The pathological standard of myocarditis refers to the infiltration of inflammatory cells, with or without myocardial necrosis in the staining of heart tissue sections according to the Dallas definition (1). A recent study estimates that the global prevalence of myocarditis is ~22 out of 100,000 patients per year (2). A variety of infectious pathogens, systemic diseases, drugs, and toxins can cause the disease (3). Myocarditis can appear with a variety of different characteristics, from mild symptoms of chest pain to ventricular arrhythmia or fatal cardiogenic shock (3). Inflammatory cells and inflammatory factors in innate immunity and adaptive immunity play important roles in the occurrence and development of myocarditis, and there has been review (4) clarifying the roles of various types of immune cells. However, the vast majority of the heart is non-inflammatory cell. As our understanding of the human immune response continues to increase, many cell types, such as CFs, ECs that were initially thought to be just bystanders have been proven to play vital roles in the inflammatory process and even determine the direction and intensity of the immune response. However, there is little research on the role of non-inflammatory cells in the process of myocarditis. Thus, in this review, we will briefly delineate the role of non-inflammatory cells, non-CMs including CFs and ECs, and focus on the everlasting cross-talk between CFs, ECs and CMs in the progression of cardiovascular diseases.

Fibroblasts in the adult mouse heart make up about 15% of the non-myocytes in the heart (5). The basic function of cardiac CFs is to synthesize a collagen-rich ECM network to provide structural integrity and biomechanical signals (6). They are dynamic participants in ventricular physiology and pathophysiology. CFs can be activated either by their infection or invasion (7, 8) or by heart stress. Activated CFs will secret inflammatory factors, chemokines, or other immunomodulatory substances and produce large amounts of ECM. In myocarditis, virus infection or cytokines induces fibroblast activation state and then CFs function through virus transmission, cell damage, chemokines, and inflammatory factors production, making heart inflammation to keep getting worse. At the same time, the extracellular matrix produced by CFs promotes myocarditis from acute inflammatory infiltration to the chronic phase (Figure 1).

ECs are arranged in the circulatory system and are essential for maintaining and regulating vascular homeostasis (27) by protecting material transport, controlling vascular permeability, and regulating vascular tension (28). They are the largest cell population in non-cardiomyocytes of the adult mouse heart, accounting for about 60% (5). Resting ECs isolate leukocyte-interacting proteins in specialized secretory vesicles (29), so they cannot interact with white blood cells. In addition, resting ECs also inhibit the transcription of other adhesion molecules, such as E-selectin, vascular cell adhesion molecule 1 (VCAM1), and to a large extent inhibit the transcription of intercellular adhesion molecule 1 (ICAM1) and pro-inflammatory cytokines (30).

However, under the influence of certain factors, ECs can be transformed into an activated state. In DCM developed from autoimmune myocardial inflammation, it is shown that endothelial activation in organ-specific diseases affects the function of blood vessels throughout the body. Vallbracht et al. proved this and found that patients with myocardial inflammation showed impaired endothelial function of radial artery, and these patients have ruled out the risk factors of classic arterial damage, such as coronary artery disease, severe left ventricular failure, diabetes or atherosclerosis (31). For myocarditis, myocardial inflammatory infiltration, endothelial activation, direct viral toxicity, and circulating cytokines cause endothelial dysfunction in patients with persistent myocardial viruses (32). The persistence of the myocardial virus can independently cause endothelial dysfunction. For patients with myocardial leukocyte infiltration, endothelial dysfunction is more obvious (32). Nearly half of patients with DCM have increased T lymphocyte density and increased immune activation of ECs and mesenchymal cells in cardiac biopsies (33).

ECs not only act as a transport device for immune cells in circulation, forming a mechanical barrier to resist invaders, but also produce chemokines, interleukins, interferons, and growth factors through paracrine after they are activated. In addition, they can induce the expression of adhesion molecules such as E-selectin, P-selectin, ICAM, or VCAM to attract and transfer immune cells to inflammatory sites (34) (Figure 1).

The functions of various cells are not independent, they share the characteristics of repetitiveness and mutual reinforcement or containment. CMs are the most important cell type for the heart to perform biological functions. So we focus on the interaction between CMs and CFs, and CMs and ECs (Figure 2). On the one hand, these interactions promote the heart's adaptive response to internal and external stimuli and compensate or overcome these pressures. On the other hand, these interactions can also cause pathological remodeling related to heart disease, speed up the disease process, and lead to heart failure and cardiogenic death. Therefore, the study of the interaction between cells will help us to employ a synergistic protective effect between cells and avoid the expansion of malignant events.

As mentioned above, fibroblasts are activated under the influence of viruses or cytokines. Therefore, reducing the invasion of viruses in fibroblasts and reducing the stimulation of fibroblasts by other inflammatory factors are important means to prevent their activation. The study by Heim et al. showed that Ribavirin, the antiviral drug, has high cell-specific activity in fibroblasts, and it can reduce the viral load in fibroblasts (92). Moreover, Kania et al. demonstrated TGF-β-mediated myofibroblast differentiation and progression of myocardial fibrosis in human and mouse myocarditis (93). Anti-TGF-β treatment prevented myocardial fibrosis in mice with autoimmune myocarditis. In addition, IL-1 can prompt uninfected fibroblasts to enhance the induction of inflammation-related genes in the presence of infected fibroblasts. Blockade of IL-1 receptor signaling with an IL-1 receptor antagonist and knockdown of IL-1 receptor using siRNA abolished cytokines in human fibroblasts during CVB3 infection (94).

