Role of Coxsackievirus B3-Induced Immune Responses in the Transition from Myocarditis to Dilated Cardiomyopathy and Heart Failure

Dilated cardiomyopathy (DCM) is a cardiac disease marked by the stretching and thinning of the heart muscle and impaired left ventricular contractile function. While most patients do not develop significant cardiac diseases from myocarditis, disparate immune responses can affect pathological outcomes, including DCM progression. These altered immune responses, which may be caused by genetic variance, can prolong cytotoxicity, induce direct cleavage of host protein, or encourage atypical wound healing responses that result in tissue scarring and impaired mechanical and electrical heart function. However, it is unclear which alterations within host immune profiles are crucial to dictating the outcomes of myocarditis. Coxsackievirus B3 (CVB3) is a well-studied virus that has been identified as a causal agent of myocarditis in various models, along with other viruses such as adenovirus, parvovirus B19, and SARS-CoV-2. This paper takes CVB3 as a pathogenic example to review the recent advances in understanding virus-induced immune responses and differential gene expression that regulates iron, lipid, and glucose metabolic remodeling, the severity of cardiac tissue damage, and the development of DCM and heart failure.


Introduction
Myocarditis is an inflammatory disease of the heart muscle commonly caused by viruses including coxsackievirus B3 (CVB3), SARS-CoV-2, parvovirus B19, influenza viruses, adenoviruses, and enteroviruses; extensive studies have been executed using CVB3 infection models [1][2][3][4]. The prognosis of myocarditis is diverse; most patients make a recovery, while up to 20% develop chronic myocarditis [5]. Dilated cardiomyopathy (DCM) is a consequence of long-term myocarditis, a severe heart disease characterized by heart enlargement, ventricle chamber dilation, and systolic dysfunction [6,7]. DCM is usually presented with progressive dyspnea, ankle swelling, arrhythmia, thromboembolism, and sudden cardiac death; it has a 5-year survival rate of approximately 50% when left untreated [8]. DCM can also be caused by drugs, toxins, genetics, and metabolic and endocrine disturbances [9]. A high prevalence of viral genomic RNA has been demonstrated in the heart tissues of patients with DCM, suggesting that viral myocarditis plays a significant role in causing DCM [10,11]. However, the molecular pathways that lead to the transition from viral myocarditis to DCM and heart failure are complex. Previous research revealed that viral pathogenesis of myocarditis is attributed to direct damage of cardiomyocytes by viral proteases and immune-and autoimmune-mediated cardiac injury. The cleavage of host immune proteins by proteases is consequential, causing several downstream effects that further progress cardiomyopathy. This review will discuss processes during CVB3 infection that damage host cells, including changes in iron homeostasis and energy metabolism, impairment of mitochondrial function, and alterations in immune profiles that promote tissue damage. More specifically, it will focus on the relevance of the immune response in these processes during the transition of CVB3-induced myocarditis to DCM, as well as recent advances in understanding these mechanisms. Unless otherwise stated, the findings reviewed were obtained from myocarditis mouse models infected with CVB3. 3C viral proteases can hinder host immune defense by cleaving mitochondrial antiviral-signaling protein (MAVS) and TIR-domain-containing adapter-inducing interferonβ (TRIF) [17]. MAVS is the adaptor molecule downstream of both melanoma differentiation-associated protein 5 (MDA5) and retinoic acid-inducible gene I (RIG-I) viral DNA detectors, which mediate the activation of nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF3) [17]. The cleavage of MAVS has been shown to diminish type 1 interferon (IFN) signaling and is important for the expression of effector proteins 2′-5′ OAS, Mx, and PKR, which collectively help degrade and inhibit viral RNA translation [17,18]. Furthermore, MAVS and TRIF are implicated in caspase-dependent apoptosis, which eliminates infected cells to suppress viral replication and prevent viral dissemination [17,[19][20][21]. By cleaving MAVS and TRIF, the immune response can be dampened and cell apoptosis can be reduced, leading to prolonged myocarditis and enhanced viral propagation [17]. 2A viral proteases can cleave nuclear factor of activated T cells 5 (NFAT5), a transcription factor in the NF-κB signaling pathway for the transcription of proteins involved in immune responses against cellular stress, including inducible nitric oxide

CVB3 Indirectly Impairs Cardiac Function by Inducing Inflammation That Results in Cardiomyocyte Necrosis and Fibrosis
Infectious pathogens can indirectly damage the myocardium by triggering and sustaining immune responses. Viral double-stranded RNA, including replication intermediates of single-stranded viral genomes, can be detected by Toll-like receptor 3 (TLR3) that is localized to cell surfaces and endosomes [26][27][28]. TLR7 and TLR8 can detect single-stranded RNA, such as the CVB3 genome, whereas TLR9 can recognize unmethylated cytosinephosphate-guanosine (CpG) DNA, which is abundant in viral genomes [29,30]. After being activated by viral components, TLRs form dimers that recruit myeloid differentiation primary response 88 (MyD88)/TRIF-related adaptor molecule (TRAM) adaptor proteins [31]. These proteins activate transcription factors specific to each pathway, including activator protein 1 (AP-1), NF-κB, and IRF3 [31]. Cytoplasmic helicases RIG-I and MDA5 can recognize distinct viruses and activate type 1 IFN production [32]. For instance, MDA5 activation induces IFN-α production, TLR3 activation induces interleukin 12 (IL-12) production, and the activation of both MDA5 and TLR3 induces IL-6 production [32]. Type 1 IFNs activate the production of ISGs to promote antimicrobial states which limit infection and recruit innate immune cells [33]. Various cytokines are important for cellular communication and viral clearance; however, they can also cause cytokine storms and significant cell stress. Consequently, studying the effective time points and interactions between cytokines will provide valuable information for understanding viral pathogenesis.
