Parkin plays a crucial role in acute viral myocarditis by regulating mitophagy activity.

Rationale: Parkin (an E3 ubiquitin protein ligase) is an important regulator of mitophagy. However, the role of Parkin in viral myocarditis (VMC) remains unclear. Methods: Coxsackievirus B3 (CVB3) infection was induced in mice to create VMC. Cardiac function and inflammatory response were evaluated by echocardiography, histological assessment, and molecular analyses. AAV9 (adeno-associated virus 9), transmission electron microscopy (TEM) and western blotting were used to investigate the mechanisms by which Parkin regulates mitophagy and cardiac inflammation. Results: Our data indicated that Parkin- and BNIP3 (BCL2 interacting protein 3 like)-mediated mitophagy was activated in VMC mice and neonatal rat cardiac myocytes (NRCMs) infected with CVB3, which blocked autophagic flux by inhibiting autophagosome-lysosome fusion. Parkin silencing aggravated mortality and accelerated the development of cardiac dysfunction in CVB3-treated mice. While silencing of Parkin did not significantly increase inflammatory response through activating NF-κB pathway and production of inflammatory cytokines post-VMC, the mitophagy activity were reduced, which stimulated the accumulation of damaged mitochondria. Moreover, Parkin silencing exacerbated VMC-induced apoptosis. We consistently found that Parkin knockdown disrupted mitophagy activity and inflammatory response in NRCMs. Conclusion: This study elucidated the important role of Parkin in maintaining cardiac function and inflammatory response by regulating mitophagy activity and the NF-κB pathway during acute VMC. Although the functional impact of mitophagy remains unclear, our findings suggest that Parkin silencing may accelerate VMC development.

Coxsackievirus group B3 regulates ASS1-mediated metabolic reprogramming and promotes macrophage inflammatory polarization in viral myocarditis.

Coxsackievirus group B3 (CVB3) belongs to the genus Enteroviruses of the family Picornaviridae and is the main pathogen underlying viral myocarditis (VMC). No specific therapeutic is available for this condition. Argininosuccinate synthase 1 (ASS1) is a key enzyme in the urea cycle that converts citrulline and aspartic acid to argininosuccinate. Here, we found that CVB3 and its capsid protein VP2 inhibit the autophagic degradation of ASS1 and that CVB3 consumes citrulline to upregulate ASS1, triggers urea cycle metabolic reprogramming, and then activates macrophages to develop pro-inflammatory polarization, thereby promoting the occurrence and development of VMC. Conversely, citrulline supplementation to prevent depletion can downregulate ASS1, rescue macrophage polarization, and alleviate the pathogenicity of VMC. These findings provide a new perspective on the occurrence and development of VMC, revealing ASS1 as a potential new target for treating this disease. IMPORTANCE: Viral myocarditis (VMC) is a common and potentially life-threatening myocardial inflammatory disease, most commonly caused by CVB3 infection. So far, the pathogenesis of VMC caused by CVB3 is mainly focused on two aspects: one is the direct myocardial injury caused by a large number of viral replication in the early stage of infection, and the other is the local immune cell infiltration and inflammatory damage of the myocardium in the adaptive immune response stage. There are few studies on the early innate immunity of CVB3 infection in myocardial tissue, but the appearance of macrophages in the early stage of CVB3 infection suggests that they can play a regulatory role as early innate immune response cells in myocardial tissue. Here, we discovered a possible new mechanism of VMC caused by CVB3, revealed new drug targets for anti-CVB3, and discovered the therapeutic potential of citrulline for VMC.

Identification of the proteolytic signature in CVB3-infected cells.

