Our main findings from the six myocardial postmortem biopsies of COVID-19 patients show myocardial interstitial edema, higher mast cell scores, and increased expression of caspase-1, ICAM-1, IL-6, CD163, MMP-9, IL-4 and type 3 collagen when compared to the control. Additionally, the TUNEL assay confirms the presence of endothelial apoptosis, and the presence of caspase-1 in the endothelial cells suggests that the mechanism is by pyroptosis. The increased expression of ICAM-1 and IL-6 indicates endothelial activation, which alongside the higher mast cell scores can explain the increased capillary permeability, microvascular leakage and the consequent formation of myocardial interstitial edema. The increased expression of MMP-9, CD163, IL-4 and IL-6 demonstrates the presence of myocardial inflammatory response in the myocardial tissue. More specifically, CD163 signalizes macrophage recruitment and MMP-9 promotes Th2 cell recruitment and matrix remodeling. The persisting Th2 (IL-4) cytokine-driven immune mechanism is relevant to the process of myocardial fibrosis. Finally, the presence of mast cells, MMP-9, IL-4, IL-6 and type 3 collagen expression suggests that the inflammatory interstitial myocardial edema may progress to remodeling and a later myocardial fibrosis. Further implications of these events will be discussed next.
The TUNEL assay resulting positive in all the COVID-19 samples proves that this disease promotes endothelial cell apoptosis (programmed cell death). The probable mechanism is by pyroptosis, a specific inflammatory form of apoptosis that occurs most frequently upon infection by intracellular pathogens (like SARS-CoV-2) and requires the function of the enzyme caspase-1 17. Caspase-1 is activated as part of a multiprotein signaling platform, the inflammasome complex, and subsequently mediates the activation and secretion of various interleukins as well as the rupture of the cell membrane 18. 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 10 and the caspase-1 identification is in accordance with Varga et al. 10, who suggested that pyroptosis might have an important role in endothelial cell injury in patients with COVID-19. These findings are also in line with previous biopsy studies which had already shown that the inflammatory process in cardiac tissue permeates the vascular wall 6,11. SARS-CoV-2 potentially causes endotheliitis 10, 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 19.
The expression of IL-6 and ICAM-1 was increased in the endothelial cells and indicates endothelial activation as well as immune cell recruitment and response. When endothelial cells are activated, they produce NF-κB and adhesion molecules such as IL-6-induced ICAM-1. These molecules have the function of attracting leukocytes to the infected region and transmitting intracellular signals, which leads to the pro-inflammatory status20. Activation of endothelial cells is also required for the regulation of vascular permeability and blood flow to the site19,21. At rest, the endothelium is highly impermeable to large molecules. However, acute changes in vascular permeability result in loss of fluid and plasma proteins from the intravascular space into the interstitium, leading to edema19,21,22.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. Therefore, the myocardial interstitial edema observed in the samples, and consequent loss of structure of the syncytium 21, may account for the cardiac dysfunction and arrhythmias associated with myocardial injury in COVID-19.
According to our findings, another mechanism besides endothelial activation could explain the increased capillary permeability and the consequent tissue edema: mast cell degranulation, as we observed higher mast cells scores in the COVID-19 patients, most of them in the degranulating process. Mast cell degranulation is associated with proinflammatory effects, primarily due to release of histamine, TNF and proteases, which functionally overlap in promoting enhanced expression of adhesion molecules on endothelial cells, as well as increased vascular permeability and blood flow22. Mast cell degranulation is an important contributor to inflammatory processes and occurs not only in the context of allergy, but also in viral infection23.
Interestingly, an increased number of mast cells in the myocardium has already been seen in autopsies of patients with Chagas' disease, an infectious disease caused by the protozoan parasite Trypanosoma cruzi.24. It is believed that the increase of mast cells in the Chagas’ patients would play an important role in containing the infection by inducing apoptosis of the infected cells. Then, the chronic phase of Chagas' disease is characterized by apoptosis, inflammatory infiltrate and collagen neoformation being associated with an increase in the number of mast cells24. Mast cells release proteases that stimulate fibroblast proliferation and collagen synthesis, being increased in numbers in fibrotic areas.23,24 Mast cells also serve as sources of TNF, which is released during degranulation and promotes cardiac fibrosis via induction of cardiomyocyte apoptosis, inflammation and MMP-9 production23. It is possible that the cardiac lesions of COVID-19 are more similar to Chagas' disease than other virus myocarditis.
A previous autopsy study showed that the presence of SARS-CoV-2 genome in the myocardial tissue was not associated with increased infiltration of mononuclear cells compared with the virus negative group7. Although most of our COVID-19 myocardium samples also showed neither inflammatory cellular infiltration nor necrosis, which would be expected in typical histological myocarditis, the high levels of MMP-9, CD163, IL-4 and IL-6 demonstrate the presence of myocardial inflammatory response in this tissue.