Collagen deposition and modification of the extracellular matrix (ECM) are compensatory repair mechanisms used to stabilize the site of injury. However, an imbalance of collagen synthesis and degradation by myofibroblasts can result in cardiac fibrosis, a condition characterized by excessive collagen deposition, which forms permanent scar tissue that disrupts cardiac function and decreases contractile efficiency [34]. These alterations contribute to cardiac remodeling, a process of interstitial changes that includes changes in the size, structure, stiffness, and functioning of the heart, all of which contribute to heart failure [35]. Altogether, the fibrotic response and the immune response work together to repair tissue damage caused by viral infection and inflammation. Significant fibrosis can be detected in mice by day 21 of CVB3 infection [36]. Granulocytes, monocytes, macrophages, and dendritic cells (DCs) contribute to cardiac fibrosis by producing profibrotic cytokines tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β) [36,37]. The differentiation of fibrocytes, which produce type 1 collagen, is largely dependent on CD4 + T cells and the conditions that activate these T cells [38]. The supporting factors supplemented by CD4 + T cells play important roles in the differentiation of monocytes into fibrocytes [38]. The presence of IL-2, IL-4, TNF, or IFN-γ due to polyclonal T-cell activation can inhibit the differentiation of murine monocytes into fibrocytes [38]. Specific combinations of those cytokines resulted in nearly complete suppression of fibrocyte differentiation and collagen deposition [38]. Since numerous immune responses are associated with fibrosis, additional fibrotic cytokines will be reviewed in Section 4.

Impaired Mitochondrial Functions and Ferroptosis Caused by CVB3 Infection and Altered Iron Metabolism
Iron is an essential mineral for energy metabolism and is a component of iron-sulfur cofactors found in several complexes of the mitochondrial respiratory chain [39]. Iron deficiency is a prevalent characteristic of cardiac disorders, such as dilation, left ventricular (LV) hypertrophy, and fibrosis; studies have demonstrated that iron deficiency can reduce cardiomyocyte contractility and relaxation, leading to complications in cardiac function [40,41]. A study on myocarditis and iron homeostasis found that serum from myocarditis patients showed decreased iron levels, along with increased levels of ferritin, an iron-storing protein [42]. This indicates an alteration in iron homeostasis and an increased iron demand during myocarditis [42]. Further, cardiomyocytes treated with serum from myocarditis patient showed elevated levels of transferrin receptor 1 (TFR1), a receptor for iron import that is upregulated in response to low intracellular iron levels [42,43]. TFR1 expression was positively correlated with IL-6 and C-reactive inflammatory protein (CRP) levels from patient sera, suggesting a relationship between inflammation and iron demand [42]. Iron metabolism also showed differences between cardiomyopathy etiologies. Virus-positive cardiomyopathy presented with greater iron demand compared to virusnegative cardiomyopathy, indicating the potential influence of inflammatory profiles on the progression of cardiomyopathy [44]. These findings suggest that inflammation can disrupt iron metabolism and cause iron deficiency, which can impair mitochondrial function and cardiac function.
Iron overload has also been associated with cardiomyopathy. Ferroptosis is a form of cell death caused by excessive amounts of iron, which interferes with the antioxidative functions of glutathione peroxidase (GPx) (Figure 2) [45]. This results in iron-dependent lipid peroxidation and leads to oxidative cell death [45]. During CVB3 infection, the myocardium showed a significant increase in ferrous iron levels, which can lead to the generation of hydroxyl radicals through Fenton reactions and damage biomolecules [46,47]. This was observed along with changes to mitochondrial morphology, such as defective cristae, condensed mitochondrial membranes, and the accumulation of malondialdehyde (MDA), which demonstrates increased lipid peroxidation [46]. Iron accumulation and increased viral RNA transcripts were detected in cardiomyocytes as early as 6 dpi [48]. Iron deposits remained in damaged cardiomyocytes 2 months after CVB3 infection, even after inflammation was diminished [48]. These findings demonstrate that both increases and decreases in iron levels occur during myocarditis and are destructive. However, the determinant of iron levels during infection-whether biological differences, diet, or pathology-is unclear. Green arrows indicate associated stimulative effects, while red arrows indicate associated inhibitory effects. CVB3 has been associated with altered iron homeostasis (both up-and down-regulated). Altered iron homeostasis can impair peroxidase activity or increase inflammation, which contributes to defective mitochondria, metabolic remodeling, and altered immune responses.
The accumulation of calpain-1, the main calpain involved in CVB3-induced myocarditis, was observed in the mitochondria during CVB3 infection [49]. Calpains are proteases activated by intracellular Ca 2+ that are involved in cell necrosis and have been implicated in the development of cardiac fibrosis and dysfunction following long-term CVB3 infection [49]. Calpains initiate pyroptosis, a form of necrotic and inflammatory programmed cell death, by activating the NLR family pyrin domain-containing 3 (NLRP3) inflammasome [49]. Calpain cleaves calcineurin A into its active form, which dephosphorylates dynamin-related protein 1 (Drp-1) and promotes the translocation of Drp-1 from the cytoplasm to the outer mitochondrial membrane [50]. The translocation of Drp-1 activates mitochondrial fission and results in the release of cytochrome c, indicating the activation of apoptosis [50]. CVB3 infection may also contribute to cardiomyocyte apoptosis by increasing Drp-1 expression [50]. Additionally, calpain-1 contributes to the loss of mitochondrial membrane potential (MMP), alterations to mitochondrial structure, and cleavage of ATP synthase-α (ATP5A1) involved in energy production, leading to a decrease in ATP-linked respiration during CVB3 infection [49]. The loss of cardiomyocyte MMP was Green arrows indicate associated stimulative effects, while red arrows indicate associated inhibitory effects. CVB3 has been associated with altered iron homeostasis (both up-and down-regulated). Altered iron homeostasis can impair peroxidase activity or increase inflammation, which contributes to defective mitochondria, metabolic remodeling, and altered immune responses.