Coxsackievirus B3 (CVB3) encodes proteinases that are essential for processing of the translated viral polyprotein. Viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. While some host protein substrates of the CVB3 3C and 2A cysteine proteinases have been identified, the full repertoire of targets is not known. Here, we utilize an unbiased quantitative proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) to conduct a global analysis of CVB3 protease-generated N-terminal peptides in both human HeLa and mouse cardiomyocyte (HL-1) cell lines infected with CVB3. We identified >800 proteins that are cleaved in CVB3-infected HeLa and HL-1 cells including the viral polyprotein, known substrates of viral 3C proteinase such as PABP, DDX58, and HNRNPs M, K, and D and novel cellular proteins. Network and GO-term analysis showed an enrichment in biological processes including immune response and activation, RNA processing, and lipid metabolism. We validated a subset of candidate substrates that are cleaved under CVB3 infection and some are direct targets of 3C proteinase in vitro. Moreover, depletion of a subset of TAILS-identified target proteins decreased viral yield. Characterization of two target proteins showed that expression of 3Cpro-targeted cleaved fragments of emerin and aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 modulated autophagy and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, respectively. The comprehensive identification of host proteins targeted during virus infection provides insights into the cellular pathways manipulated to facilitate infection. IMPORTANCE: RNA viruses encode proteases that are responsible for processing viral proteins into their mature form. Viral proteases also target and cleave host cellular proteins; however, the full catalog of these target proteins is incomplete. We use a technique called terminal amine isotopic labeling of substrates (TAILS), an N-terminomics to identify host proteins that are cleaved under virus infection. We identify hundreds of cellular proteins that are cleaved under infection, some of which are targeted directly by viral protease. Revealing these target proteins provides insights into the host cellular pathways and antiviral signaling factors that are modulated to promote virus infection and potentially leading to virus-induced pathogenesis.

CaMKIIδ, Stabilized by RNA N6-Methyladenosine Reader IGF2BP2, Boosts Coxsackievirus B3-Induced Myocardial Inflammation via Interacting with TIRAP.

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to be aberrantly activated in viral myocarditis (VMC), but the role of its subtype CaMKIIδ in VMC remains unclear.VMC mice and cardiomyocytes models were induced by Coxsackievirus B3 (CVB3) treatment. Mice that underwent sham surgery and saline-treated cardiomyocytes served as controls. Body weight, survival, left ventricular ejection fraction (LVEF), and fractional shortening (LVFS) were measured, and HE staining was performed to evaluate heart function in VMC mice model and sham control. Inflammation factors in serum or cell supernatant were detected by ELISA. Expressions of CaMKIIδ, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), nuclear factor NF-kappaB (NF-κB) signals, and inflammation factors were examined by quantitative real time polymerase chain reaction (qRT-PCR) or western blot. CCK-8, EdU, and flow cytometry were used to evaluate cell behaviors. Co-immunoprecipitation (Co-IP), RNA immunoprecipitation (RIP), and RNA pull-down were utilized to validate molecule interaction. Methylated RNA immunoprecipitation (MeRIP) was performed to measure N6-methyladenosine (m6A) level of specific molecule.CaMKIIδ was upregulated in VMC mice and CVB3-treated primary cardiomyocytes, of which knockdown improved cell viability, proliferation, and suppressed cell apoptosis in vitro, thereby alleviating myocarditis in vivo. The stability of CaMKIIδ was attributed to the presence of IGF2BP2 through m6A modification. Loss of CaMKIIδ repressed NF-κB pathway via negatively and directly regulating TIRAP to be involved in inflammatory damage.CaMKIIδ, stabilized by m6A reader IGF2BP2, modulated NF-κB pathway via interacting with TIRAP to alter cell viability, proliferation, and apoptosis, thereby affecting VMC outcome.

Acetylation of FOXO1 activates Bim expression involved in CVB3 induced cardiomyocyte apoptosis.

Viral myocarditis (VMC) is the major reason for sudden cardiac death among both children and young adults. Of these, coxsackievirus B3 (CVB3) is the most common causative agent of myocarditis. Recently, the role of signaling pathways in the pathogenesis of VMC has been evaluated in several studies, which has provided a new perspective on identifying potential therapeutic targets for this hitherto incurable disease. In the present study, in vivo and in vitro experiments showed that CVB3 infection leads to increased Bim expression and triggers apoptosis. In addition, by knocking down Bim using RNAi, we further confirmed the biological function of Bim in apoptosis induced by CVB3 infection. We additionally found that Bim and forkhead box O1 class (FOXO1) inhibition significantly increased the viability of CVB3-infected cells while blocking viral replication and viral release. Moreover, CVB3-induced Bim expression was directly dependent on FOXO1 acetylation, which is catalyzed by the co-regulation of CBP and SirTs. Furthermore, the acetylation of FOXO1 was an important step in Bim activation and apoptosis induced by CVB3 infection. The findings of this study suggest that CVB3 infection induces apoptosis through the FOXO1 acetylation-Bim pathway, thus providing new insights for developing potential therapeutic targets for enteroviral myocarditis.