Fibroblasts can produce a variety of inflammatory factors and chemokines after activation. Among them, CCL20 is a chemokine that can effectively recruit Th17 cells. The neutralization of CCL20 can effectively reduce cardiac inflammation (17). In addition, blocking HMGB1 secreted by fibroblasts can effectively improve cardiac fibrosis in EAM mice (12). Studies have shown that fibroblasts in the heart are a heterogeneous cell population, in which Sca-1+ cardiac fibroblasts are not only efficient GM-CSF producers, but also have certain plasticity (95). They have a certain degree of plasticity to alter their own cytokine production under the influence of local microenvironment. Therefore, exploring the characteristics of different subpopulations of fibroblasts in myocarditis and finding targeted interventions based on their characteristics can better inform the treatment of patients with myocarditis.

After endothelial cells are infected by virus, the expressions of cell adhesion molecules such as ICAM-1, integrin αvβ3, P- and E-selectin are increased and the release of coagulation factor-von Willebrand factor (VWF) is also increased (96). The increase of adhesion molecules makes the circulating inflammatory cells more likely to adhere to blood vessels, while the increase of coagulation factors aggravates the retention of inflammatory cells and affects the execution of vascular function through the coagulation pathway. Telbivudine, an antiviral drug clinically used to treat hepatitis B, exerts endothelial protection in HPV B19 infected endothelial cells and can improve HPV B19-associated inflammatory cardiomyopathy (97). In addition, CVB3 virus can also infect endothelial cells through Coxsackie and adenovirus receptor (CAR). Bosentan and valsartan are angiotensin II receptor antagonists for the treatment of hypertension. In CVB3-induced myocarditis, bosentan and valsartan treatment can dose-dependently downregulate the levels of CAR mRNA and protein which is required for viral entry into cells. Reduction of CAR significantly reduced viral load in CVB3-infected Human Umbilical Vein Endothelial Cells (HUVECs) (98, 99). What's more, House Dust Mite Specific Antibodies, which can bind to cardiac vascular endothelial cells to weaken their barrier function, have been detected in the serum of patients with myocarditis. Since barrier dysfunction may lead to local tissue inflammation, removal of this antibody may be effective in relieving myocarditis (100). Moreover, compared with healthy rats, rats with autoimmune myocarditis exhibited marked mobilization of Endothelial Progenitor Cells (EPCs) from the bone marrow to the periphery, and their ability to adhere to fibronectin, mature endothelial cells, and cultured cardiomyocytes was significantly reduced. Metastasis of splenocyte-derived EPCs partially attenuates myocardial injury induced by experimental myocarditis. Of these, the transformation of EPCs into resident mature endothelial cells appears to be the most important reason (101). Therefore, augmentation of damaged endothelial cells by cell therapy could also serve as a promising therapeutic approach.

Among endothelial cell-derived proteins with altered secretion or expression in myocarditis, depletion of functional selectin ligand expression reduces the migration of pathogenic CD8⁺ T cells to the heart, which in turn reduces cardiac inflammatory infiltration and cardiomyocyte damage in myocarditis. In addition, the expression of MHC class II in myocarditis endothelial cells with dilated cardiomyopathy is increased (102). Studies have shown that loss of MHC class II expression on endothelial cells protects mice from experimental autoimmune myocarditis (103). Cyclosporine and statins reduce endothelial MHC class II levels and may prevent diffuse endothelial dysfunction in myocarditis (98). What's more, the antisense ICAM-1 inhibitor alicaforsen has been studied in four Phase 2 studies in ulcerative colitis (UC), and analysis suggests that alicaforsen enema may provide an effective and potential lasting response (104). However, it still needs more evidence to confirm whether these drugs can affect the expression of ICAM-1 in endothelial cells in myocarditis and exert their therapeutic effect on myocarditis.

In conclusion, the factors that cause the activation of fibroblasts and endothelial cells or the active substances produced by fibroblasts or endothelial cells that affect the occurrence and development of myocarditis can be used as potential targets for clinical treatment of myocarditis.

CFs and ECs can be infected by pathogens and release immunologically active substances to participate in the inflammatory response. In addition, CFs affect the fibrosis process and long-term prognosis of the heart through ECM changes. At the same time, through the interaction with the CMs, they may play a synergistic or antagonistic effect during the disease process.

However, there are still many problems. First of all, many of the interactions between cells are experiments conducted in vitro, such as the use of conditioned media or co-cultivation. It's difficult to perform in vivo experiments due to technical problems. However, the in vitro environment is quite different from the in vivo environment which has a more abundant neurohumoral regulation process.

In addition, a problem faced by cell experiments under in vitro conditions is that the conditioned medium may not be able to reproduce the observed effect. The composition and content of the conditioned medium will vary with the intervention environment, such as cell content, cell viability, cell intervention time, and the selection of cell lines, etc. That will make the results unstable.

Finally, the body is in a complex internal environment. The virus infection or the release of inflammatory factors both have beneficial and harmful aspects. This is closely related to the time and space relationship between cells. How to balance the pros and cons, and how to determine the correct intervention intensity, intervention time, and intervention methods is an important issue currently facing.

In general, a correct understanding of the function and mode of action of various cells, especially the non-inflammatory cells of the heart, such as CFs and ECs, will be more conducive to our understanding of diseases. And it contributes to the development of new myocarditis pathogenesis and corresponding treatment strategies.

DWW conceived this review, organized, and critically revised the manuscript. YX did major work of writing the manuscript. CC made contributions to editing the manuscript. ZW gave final approval of the version to be published. All authors were fully aware of and approved the submission of this manuscript.

This work was supported by grants from the National Natural Science Foundation of China (81630010 and 81790624 to DWW, 82070395 to ZW), the Natural Science Foundation of Hubei Province (2020CFA016 to CC), and the Tongji Hospital Clinical Research Flagship Program (2019CR207 to DWW). No funding bodies had any role in the program design, preparation of the manuscript, or decision to publish.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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