The accumulation of calpain-1, the main calpain involved in CVB3-induced myocarditis, was observed in the mitochondria during CVB3 infection [49]. Calpains are proteases activated by intracellular Ca 2+ that are involved in cell necrosis and have been implicated in the development of cardiac fibrosis and dysfunction following long-term CVB3 infection [49]. Calpains initiate pyroptosis, a form of necrotic and inflammatory programmed cell death, by activating the NLR family pyrin domain-containing 3 (NLRP3) inflammasome [49]. Calpain cleaves calcineurin A into its active form, which dephosphorylates dynamin-related protein 1 (Drp-1) and promotes the translocation of Drp-1 from the cytoplasm to the outer mitochondrial membrane [50]. The translocation of Drp-1 activates mitochondrial fission and results in the release of cytochrome c, indicating the activa-tion of apoptosis [50]. CVB3 infection may also contribute to cardiomyocyte apoptosis by increasing Drp-1 expression [50]. Additionally, calpain-1 contributes to the loss of mitochondrial membrane potential (MMP), alterations to mitochondrial structure, and cleavage of ATP synthase-α (ATP5A1) involved in energy production, leading to a decrease in ATP-linked respiration during CVB3 infection [49]. The loss of cardiomyocyte MMP was positively correlated with the amount of CVB3 virions, suggesting that CVB3 infection promotes mitochondrial membrane depolarization in cardiomyocytes [51]. Mitochondria were found to co-localize with lysosomes in CVB3-infected cardiomyocytes and were degraded by PTEN-induced putative kinase protein 1 (PINK1)/Parkin-mediated mitophagy; this finding is consistent with previous observations of mitophagy induced by the loss of MMP [51]. Mitochondrial fragmentation during CVB3 infection resulted in the formation of mitophagosomes that can aid in viral dissemination [51,52]. Additionally, the degradation of RIG-I and MDA5 was observed along with impaired recruitment of MAVS due to Parkin-mediated K48-linked polyubiquitination [51,53]. Since MAVS is essential for mediating interactions with the innate antiviral response kinase TANK-binding kinase 1 (TBK1) and IRF3, Parkin-mediated mitophagy can also result in the suppression of type 1 and 3 IFN production [51,53].

Impaired Lipid and Glucose Metabolism Mediated by CVB3-Induced Inflammation
Anomalies in energy metabolism caused by inflammation can compromise cell and organ function [54]. During acute CVB3-induced myocarditis, a transcriptomic analysis of patients suggests that genes belonging to several metabolic pathways, including fatty acid β-oxidation, the TCA cycle, and the electron transport chain (ETC), are downregulated [54]. ETC complex protein levels and cytochrome c oxidase enzyme activity were decreased, indicating anomalies in mitochondrial oxidative phosphorylation [54]. The infected hearts also expressed decreased levels of mitochondrial transcription factor A (TFAM), which is a mitochondrial biogenesis regulator that enables mitochondrial DNA transcription and maintenance [54][55][56][57]. Since the heart is an energy-demanding organ, impairments in mitochondrial function can significantly contribute to the cardiac diseases [49]. Cardiac metabolic remodeling was supported by the evidence of decreased ATP, ADP, AMP, NAD, and cardiolipin, and an increase in UDP-GlcNAc and arachidonic acid in CVB3-myocarditis hearts [54]. UDP-GlcNAc modifies proteins by O-GlcNAcylation, which can alter protein function and key cellular processes [58]. Cardiolipin is a phospholipid found mainly in the inner mitochondrial membrane; modified species of cardiolipin from remodeling can cause oxidative stress and mitochondrial dysfunction [59]. The oxygenation of arachidonic acid through cyclooxygenases or lipoxygenases leads to the production of prostaglandins and leukotrienes, respectively, which are known mediators of inflammation [60]. Thus, these cardiac metabolites are potential contributors to cardiac remodeling following CVB3 infection.
Adiponectin (APN) is a cytokine secreted by adipose tissues and a regulator of homeostatic pathways such as lipid and glucose metabolism in distant tissues [61]. However, emerging studies have demonstrated higher levels of APN in the coronary sinus compared to the aortic root, suggesting that APN is also synthesized within the heart and may contribute to the development of cardiovascular diseases [62]. APN interrupts TLR3 signaling in cardiac and immune cells by inhibiting the expression of CD14, which is the co-receptor crucial for TLR signaling [63]. In turn, T cell responses are reduced during the sub-acute phase of myocarditis along with diminished viral clearance [63]. In APN knockout mice, the upregulations of IFN-β, IFN-γ, TNF-α, IL-1β, IL-6, and IL-12 were restored to levels comparable to those observed in non-infected mice [63]. During myocarditis, IL-12 signaling increases IL-1β and IL-18, while IL-12 deficiency decreases inflammation and viral replication during myocarditis [64]. In mice, TLR4 deficiency has been shown to decrease levels of IL-1β and IL-18, as well as viral replication and myocarditis [65]. This suggests that TLR4 may share a downstream pathway with IL-12, given the similarities in the pathogenicity between these phenotypes [65]. IL-13 also regulates IL-1β and IL-18 levels by decreasing CASP1 activation, which downregulates the conversion of inactive precursors to active forms of IL-1β and IL-18 [66]. Upregulated T cell proliferation and expression of the pro-inflammatory cytokines IL-1β, IL-18, IFN-γ, TGF-B1, and IL-4 can suppress regulatory T cells, leading to increased anti-cardiac myosin autoantibodies, inflammation, and cardiac fibrosis [66]. APN deficiency accelerates viral clearance while reducing inflammation, necrotic lesions, and the release of troponin I, ultimately maintaining left ventricular function [63].