Coxsackievirus A6 2C protein antagonizes IFN-ß production through MDA5 and RIG-I depletion.

IMPORTANCE: Coxsackievirus A6 (CV-A6) is a major emerging pathogen associated with atypical hand, foot, and mouth disease and can cause serious complications such as encephalitis, acute flaccid paralysis, and neurorespiratory syndrome. Therefore, revealing the associated pathogenic mechanisms could benefit the control of CV-A6 infections. In this study, we demonstrate that the nonstructural 2CCV-A6 suppresses IFN-ß production, which supports CV-A6 infection. This is achieved by depleting RNA sensors such as melanoma differentiation-associated gene 5 and retinoic acid-inducible gene I (RIG-I) through the lysosomal pathway. Such a function is shared by 2CEV-A71 and 2CCV-B3 but not 2CCV-A16, suggesting the latter might have an alternative way to promote viral replication. This study broadens our understanding of enterovirus 2C protein regulation of the RIG-I-like receptor signaling pathway and reveals a novel mechanism by which CV-A6 and other enteroviruses evade the host innate immune response. These findings on 2C may provide new therapeutic targets for the development of effective inhibitors against CV-A6 and other enterovirus infections.

Macrophage CAPN4 regulates CVB3-induced cardiac inflammation and injury by promoting NLRP3 inflammasome activation and phenotypic transformation to the inflammatory subtype.

Exploring the immune mechanism of coxsackievirus B3 (CVB3)-induced myocarditis may provide a promising therapeutic strategy. Here, we investigated the regulatory role of macrophage CAPN4 in the phenotypic transformation of macrophages and NOD-like receptor protein 3 (NLRP3) inflammasome activation. We found that CAPN4 was the most upregulated subtype of the calpain family in CVB3-infected bone marrow-derived macrophages (BMDMs) and Raw 264.7 cells after CVB3 infection and was upregulated in cardiac macrophages from CVB3-infected mice. Conditional knockout of CAPN4 (CAPN4flox/flox; LYZ2-Cre, CAPN4-cKO mice) ameliorated inflammation and myocardial injury and improved cardiac function and survival after CVB3 infection. Enrichment analysis revealed that macrophage differentiation and the interleukin signaling pathway were the most predominant biological processes in macrophages after CVB3 infection. We further found that CVB3 infection and the overexpression of CAPN4 promoted macrophage M1 polarization and NLRP3 inflammasome activation, while CAPN4 knockdown reversed these changes. Correspondingly, CAPN4-cKO alleviated CVB3-induced M1 macrophage transformation and NLRP3 expression and moderately increased M2 transformation in vivo. The culture supernatant of CAPN4-overexpressing or CVB3-infected macrophages impaired cardiac fibroblast function and viability. Moreover, macrophage CAPN4 could upregulate C/EBP-homologous protein (chop) expression, which increased proinflammatory cytokine release by activating the phosphorylation of transducer of activator of transcription 1 (STAT1) and 3 (STAT3). Overall, these results suggest that CAPN4 increases M1-type and inhibits M2-type macrophage polarization through the chop-STAT1/STAT3 signaling pathway to mediate CVB3-induced myocardial inflammation and injury. CAPN4 may be a novel target for viral myocarditis treatment.

Effect of FoxO1 on Cardiomyocyte Apoptosis and Inflammation in Viral Myocarditis via Modultion of the TLR4/NF-κB Signaling Pathway.