MMP-9 is an endopeptidase which cleaves structural elements of the extracellular matrix and also plays important roles in immune cell function 25. MMP-9 promotes Th2 cell recruitment and has been shown to be significantly increased during several cardiovascular diseases, including hypertension, atherosclerosis and myocardial infarction 25. This, along with the high CD163 expressing macrophages on the tissue samples, are signs of cell recruitment, which is characteristic of immune inflammatory response 26. Recruited monocytes and macrophages are capable of producing and secreting large amounts of pro-inflammatory mediators and pro-fibrotic growth factors, promoting remodeling 27. In fact, SARS-CoV-2 particles have already been observed in a cytopathic interstitial inflammatory cell in myocardial tissue 9, and other autopsies of patients with COVID-19 revealed infiltration of the myocardium by interstitial mononuclear inflammatory cells 6,9.
Additionally, the persisting Th2 (IL-4) cytokine-driven immune mechanism is relevant to the process of myocardial fibrosis 28. In fact, IL-6 and IL-4, which were increased in the COVID-19 samples, have already been shown to be two profibrotic cytokines, as they induce MMP-9 expression and collagen synthesis through gene transcription modulation 28–30. MMP-9 also stimulates cardiac fibroblast migration, increases collagen synthesis, upregulates angiogenic factors, and induces the transition of cardiac fibroblasts to myofibroblasts 14,27.
As expected, we found no difference in TNF-α between cases and control, since this nonspecific proinflammatory cytokine is involved in several pathological processes such as acute and chronic inflammation, autoimmunity and malignant disease 31. We also observed no difference in TGF-β. This is a cytokine with major roles in cardiac fibrogenesis 27,32,33 which activates SMAD2/3 pathways, stimulating alternative pathogenetic pathways and regulating cell synthesis and differentiation, promoting fibrogenesis 33. 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 27,33 or mast cell degranulation 27, might be involved in the pathophysiology of COVID-19. Mast cell tryptase is a protease that can directly induce fibroblast activation, myofibroblast differentiation and collagen synthesis independently of TGF-β 34.
Myocardial fibrosis is characterized by dysregulated collagen turnover and excessive fibrillar collagen accumulation in the interstitial and perivascular spaces 27,32. Synthesis of both type 1 and type 3 collagen is markedly increased in the remodeling fibrotic heart regardless of the etiology of fibrosis 27. 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 and type 4 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 27,35. Type 3 collagen, on the other hand, typically forms thin fibers and, because of that, it takes less time to build 27,35. Type 4 collagen is structural and was observed in the samples to validate the collagen assays, as a control.
Our observation of type 3 collagen in the COVID-19 samples, but not in the control, along with the presence of mast cells and the increased expression of MMP-9, IL-6, IL-4, is consistent with the hypothesis that COVID-19 acute myocardial injury may cause myocardial fibrosis in the long term. This is further supported by the observation of chronic myocardial interstitial edema in the COVID-19 samples but not in the control, since it results in deposition of interstitial collagen, causing interstitial fibrosis 13. Also, these findings may indicate an early stage myocardial fibrotic response as opposed to a pre-existing fibrosis, which would be marked by a higher level of type 1 collagen expression and little IL-4 and MMP-9 expression. A genetic response study on experimental autoimmune myocarditis showed that myocardial fibrosis had formed on day 21, but not before 36. In addition, advanced age and chronic illnesses are known to lead to myocardial fibrosis, and both our control and COVID-19 samples were mainly elderly.
Taken together our findings indicate that the microvascular dysfunction may lead to thrombosis and justifies the rational use of anticoagulant and anti-aggregating therapy 37, and myocardial interstitial edema presented here may be one explanation for the high prevalence of cardiac arrhythmia in COVID-19 patients. 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 effect38,39. However, further studies evaluating cardiac sequelae and mortality following hospital discharge are needed.
We present a panel of immunohistochemical markers with high expression in COVID-19 myocardium samples and, to the best of our knowledge, this is the first study with TUNEL assay which proves endothelial cell apoptosis in COVID-19 myocardial tissue. Moreover, our study brings light to the pathogenesis of the disease by putting all the biomarkers together and showing mast cell degranulation in myocardium samples. There are, however, a few limitations to our study. The COVID-19 patients were mainly elderly 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 therefore cannot predict the evolution of the disease.
In conclusion, the multifactorial pathogenesis of COVID-19 myocardial injury seems to be related with pyroptosis leading to endothelial cell injury and disfunction, which contributes to the impaired microcirculatory function. Subsequently, the inflammation with associated interstitial edema would explain the myocardial disfunction and arrythmias in COVID-19 patients. These patients could be more prone to thrombotic diseases, heart failure and even death. Our hypothesis seems to be an explanation as to why patients with underlying heart conditions are predisposed to developing more severe manifestations of COVID-19. Finally, our findings show that COVID-19 myocardial injury may cause myocardial fibrosis in the long term. These patients should be monitored for systolic and diastolic dysfunction and arrythmias leading to heart failure after the acute phase of COVID-19.