CVB3 may indirectly cause a reduction in energy metabolism during myocarditis. Notably, oxidative phosphorylation or fatty acid β-oxidation gene expression, including ETC complexes I and III and very long-chain acyl-CoA dehydrogenase (VLCAD), remain unchanged during CVB3 infections lasting up to 72 h [54]. Instead, CVB3 infection increases the expression of cardiac TNF-α, an activator of the NF-κB immunoregulatory pathway, and NF-κB pathway proteins, including NF-κB inhibitor alpha (IκBα) and transcription factor p65 [54]. TNF-α levels were inversely correlated with the expression of VLCAD, ETC complexes I, II and III, and peroxisome proliferator-activated receptor (PPAR) gamma 1 alpha (PGC-1α) [54]. Together, the transcriptomic analyses suggest that CVB3 infection induces the release of cytokines that contribute to energy metabolism anomalies. Increased anaerobic glycolysis is evidenced by increased expressions of glucose transporter-1 (Glut1), lactate dehydrogenase-1 (LDH-1), lactate transporter monocarboxylate transporter-4 (MCT-4), and other glycolytic enzymes [54]. The increase in anaerobic glycolysis suggests that energy production is diverted from the normal aerobic pathway in the mitochondria. This can be a result of insufficient oxygen supply, damage to mitochondrial respiratory chain proteins, or increased energy demand, which can occur when mitochondrial functions are impaired.
The transcripts of fatty acid metabolism regulators PGC-1α, PGC-1β, and PPAR-α were found to be decreased in viral myocarditis hearts [54,57]. PPARs are anti-inflammatory nuclear receptors that can be expressed in immune cells [67]. They function as a transcription factor and can regulate the activity of other transcription factors, such as the inhibition of NF-κB [67]. The activation of PPAR-α inhibits Th17 cell differentiation by suppressing IL-17, IL-6, TGF-B, p-STAT3, and ROR-γt expression, which are critical for Th17 differentiation [68,69]. PPAR-α and its coactivators stimulate lipid catabolism by increasing fatty acid uptake, fatty acid oxidation, and lipoprotein assembly; they are highly expressed in organs that depend largely on oxidative metabolism for energy [70][71][72]. The deficiency of PGC-1α and PGC-1β has been shown to accelerate heart failure, leading to oxidative stress, decreased cardiac deficiency, and reduced glucose metabolism [54,73,74]. These observations coincided with decreased ejection fraction and fractional shortening. Moreover, these findings suggest that metabolic remodeling begins as early as acute myocarditis, it occurs before structural remodeling, and it contributes to cardiomyocyte dysfunction by driving cell death [54].

Immune-Associated Cells and Genes That Influence Cardiac Remodeling and Dilated Cardiomyopathy (DCM) Development
In response to infection, innate immune cells are recruited to tissues to phagocytose pathogens and mediate pro-inflammatory and antiviral responses [75]. Adaptive immune cells include T cells and B cells with different subsets, cytokine profiles, and memory and effector functions [76,77]. Fibroblasts are involved in injury repair and produce connective tissue such as collagen to provide the structural framework for organs and tissue [78]. This section will review various genes expressed by immune cells and fibroblasts that contribute to the duration and severity of myocarditis and development of DCM (Table 1).

Monocytes
Monocytes are recruited to infected tissues and can differentiate into macrophages or DCs to phagocytose pathogens and mediate pro-inflammatory and antiviral responses. Heart tissue biopsies from patients with myocarditis revealed an increase in the number of monocytes that express CCR2 [90]. CCR2 facilitates monocyte egression from the bone marrow into the blood and their subsequent migration into inflammatory sites [91]. The silencing of CCR2 in CVB3-infected mice significantly was shown to reduce the accumulation of lymphocyte antigen 6 complex (Ly6C) high monocytes in cardiac tissue during acute myocarditis [90]. The presence of Ly6C high monocytes is crucial for CD8 + T cell responses, as demonstrated by a shift in immune responses when these monocytes are absent; this shift is characterized by increased bone marrow neutrophil expansion and preserved cardiac function, as measured by left ventricular ejection fraction [36,90]. Delayed silencing of CCR2 improved late-stage disease outcomes for myocarditis in mice at 60 dpi by reducing inflammation and fibrosis [90]. Monocyte recruitment is also mediated by stabilin-1, a receptor expressed on myeloid cells that facilitates adhesion with extracellular fibronectin [88]. Stabilin-1 deficiency led to a significant reduction in the recruitment of both pro-inflammatory (Ly6C high ) and anti-inflammatory (Ly6C low ) monocytes [88,89]. Since stabilin-1 regulates monocyte differentiation into regulatory macrophages, a deficiency in stabilin-1 drastically reduces the number of cardiac anti-inflammatory macrophages, which are crucial for suppressing T cell responses [88]. Thus, stabilin-1-KO mice showed increased pro-inflammatory responses, cardiac necrosis, and mortality during myocarditis [88].
Following CVB3 infection, monocytes displayed differentially expressed genes in three different clusters. Monocyte cluster A had anti-inflammatory and tissue repair functions with upregulated levels of M2 macrophage markers [113]. This cluster helped with myocardial repair and cardiomyocyte cell survival by expressing increased transcript levels of transforming growth factor-beta-induced protein (TGFBI) and S100 calcium binding protein A4 (S100A4) [113]. Monocyte cluster B regulated pathways involved in cardiac muscle contraction and hypoxia by expressing upregulated levels of CCL24, CCL2, galectin 1 (Gal-1), and WAP four-disulfide core domain 17 (WFDC17) transcripts [113]. Monocyte cluster C also had an anti-inflammatory phenotype, expressing increased levels of the inhibitory Fc receptor Fc gamma receptor IIb (FCGR2B), IL-1β suppressor membrane-spanning 4-domains (MS4A6D), and arginine metabolism activator glycine amidinotransferase (GATM) [113]. Thus, the different populations of monocytes that infiltrate the heart at 21 dpi have increased expressions of genes, contributing to an anti-inflammatory state [113].