To investigate the possible effect of FoxO on coxsackievirus B3 (CVB3) -induced cardiomyocyte inflammation and apoptosis via modulation of the TLR4/NF-κB signaling pathway.Viral myocarditis (VMC) models were establied via CVB3 infection both in vivo and in vitro. Western blotting was adopted to detect FoxO1 and TLR4 expressions in myocardial tissues and cells. Cardiomyocytes of suckling mouse were divided into the control, CVB3, CVB3 + pcDNA, CVB3 + pcDNA-FoxO1, CVB3 + TLR4 siRNA, and CVB3 + pcDNA-FoxO1 + TLR4 siRNA groups. Flow cytometry was employed to evaluate cell apoptosis. The expressions of inflammatory factors including TNF-α, IL-1ß, and IL-6 were detected via quantitative reverse transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. Then, TLR4/NF-κB pathway-related proteins were determined via Western blotting.VMC mice had increased FoxO1 and TLR4 expressions in myocardial tissues. Cardiomyocytes with CVB3 infection also had upregulated protein expressions of p-FoxO1/FoxO1 and TLR4. Compared with those in the control group, the cardiomyocytes in the CVB3 group were increased in LDH and CK-MB levels, cell apoptosis rate and inflammatory factors (TNF-α, IL-1ß and IL-6), as well as protein expressions of TLR4 and p-p65/p65. Compared with those in the CVB3 group, the cardiomyocytes in the CVB3 + pcDNA-FoxO1 group were further upregulated whereas those in the CVB3 +TLR4 siRNA group were downregulated in the aforementioned indicators. Furthermore, TLR4 siRNA can reverse the effect of pcDNA-FoxO1 on the aggravation of cardiomyocyte injury induced by CVB3 infection.FoxO1 can upregulate the TLR4/NF-κB signaling pathway to promote cardiomyocyte apoptosis and inflammatory injury in CVB3-induced VMC.

Sema3A alleviates viral myocarditis by modulating SIRT1 to regulate cardiomyocyte mitophagy.

Viral myocarditis (VMC) is a common myocardial inflammatory disease characterized by inflammatory cell infiltration and cardiomyocyte necrosis. Sema3A was reported to reduce cardiac inflammation and improve cardiac function after myocardial infarction, but its role in VMC remains to be explored. Here, a VMC mouse model was established by infection with CVB3, and Sema3A was overexpressed in vivo by intraventricular injection of an adenovirus-mediated Sema3A expression vector (Ad-Sema3A). We found that Sema3A overexpression attenuated CVB3-induced cardiac dysfunction and tissue inflammation. And Sema3A also reduced macrophage accumulation and NLRP3 inflammasome activation in the myocardium of VMC mice. In vitro, LPS was used to stimulate primary splenic macrophages to mimic the macrophage activation state in vivo. Activated macrophages were co-cultured with primary mouse cardiomyocytes to evaluate macrophage infiltration-induced cardiomyocyte damage. Ectopic expression of Sema3A in cardiomyocytes effectively protected cardiomyocytes from activated macrophage-induced inflammation, apoptosis, and ROS accumulation. Mechanistically, cardiomyocyte-expressed Sema3A mitigated macrophage infiltration-caused cardiomyocyte dysfunction by promoting cardiomyocyte mitophagy and hindering NLRP3 inflammasome activation. Furthermore, NAM (a SIRT1 inhibitor) reversed the protective effect of Sema3A against activated macrophage-induced cardiomyocyte dysfunction by suppressing cardiomyocyte mitophagy. In conclusion, Sema3A promoted cardiomyocyte mitophagy and suppressed inflammasome activation by regulating SIRT1, thereby attenuating macrophage infiltration-induced cardiomyocyte injury in VMC.

Umifenovir Epigenetically Targets the IL-10 Pathway in Therapy against Coxsackievirus B4 Infection.

