Pyroptosis is an inflammatory form of programmed cell death that occurs most frequently upon infection by intracellular pathogens, and requires the function of the enzyme caspase-1 16. Caspase-1 is activated during pyroptosis as part of a multiprotein signaling platform, the inflammasome complex, and subsequently mediates the activation and secretion of various interleukins, such as IL-1β, IL-4 and IL-11, as well as the rupture of the cell membrane 17. We observed higher levels of caspase-1 adjacent to endothelial cells in the COVID-19 samples demonstrating endothelial infection, pyroptosis and injury in these patients. Moreover, SARS-CoV-2 particles have been described in endothelial cells by electron microscopy 9 and the caspase-1 identification is in accordance with Varga et al. 9, who suggested that pyroptosis might have an important role in endothelial cell injury in patients with COVID-19.
The IHC analysis also showed increased expression of ICAM-1, IL-6, IL-4, CD163, MMP-9 and type 3 collagen in the COVID-19 samples when compared to the control. The first two biomarkers, ICAM-1 and IL-6, were present in large quantities in the endothelial cells and indicate endothelial activation as well as immune cell recruitment and response. These findings are in line with the observations on caspase-1, and with previous biopsies studies which had already shown that the inflammatory process in cardiac tissue permeates the vascular wall 6,10. SARS-CoV-2 potentially causing endotheliitis 9, which is determinant of microvascular dysfunction by shifting the vascular equilibrium towards more vasoconstriction with subsequent organ ischemia, inflammation with associated tissue edema, and a procoagulant state 18.
The endothelial injury in COVID-19, which was demonstrated to occur via pyroptosis and interleukin secretion, would increase capillary permeability and cause tissue edema 15,17,18. When comparing the sample tissues from the COVID-19 patients to the control, we observed severe pericellular interstitial edema in between the cardiomyocytes, causing them to separate. The maintenance of cytoarchitecture and extracellular environment of the myocardium is fundamental for the electrical and contractile function of the heart 15. Thus, the cardiac dysfunction and arrhythmias associated with myocardial injury in COVID-19 may be related to the myocardial interstitial edema and consequent loss of structure of the syncytium 19.
Although studies including post mortem biopsies are still scarce, the literature converges into a few key aspects regarding the lesions caused by SARS-CoV-2, those being mainly the hyaline membrane formation alongside diffuse alveolar damage findings, the myocardial interstitial edema and the endothelium inflammation 20–22. Our results show myocardial interstitial edema, which alongside the endothelial inflammation caused by SARS-CoV-2, may progress to a later fibrotic myocardial reorganization, as evidenced by the presence of IL-4, ICAM-1, MMP-9 and type 3 collagen expression. These findings may indicate an early stage myocardial fibrotic response as opposed to a pre-existing fibrosis, that would be marked by a higher level of type 1 collagen expression and little IL-4 and MMP-9 expression.
The high levels of MMP-9, CD163, IL-4 and IL-6 demonstrate myocardial inflammatory stress in the COVID-19 tissue. SARS-CoV-2 particles have been already observed in a cytopathic interstitial inflammatory cell in myocardial tissue 8 and other autopsies of patients with COVID-19 also revealed infiltration of the myocardium by interstitial mononuclear inflammatory cells 6,8. MMP-9 is a endopeptidase which cleaves structural elements of the extracellular matrix and also plays important roles in immune cell function 20. MMP-9 promotes Th2 cells recruitment and it has been shown to be significantly increased during several cardiovascular diseases, including hypertension, atherosclerosis and myocardial infarction 20. This, along with the high CD163 expressing macrophages on the tissue samples, are signs of cell recruitment, which is characteristic of immune inflammatory response 21. Monocytes and macrophages recruited are capable of producing and secreting large amounts of pro-inflammatory mediators and pro-fibrotic growth factors 22.
The persisting Th2 (IL-4) cytokine-driven immune mechanism is relevant to the process of myocardial fibrosis 23. In fact, IL-6 and IL-4 have already been shown to be two profibrotic cytokines, as they induce MMP-9 expression and collagen synthesis through gene transcription modulation 23–25. MMP-9 also stimulates cardiac fibroblast migration, increases collagen synthesis, upregulates angiogenic factors, and induces the transition of cardiac fibroblasts to myofibroblasts 14,22.