Macrophages
Interleukin-1 receptor-associated kinase 4 (IRAK4) is a signal transducer that mediates the translocation of NF-κB after TLR or T cell receptor (TCR) activation [100]. While IRAK4 supports the production of pro-inflammatory cytokines, it also promotes CVB3 infection by blocking the migration of early protective macrophages and the production of IFNs [100]. In mice with myocarditis, IRAK4 deficiency improves heart functions and partially reverses the decrease in fractional shortening caused by CVB3 infection [100]. Since IRAK4 negatively regulates Stat5, the major transcription factor of CCR5, it limits CCR5dependent migration of monocytes to the heart during myocarditis [100]. Interestingly, IRAK4 only modulates CCR5 expression in monocytes and macrophages; it does not modulate T cells which also express CCR5 [100]. The absence of CCL5 and CCR5 in mice led to higher mortality rates and enhanced viral proliferation, which demonstrates that infiltrating CD11b + F4/80 + monocytes and macrophages at 2 dpi play a crucial role for containing the infection [100]. IRAK4 also contributed to the dimerization of IRFs, which are transcription factors involved in the synthesis of type 1 IFNs [30,114]. IRF5/IRF7 heterodimers inhibit the transcription of IFN-α, while IRF5 homodimers can activate IFN-α transcription [114]. IRAK4 has been shown to favor IRF5/IRF7 heterodimerization in macrophages, thus inhibiting type 1 IFN-mediated antiviral responses [100]. Altogether, IRAK4 exacerbates myocarditis by decreasing the early influx of protective monocytes and macrophages to the heart, as well as inhibiting type 1 IFN production, which ultimately benefits viral replication. Type 1 IFN signaling is also inhibited through the STAT3 signaling pathway by TRIM18, an E3 ubiquitin ligase that mediates the ubiquitination of protein phosphatase 1A (PPM1A) [112]. TRIM18 recruits PPM1A to dephosphorylate and inactivate TBK1 [112]. Thus, TBK1 can no longer interact with its upstream adaptors MAVS and STING and attenuates type 1 IFN signaling [112]. The knockdown of TRIM18 considerably increases type 1 IFN signaling and has been shown to increase IFN-α and IFN-β production by macrophages during viral myocarditis [112].
Substantial amounts of infiltrating macrophages can be observed in the myocardium during CVB3 infection [97]. These macrophages secrete IL-1β, which is a cytokine implicated in the transition from acute myocarditis to inflammatory cardiomyopathy [97,115,116]. During acute myocarditis, IL-1β elevates the expression of matricellular proteins tenascin C (TN-C) and osteopontin (OPN) [97]. TN-C is an activator of pro-fibrotic responses in fibroblasts and increases the rate of fibroblast migration across injured tissues and collagen deposition [107]. Additionally, OPN induces fibrotic tissue development and cardiac remodeling by promoting the formation of insoluble collagen, which contributes to LV stiffness and the development of systolic dysfunction [101]. IL-1β also decreases cardiac lymphatic muscle cell contractility in a dose-dependent manner [98]. This may hinder the clearance of inflammatory cells and cytokines and prolong tissue damage, leading to cardiomyopathy [98]. While the impediment of viral clearance is a concern when using antiinflammatory therapies, the neutralization of IL-1β can prevent fibrosis without increasing viral load in the heart and presents a potential therapeutic target [97].
Macrophages secrete large amounts of Gal-3 during acute and chronic CVB3-induced myocarditis to mediate both the migration of macrophages to fibroblasts and the transformation of fibroblasts into matrix-secreting myofibroblasts [93,117,118]. Although the inhibition of Gal-3 may not prevent viral clearance, it reduces acute inflammation, collagen secretion, and the expression of A-and B-type natriuretic peptide (ANP and BNP) mRNA, which are markers of cardiac dysfunction [93]. The absence of macrophages leads to a reduction in Gal-3 + cells in the heart normally observed during CVB3 infection, a decline in myofibroblast activation, and fewer collagen deposits [93]. This resulted in viral clearance by 30 dpi and the attenuation of acute myocarditis [93]. These findings demonstrate that inflammatory macrophages encourage the development of fibrosis following myocarditis by secreting Gal-3. Cardiac-infiltrating macrophages express upregulated levels of dipeptidase 2 (Dpep2) during myocarditis [79]. Dpep2 protects mice from inflammatory macrophages in the acute phase by suppressing the activation of the NF-κB pathway to limit the expression of TNF-α, IL-6, and monocyte chemoattractant protein 1 (MCP-1) in response to infection [79].

Neutrophils
The increase in neutrophils circulating in both the blood and heart can be detected within 24 h of infection [36]. On day 3, neutrophils were found to be more abundant in the heart compared to macrophages and monocytes, making them the predominant immune cell present [36]. Gene ontology analyses identified the upregulation of pathways involved with IL-17, NF-κB, TNF, IL-1β and IL-4 signaling, cell death, and viral life cycle regulators in neutrophils during myocarditis [113].