Umifenovir, a broad-spectrum nonnucleoside antiviral drug, has a promising efficacy against coxsackievirus B4 (CVB4) infection, but its mechanism remains unclear. CVB4 is a common human single-stranded RNA virus that belongs to the Picornaviridae family and the Enterovirus genus. Enterovirus can cause severe diseases, such as meningitis, myocarditis, pancreatitis, insulin-dependent diabetes, and several other diseases, in both adults and children. We have previously demonstrated the critical role of interleukin 10 (IL-10) in promoting CVB4 infection and the downregulation of IL-10 by umifenovir. To further explore the underlying mechanisms of umifenovir, we characterized the epigenetic regulation of IL-10 in IL-10 knockout RAW264.7 cells and a BALB/c mouse splenocyte model. Mechanistically, we found that umifenovir inhibited CVB4-activated IL-10 by enhancing the methylation level of the repressive histones H3K9me3 and H3K27me3 while reducing the acetylation level of the activating histone H3K9ac in the promoter region of the IL-10 gene. Furthermore, using a chromosome conformation capture approach, we discovered that CVB4 infection activated the IL-10 gene by forming an intrachromosomal interaction between the IL-10 gene promoter and an intronic enhancer of the downstream MK2 (mitogen-activated protein kinase [MAPK]-activated protein kinase 2 [MAPKAPK2]) gene, a critical component of the p38-MAPK signaling pathway, which is required for IL-10 gene expression. However, umifenovir treatment abolished this spatial conformation and chromatin interaction, thus reducing the continuous expression of IL-10 and subsequent CVB4 replication. In conclusion, this study reveals a novel epigenetic mechanism by which umifenovir controls CVB4 infections, thus laying a theoretical foundation for therapeutic use of umifenovir. IMPORTANCE Viral infections are major threats to human health because of their strong association with a variety of inflammation-related diseases, especially cancer. Many antiviral drugs are performing poorly in treating viral infections. This is probably due to the immunosuppressive effect of highly expressed IL-10, which is caused by viral infection. Umifenovir is a broad-spectrum antiviral drug. Our recent studies showed that umifenovir has a significant inhibitory effect on CVB4 infection and can reduce IL-10 expression caused by CVB4. However, another antiviral drug, rupintrivir, showed good antiviral activity but had no effect on the expression of IL-10. This suggests that the regulation of IL-10 expression is a key part of the antiviral mechanism of umifenovir. Therefore, due to the dual function of the inhibition of CVB4 replication and the regulation of immune antiviral pathway, the mechanism of umifenovir is of great value to study.

LncGBP9 knockdown alleviates myocardial inflammation and apoptosis in mice with acute viral myocarditis via suppressing NF-κB signaling pathway.

BACKGROUND: Myocardial inflammation and apoptosis are key processes in coxsackievirus B3 (CVB3)-induced acute viral myocarditis (AVMC). Accumulating evidence reveals the essential roles of long noncoding RNAs (lncRNAs) in the pathogenesis of AVMC. Here, we aimed to evaluate the biological functions of a novel lncRNA guanylate-binding protein 9 (lncGBP9) in AVMC progression and further explore its underlying mechanisms. METHODS: Initially, mouse models of AVMC were constructed by CVB3 infection. The expression and localization of lncGBP9 in heart tissues were analyzed using RT-qPCR and FISH. Adeno-associated virus serotype 9 (AAV9)-mediated lncGBP9 knockdown was then employed to clarify its roles in survival, cardiac function, and myocardial inflammation and apoptosis. Moreover, the mRNA and protein levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) were detected by RT-qPCR and ELISA, and the regulation of lncGBP9 knockdown on the NF-κB signaling pathway was investigated by Western blotting. Using an in vitro model of HL-1 cardiomyocytes exposed to CVB3 infection, the effects of lncGBP9 knockdown on cell viability, inflammation, and apoptosis were further evaluated in vitro. RESULTS: Increased lncGBP9 expression was detected in the heart tissues of AVMC mice and CVB3-infected HL-1 cells, and was mainly located in the cytoplasm. Knockdown of lncGBP9 remarkably alleviated the severity of AVMC in CVB3-infected mice, as verified by improved cardiac function, and reduced myocardial inflammation and apoptosis. Additionally, lncGBP9 knockdown suppressed the NF-κB signaling pathway and consequently reduced productions of pro-inflammatory cytokines in vivo. In vitro functional assays further confirmed that lncGBP9 knockdown promoted cell viability, inhibited cell apoptosis, and reduced pro-inflammatory cytokines release in CVB3-infected HL-1 cells through suppressing NF-κB activation. CONCLUSIONS: Collectively, lncGBP9 knockdown exerts anti-inflammatory and anti-apoptotic effects in CVB3-induced AVMC, which may be mediated in part via NF-κB signaling pathway. These findings highlight lncGBP9 as an attractive target for therapeutic interventions.