As expected, we found no difference in TNF-α between cases and control. We also observed no difference in TGF-β. This is a cytokine with major roles in cardiac fibrogenesis 22,26,27 which activates SMAD2/3 pathways, stimulating alternative pathogenetic pathways and regulating cell synthesis and differentiation, promoting fibrogenesis 27. We hypothesize that the TGF-β pathway was still not activated in these cases. If not TGF-β, an alternative pathway for myocardial fibrosis, such as the activation of macrophages via IL-4 22,27 or mast cell degranulation 22, might be involved in the pathophysiology of COVID-19.
Myocardial fibrosis is characterized by a dysregulated collagen turnover and excessive fibrillar collagen accumulation in the interstitial and perivascular spaces 22,26. Synthesis of both type 1 and type 3 collagen is markedly increased in the remodeling fibrotic heart regardless of the etiology of fibrosis 22. In our study, type 3 collagen was observed in large quantities in the interstitial and perivascular spaces in the COVID-19 samples when compared to the control. Type 1 collagen, in contrast, showed no difference between cases and control.
Type 1 collagen cross links with type 3 collagen to form the final fibers in myocardial fibrosis which is primarily associated with thick fibers that confer tensile strength, and because of that, takes longer to form 22,28. A genetic response study on experimental autoimmune myocarditis showed that myocardial fibrosis had formed on day 21, but not before 29. In addition, advanced age and chronic illnesses are known to lead to myocardial fibrosis, and both our control and COVID-19 samples were patients well over 70 years old.
Type 3 collagen, on the other hand, typically forms thin fibers and, because of that, it takes less time to build 22,28. Our observation of type 3 collagen in the COVID-19 samples, but not in the control, along with the increased expression of CD163, IL-4, IL-6 and MMP-9, is consistent with the hypothesis that COVID-19 acute myocardial injury may cause myocardial fibrosis in the long term. In addition, formation of chronic myocardial interstitial edema, observed in the COVID-19 samples but not in the control, also results in deposition of interstitial collagen, which causes interstitial fibrosis 13.
An important finding is that the echocardiogram from our COVID-19 patients 24 hours before death showed no changes in ejection fraction or in left ventricle before or after the disease. Patient 1 had mid-range ejection fraction (43%) and mild eccentric hypertrophy before the onset of COVID-19 symptoms and maintained the pattern during hospitalization. Patient 2 had preserved ejection fraction (60%) and preserved dimensions of the left ventricle. At that time, the IHC biomarkers had probably already been activated. Our findings could mean that biomarker changes appear much earlier than echocardiographic changes and, therefore, we should not wait for alterations in echocardiogram to infer cardiac involvement of COVID-19.
The microvascular dysfunction may lead to thrombosis and justifies the rational use of anticoagulant and anti-aggregating therapy 30. The myocardial interstitial edema presented here may be one explanation for the high prevalence of cardiac arrhythmia in COVID-19 patients despite other drug factors that may be involved, such as the use of hydroxychloroquine and azithromycin31. Furthermore, our findings suggest that COVID-19 myocardial injury may cause myocardial fibrosis in the long term. Based on laboratory tests, individualized cardiac magnetic resonance could be useful to assess patients’ cardiac involvement, and thus guide treatment. Additionally, drugs which act in cardiac remodeling, such as angiotensin-converting enzyme inhibitors or mineralocorticoid receptor antagonists, could be useful in a long-term myocardial protective effect32,33. However, further studies evaluating cardiac sequelae and mortality following hospital discharge are needed.
We present a panel of immunohistochemical markers showing different and intricated mechanisms of myocardial injury. However, our study has a few limitations. The two COVID-19 patients were elderly with more than 70 years of age and had underlying conditions that could be confounders. Also interpretation of our findings should take into account that autopsies do not allow the observation of the entire pathological process, and cannot predict the evolution of the disease.