Damage-associated molecular patterns (DAMPs) are immune-activating proteins that are secreted after cell injury or infection and include interleukins, heat-shock proteins, highmobility group box 1 (HMGB1), and S100 proteins [119,120]. S100A8 and S100A9 are mainly expressed and secreted by neutrophils and monocytes upon cellular activation [121][122][123]. They are implicated in inflammation, including the release of IL-6, IL-8, CCL2, CCL20, and CXCL10 in tendinopathy and the activation of neutrophils in COVID-19 patients [124,125]. The activation of NADPH oxidase 1 (Nox1) to produce reactive oxygen species (ROS) can be initiated by the binding of S100A9 to the receptor for advanced glycation end products (RAGE) [104]. Monocyte-derived macrophages overexpressing S100A8 and S100A9 show increased ROS production and IL-10 mRNA expression [123]. The increase in IL-10 may be a defensive mechanism since controlled macrophage activation is influenced by the levels of IFN-γ and IL-10 [126]. IL-10 deficiency is associated with the uncontrolled production of iNOS, which catalyzes the production of nitric oxide (NO) and contributes to ongoing tissue damage [126]. S100A9 deficiency in CVB3-infected mice leads to a significant decrease in RAGE-dependent ROS production in the left ventricle, which can be reversed with S100A9 supplementation [104]. Moreover, S100A9 deficiency is associated with reduced numbers of neutrophils and monocytes in the left ventricle, as well as decreased CXCL2 secretion by monocytes and neutrophils at inflammation sites [104]. This finding suggests that S100A9 promotes the accumulation of monocytes and neutrophils in the left ventricle during myocarditis. S100A9 may also contribute to tissue damage by increasing granulocyte activity or causing mitochondrial dysfunction. A recent study has demonstrated that S100A8 and S100A9 are released by neutrophils recruited to infarcts in the heart [127]. S100 proteins can bind to TLR4s on naïve neutrophils to stimulate IL-1β secretion and induce granulocyte production [127]. S100A8 and S100A9 have been demonstrated to cause mitochondrial respiratory dysfunction by TLR4 signaling that inhibits mitochondrial complex 1, resulting in the death of cardiomyocytes in mice with ischemic/reperfusion injury [105]. After CVB3 infection, the absence of S100A9 was associated with improved global heart function, including ejection fraction, cardiac output, stroke work function, and systolic and diastolic functions [104]. Endomyocardial biopsies from patients with CVB3-positive myocarditis expressed a significant increase in S100A8 and S100A9 compared to healthy controls [104]. These levels were reduced once the virus was cleared, suggesting that S100A8 and S100A9 expressions are induced by CVB3 infection [104]. CVB3 infection notably results in increased levels of S100A8 and S100A9 in the left ventricles of mice [104]. However, S100A8 and S100A9 are not differentially expressed in CVB3-negative DCM patients, further suggesting that their expression levels are dependent on the viral load [104]. Interestingly, S100A9 has been associated with higher expression of coxsackievirus and adenovirus re-ceptor (CAR) mRNA and decreased IFN-β in CVB3-infected mice, which can contribute to the increase in CVB3 RNA copy numbers in mice [104]. IFN-β deficiency increases susceptibility to disease by downregulating the expression of ISGs, including 2 -5 OAS, which degrades both viral and host RNA, and GTPase Mx, which binds to the viral components of various viruses to block their functions [18,128]. The current findings suggest that S100A8 and S100A9 aggravate CVB3-induced myocarditis by increasing the accumulation of myeloid cells that can release tissue-damaging granules, are mainly involved in the earlier stages of disease that leads to impaired heart functions, and do not appear to play a role in DCM when the virus is absent [104].
Both Th1 and Th17 cells infiltrate the heart during myocarditis. However, TH17 responses result in more tissue damage than Th1 cell responses [96,136]. Compared to Th1 cells, a greater number of Th17 cells co-secrete TNF-α and IL-6, which are associated with heart failure [96,136]. IL-10 secretion is also reduced in Th17 cells compared to Th1 cells [96,136]. CD80 and CD86 are costimulatory molecules expressed by antigenpresenting cells; both molecules can interact with CD28 and CTLA-4 receptors on T cells to regulate T cell activation and cytokine production [137]. Both CD80 and CD86 arose from gene duplication and have shared functions; however, only CD80 exacerbates myocarditis [92]. CD80 was found to upregulate ROR-γt expression in heart tissue, leading to increased production of IL-17 by CD4 + T cells and promoting Th17 cell differentiation [92,138]. Although CD86 alone does not affect Th17 cell populations, it is involved in the balance of Th17 cells by neutralizing the effects of CD80 [92]. IL-17A, which is largely secreted by Th17 cells, affects the proportions of infiltrating cell populations [96]. It mediates the recruitment of monocytes, eosinophils, and neutrophils to the heart by upregulating several chemokines that direct their migration, including CCL2, CCL3, CCL5, CXCL1, and CXCL10 [96]. Interestingly, while the secretion of IL-17A by Th17 cells may not contribute to the severity of CVB3-induced myocarditis, it plays a role in the development of DCM [96]. The pathogenic effects of IL-17A may depend on the induction of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, as well as pro-fibrotic cytokine TGF-β1, which were abrogated in the absence of IL-17A [96]. Continual TNF-α secretion can negate improvements in both cardiomyocyte contraction and relaxation mediated by cardiac microvascular endothelial cells (CMECs) [108]. Elevated levels of circulating TNF-α are detected in patients with DCM and have been associated with cachexia [139,140]. The elevated TNF-α levels coincided with inflammatory cell accumulation and fibrogenic responses, contributing to cardiac dysfunction [109]. In mice, TNF-α deficiency attenuates cardiac CASP8 activation and apoptosis in response to pressure overload, demonstrating that TNF-α contributes to cardiac remodeling through caspase-dependent myocyte apoptosis [109]. IL-6 receptor blockade has been shown to attenuate CVB3-induced production of TNF-α [99]. IL-17A mediates left ventricular dilation and is essential for increasing left ventricular end-systolic and end-diastolic diameters [96]. This cytokine also compromises myocardial contractility and contributes to decreased fractional shortening and ejection fraction [96]. IL-17A is associated with fibrotic tissue development and coincides with increasing type 1 and type 3 collagen deposition, gelatinase, matrix metalloproteinase 2 (MMP-2), and MMP-9 biomarkers for severe cardiac remodeling in patients; it is also associated with decreasing tissue inhibitor of metalloproteinases 1 (Timp1) and Timp4 [96,141]. MMPs are enzymes involved in the degradation of ECM components, such as signaling molecules, for release, activation, and inactivation of cytokines, as well as the migration of immune cells [142]. MMP-9 was shown to be necessary for attenuating acute CVB3 infection, presumably by mediating the degradation of IFN-β and IFN-γ [143].
