patients with LM (n=7) and with DCI (n=8) and second wave (Wave 2) COVID-19 patients with DCI (n=10). Each point in the graphs represents the value of one individual patient, the bars represent mean ± SD. The bars represent mean ± SD. For comparisons Kruskal-Wallis tests with Dunn’s multiple comparison tests were used. *p<0.05, **p<0.01 (exact p-values are given in the text).