Dapagliflozin Alleviates Coxsackievirus B3-induced Acute Viral Myocarditis by Regulating the Macrophage Polarization Through Stat3-related Pathways.

Viral myocarditis (VMC), which is most prevalently caused by Coxsackievirus B3 (CVB3) infection, is a serious clinical condition characterized by cardiac inflammation. Dapagliflozin, a kind of sodium glucose co-transporters 2(SGLT-2) inhibitor, exhibited protective effects on plenty of inflammatory diseases, while its effect on viral myocarditis has not been studied. Recently, we found the protective effect of dapagliflozin on VMC. After CVB3 infection, dapagliflozin and STATTIC (a kind of stat3 inhibitor) were given to Balb/c male mice for 8 days, and then the severity of myocarditis was assessed. Our results indicated that dapagliflozin significantly alleviated the severity of viral myocarditis, elevated the survival rate, and ameliorated cardiac function. Besides, dapagliflozin can decrease the level of pro-inflammatory cytokines including IL-1ß, IL-6, and TNF-α. Furthermore, dapagliflozin can inhibit macrophages differentiate to classically activated macrophages (M1) in cardiac tissue and activate the Stat3 signal pathway which is reported to promote polarization of the alternatively activated macrophage (M2). And STATTIC can reverse these changes caused by dapagliflozin. In conclusion, we found that dapagliflozin treatment increased anti-inflammatory macrophage polarization and reduced cardiac injury following VMC via activating Stat3 signal pathway.

Molecular basis of differential receptor usage for naturally occurring CD55-binding and -nonbinding coxsackievirus B3 strains.

Receptor usage defines cell tropism and contributes to cell entry and infection. Coxsackievirus B (CVB) engages coxsackievirus and adenovirus receptor (CAR), and selectively utilizes the decay-accelerating factor (DAF; CD55) to infect cells. However, the differential receptor usage mechanism for CVB remains elusive. This study identified VP3-234 residues (234Q/N/V/D/E) as critical population selection determinants during CVB3 virus evolution, contributing to diverse binding affinities to CD55. Cryoelectron microscopy (cryo-EM) structures of CD55-binding/nonbinding isolates and their complexes with CD55 or CAR were obtained under both neutral and acidic conditions, and the molecular mechanism of VP3-234 residues determining CD55 affinity/specificity for naturally occurring CVB3 strains was elucidated. Structural and biochemical studies in vitro revealed the dynamic entry process of CVB3 and the function of the uncoating receptor CAR with different pH preferences. This work provides detailed insight into the molecular mechanism of CVB infection and contributes to an in-depth understanding of enterovirus attachment receptor usage.

Knockdown of LncRNA MALAT1 Alleviates Coxsackievirus B3-Induced Acute Viral Myocarditis in Mice via Inhibiting Th17 Cells Differentiation.

Acute viral myocarditis (AVMC), most often caused by coxsackievirus B3 (CVB3) infection, is characterized by myocardial inflammation associated with high morbidity and mortality. A pathogenic role for T helper (Th) 17 cells in AVMC is well established. Long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been shown to play a key role in various inflammatory diseases. However, the expression of MALAT1 and its impact on Th17 cells differentiation in AVMC remain unclear. In the present study, we found that MALAT1 was highly expressed in mice with AVMC, and the expression was correlated positively with cardiac pathological scores, cardiac IL-17 mRNA expression, and the percentages of splenic Th17 cells. We further demonstrated that MALAT1 knockdown could significantly alleviate the severity of disease and inhibit the differentiation of Th17 cells, accompanying the reduced mRNA expression of RORγt and productions of Th17-related pro-inflammatory cytokines in vivo. Additionally, in vitro analysis showed that MALAT1 knockdown suppressed naïve CD4+ T cells differentiation towards Th17 cells. In conclusion, our results suggest that MALAT1 knockdown alleviates CVB3-induced AVMC in mice, which may be partially attributable to the decline in Th17 cells responses. MALAT1 may serve as a novel therapeutic option in AVMC.