IL-9 is largely secreted by CD8 + T cells on day 5 and by CD4 + T cells on day 7 of infection [81]. IL-9 inhibits viral replication by reducing CAR expression on cardiomyocytes [81]. The knockout of IL-9 decreases TGF-B secretion while increasing IL-17a secretion [81]. The synergistic enrichment of IL-9, IL-3, IL-4, IL-13, and IL-15 during acute myocarditis can be cardioprotective by significantly increasing the number of cardiac helper T cells, shrinking necrotic lesion sizes, and limiting CVB3 genomes [80]. These synergistic changes can prevent cardiac remodeling and fibrosis, decrease end-diastolic dilatation, and improve systolic function 5 weeks post-infection [80]. IL-4 and IL-13 promote CCL11 secretion by fibroblasts [144]. The overexpression of IL-9 significantly reduces the percentage of Th17 and Th22 cells, which are adversely linked to cardiac remodeling and increased anti-inflammatory Th2 cells at 14 dpi [80].
During myocarditis, signal transducer and activator of transcription 4 (STAT4) promotes the NF-κB pathway by increasing IKBα and p65 phosphorylation in myocardial cells [106]. STAT4-driven NF-κB activity upregulates Th1 inflammatory cytokines IFN-γ and IL-2, while downregulating Th2 anti-inflammatory cytokines IL-6 and IL-10 [106]. The overexpression of STAT4 results in increased focal necrosis, fibrosis, and interstitial hyperplasia, while the silencing of STAT4 and the NF-κB pathway represses the development of myocarditis [106]. STAT4 and Janus kinase 2 (JAK2) phosphorylation can be decreased by progranulin (PGRN), which is a pleiotropic growth factor and adipokine that is constitutively expressed in certain epithelial cells and myeloid-and lymphoid-derived cells [87,145]. PGRN is significantly increased in plasma and cardiac tissues during sub-acute (7 dpi) myocarditis to attenuate CVB3 replication [87]. PGRN is also negatively correlated with cardiac inflammation and the loss of body weight in mice [87]. PGRN directly inhibits Th1 differentiation by decreasing the phosphorylation of JAK2 and STAT4; it also inhibits Th17 cell differentiation by decreasing JAK3 and STAT3 phosphorylation [87]. Mice with PGRN deficiency show significant upregulation in Th1 and Th17 phenotypes and severe myocarditis with necrosis [87]. The pro-inflammatory cytokines IFN-γ, TNF-α, IL-17A, and IL-21 were also found to be upregulated in the absence of PGRN [87]. During CVB3induced myocarditis, IL-21 promotes the activation of CD8 + T cells, which may include autoimmune CD8 + T cells that target cardiac tissue [146]. Upregulation of CD1d recognized by γδ T cells to activate autoimmune CD8 + T cells requires both an active infection and the presence of TNF-α signaling [110].

B Cells
B cells are important regulators of left ventricular contractility and myocardial leukocytes, including the balance of different myeloid-derived cells and T cell populations [147]. Myocarditis significantly increases B cell activation and antigen presentation, which was observed alongside increased CD69, CD40, CD80, and MHCII expression 7 dpi [148]. B cells can contribute to the development of viral myocarditis through antigen presentation and cytokine production to alter the balance of T cell populations. TNF-α, IL-6, and IL-17 secretion by B cells promoted Th1 and Th17 differentiation during myocarditis, while the downregulation of IL-4 significantly limited Th2 differentiation [148].
B10 cells are regulatory B cells that can limit inflammation by producing IL-10. A significant increase in B10 cells has been observed in the heart at around day 7 of myocarditis [86]. B10 cells can alleviate myocardial damage by decreasing the amount of Th17 infiltrates [86,149]. This population of B cells can downregulate the expression of T-bet and ROR-γt, which are transcription factors for Th1 and Th17 phenotypes, respectively [86,149]. The suppression of Th17 differentiation by B10 cells can be enhanced by prostaglandin E2 (PGE2) treatment [86]. PGE2 is a lipid molecule produced by fibroblasts and macrophages that has anti-inflammatory effects on immune cells, including neutrophils and macrophages [83][84][85]. PGE2 induces the expansion of B10 cells by activating AP-1 and AhR, which are transcription factors that promote the expression of IL-10 [86]. Treatment with PGE2 enhances B10 cell suppression of Th17 differentiation, reduces inflammatory infiltrates, and restrains cardiac hypertrophy during myocarditis [86]. However, B cells in DCM patients were found to secrete elevated levels of TNF-α in comparison to antiinflammatory cytokines, such as IL-10 [150].
IL-10 is an anti-inflammatory cytokine that inhibits the release of pro-inflammatory cytokines, including TNF-α; thus, it also inhibits myocyte apoptosis induced by TNF-α [82]. TNF-α-induced apoptosis involves the production of ROS by sphingomyelinases through a ceramide-dependent signaling pathway [111]. ROS products can act upon cellular macromolecules and promote lipid degradation and enzyme inactivation [151]. Consequently, cellular respiration and DNA synthesis are inhibited in response to the damage of macromolecules, leading to apoptosis [151]. Nevertheless, superoxides, a major ROS species, can react with NO and cause its depletion from cardiac microvascular endothelial cells, which require NO to favorably enhance cardiomyocyte contraction and relaxation [108]. Patients with DCM and mild congestive heart failure show significant oxidative stress, which can contribute to cardiomyopathic changes [152]. IL-10 interrupts the pro-apoptotic pathway induced by TNF-αby downregulating IKK phosphorylation in manner dependent on the phosphorylation of ERK1/2 MAPK [82].

Fibroblasts
Fibrotic and inflammatory responses work together to promote the healing of damaged tissue caused by pathogen infection and subsequent clearance. Several populations of fibroblasts express elevated levels of C3 and C4b complement proteins; serine protease inhibitor A3N (Serpina3n), which promotes wound healing; and IFN-induced transmembrane protein (Ifitm) during myocarditis [113]. CVB3 triggers the release of TNF-α, IL-1β, IL-6, and CCL2 by fibroblasts; however, it does trigger the release of profibrotic mediators, such as Col I, Col III, or TGF-β [153]. Instead, fibroblasts secrete early stem cell factor (SCF), which triggers mast cells to degranulate and secrete more TNF-α [153]. In return, TNF-α stimulates the production of Col I, Col III, and TGF-β by fibroblasts [153]. The absence of mast cells is associated with reduced fibrosis and improved cardiac dysfunction [153]. Thus, the interaction between mast cells and fibroblasts is crucial for activating fibroblast functions and promoting the generation of fibrotic tissue.
Cardiomyocytes and cardiac fibroblasts were identified as the main producers of IL-6 during myocarditis [99]. IL-6 signaling is highly correlated with ICAM expressions that are important for adhesion and recruitment [99]. When stimulated by IL-6, cardiac fibroblasts produce more IL-6 in a positive feedback loop, which can lead to increased cell necrosis and immune cell infiltration [99]. Mice treated with IL-6 receptor blockade have reduced numbers of infiltrating T cells and macrophages, potentially due to the downregulation of the adhesion molecules that mediate lymphocyte migration [99]. IL-6 receptor blockade during CVB3 infection has been observed to alter titin phosphorylation. Sarcomeres are the basic contractile unit that forms muscle fibers and are composed of different structural protein filaments, including titin. Titin functions like a molecular spring and contains numerous phosphorylation sites that can increase or decrease myocyte stiffness [154]. Phosphorylation of the cardiac-specific N2-B unique sequence (N2-Bus) decreases myocyte stiffness, while phosphorylation of the titin domain rich in proline, glutamate, valine, and lysine (PEVK) increases stiffness [155]. The differing effects are caused by the amino acid charges in the sequence. Phosphorylation of the basic regions in the PEVK domain reduces electrostatic repulsion, which leads to increased intramolecular interactions, thus increasing stiffness [156]. Failing hearts of DCM and hypertrophic cardiomyopathy (HCM) patients displayed N2-Bus hypophosphorylation and PEVK hyperphosphorylation, both of which can contribute to increased myocyte stiffness [156]. IL-6 has been observed to increase myocyte stiffness by inducing titin phosphorylation at the PEVK region [155]. The neutralization of IL-1β markedly reduces the expression of IL-6, which is essential for inducing Th17-mediated pro-inflammatory responses [97,157].
Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated by proteases cleaving the extracellular N-terminus of PAR, which in turn produces a tethered activating ligand [158,159]. Depending on the protease and the resulting ligand sequence produced, activated PARs can couple with different G protein α-subtypes that determine the downstream signaling pathway, such as inflammatory responses, by activating NF-κB [159]. Activated PAR1 in cardiac fibroblasts can stimulate TLR3 to upregulate the phosphorylation of P38 [102]. Phosphorylated P38 promotes the production of CXCL10 [102]. CXCL10 is an ISG that recruits NK cells and CD3 + leukocytes to eliminate infected cells and contain the viral infection [160]. However, PAR1 activity may enhance viral replication by reducing autophagic flux [161,162]. The reduction in autophagic flux prevents the degradation of viral RNA, viral proteins, and autophagosomes, leading to an accumulation of autophagosome membranes that can be exploited as scaffolds for virus assembly [162]. Activated PAR2 can directly interact with TLR3 to inhibit TLR3-mediated IFN-β production, which normally stimulates antiviral responses [103]. Thus, the decrease in IFN-β signaling due to PAR2 activation coincides with increased viral replication [103]. PAR2 is also associated with increased expressions of CAR and decay-accelerating factor (DAF), which is the coreceptor of CAR and can increase the rate of viral infection [103]. Mice expressing PAR2 present persisting levels of viral titer, increased CD68 + and CD3 + cell infiltrates in the heart, more severe tissue damage, significantly higher expressions of TNF-α, IL-6, IL-1β, CCL2, CCL5, and IFN-γ at 8 dpi, and a significant reduction in left ventricular function at 28 dpi compared to PAR2-KO mice [103]. These results suggest that PAR2 impedes the innate immune response and viral clearance by blocking TLR3-signaling, leading to more aggressive adaptive immune responses. These findings were consistent with clinical studies in which patients who expressed higher levels of PAR2 secreted lower levels of IFN-β, had more CD3 + , CD45 + , and myocardial infiltrates, and showed impaired heart function measured by left ventricular ejection fraction (LVEF) [103].

Perspectives
The collection of evidence demonstrates that CVB3-induced myocarditis can impair cardiac function by cleaving host proteins involved in the immune response, including MAVs, TRIF, NFAT5, SQSTM1, and CARD8, to impair host defense mechanisms and promote pyroptosis and apoptosis. CVB3 infection impairs mitochondrial functions through multiple pathways, including altered iron homeostasis and inflammation. Mitophagy following CVB3 infection is inter-related with the immune response, including the inhibition of IFNs. Differential cytokine expression and immune cell phenotypes play an extensive role in determining the outcome of myocarditis. The shift in immune profiles during infection can promote tissue damage, as well as the development of DCM and heart failure. Given that some patients remain asymptomatic while others develop heart failure, biological differences in immune profiles can be significant contributors to disease outcomes. Thus, recently discovered immune-associated genes that mediate the transition from myocarditis to cardiac dysfunction are critical targets for pathology and therapeutic development. As immunostimulant and suppressive therapies have shown inconsistencies due to varying time points of administration and patient variance, it is crucial to understand the complex network of immune cells and cytokines to personalize therapy based on factors including a patient's genetic profile or the severity of DCM. In genetics research, a focus on identifying new biomarkers may help identify early signs of myocarditis and DCM progression for diagnosis and treatment purposes. Moreover, future research could focus on identifying new genes that lead to DCM and how different mutations in the same gene can impact the severity of DCM. The use of bioinformatics analyses will lead to a better understanding of the relationships between an array of networks that impact the outcomes of myocarditis, including iron, lipid, and glucose metabolism, viral protease activity, cytokine expression, and immune cell responses.