Inhibition of calpain reduces cell apoptosis by suppressing mitochondrial fission in acute viral myocarditis.

Cardiomyocyte apoptosis is critical for the development of viral myocarditis (VMC), which is one of the leading causes of cardiac sudden death in young adults. Our previous studies have demonstrated that elevated calpain activity is involved in the pathogenesis of VMC. This study aimed to further explore the underlying mechanisms. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin were infected with coxsackievirus B3 (CVB3) to establish a VMC model. Apoptosis was detected with flow cytometry, TUNEL staining, and western blotting. Cardiac function was measured using echocardiography. Mitochondrial function was measured using ATP assays, JC-1, and MitoSOX. Mitochondrial morphology was observed using MitoTracker staining and transmission electron microscopy. Colocalization of dynamin-related protein 1 (Drp-1) in mitochondria was examined using immunofluorescence. Phosphorylation levels of Drp-1 at Ser637 site were determined using western blotting analysis. We found that CVB3 infection impaired mitochondrial function as evidenced by increased mitochondrial ROS production, decreased ATP production and mitochondrial membrane potential, induced myocardial apoptosis and damage, and decreased myocardial function. These effects of CVB3 infection were attenuated by inhibition of calpain both by PD150606 treatment and calpastatin overexpression. Furthermore, CVB3-induced mitochondrial dysfunction was associated with the accumulation of Drp-1 in the outer membrane of mitochondria and subsequent increase in mitochondrial fission. Mechanistically, calpain cleaved and activated calcineurin A, which dephosphorylated Drp-1 at Ser637 site and promoted its accumulation in the mitochondria, leading to mitochondrial fission and dysfunction. In summary, calpain inhibition attenuated CVB3-induced myocarditis by reducing mitochondrial fission, thereby inhibiting cardiomyocyte apoptosis. Calpain is activated by CVB3 infection. Activated calpain cleaves calcineurin A and converts it to active form which could dephosphorylate Drp-1 at Ser637 site. Then, the active Drp-1 translocates from the cytoplasm to mitochondria and triggers excessive mitochondrial fission. Eventually, the balance of mitochondrial dynamics is broken, and apoptosis occurs.

Cellular Protein HuR Regulates the Switching of Genomic RNA Templates for Differential Functions during the Coxsackievirus B3 Life Cycle.

Coxsackievirus B3 (CVB3) is an enterovirus belonging to the family Picornaviridae. Its 5' untranslated region (UTR) contains a cloverleaf structure followed by an internal ribosome entry site (IRES). The cloverleaf forms an RNA-protein complex known to regulate virus replication, translation, and stability of the genome, and the IRES regulates virus RNA translation. For positive-strand RNA-containing viruses, such as members of the flaviviruses or enteroviruses, the genomic RNA is used for translation, replication, and encapsidation. Only a few regulatory mechanisms which govern the accessibility of genomic RNA templates for translation or replication have been reported. Here, we report the role of human antigen R (HuR) in regulating the fate of CVB3 positive-strand RNA into the replication cycle or translation cycle. We have observed that synthesis of HuR is induced during CVB3 infection, and it suppresses viral replication by displacing PCBP-2 (a positive regulator of virus replication) at the cloverleaf RNA. Silencing of HuR increases viral RNA replication and consequently reduces viral RNA translation in a replication-dependent manner. Furthermore, we have shown that HuR level is upregulated upon CVB3 infection. Moreover, HuR limits virus replication and can coordinate the availability of genomic RNA templates for translation, replication, or encapsidation. Our study highlights the fact that the relative abundance of translation factors and replication factors in the cell decides the outcome of viral infection. IMPORTANCE A positive-strand RNA virus must balance the availability of its genomic template for different viral processes at different stages of its life cycle. A few host proteins are shown to be important to help the virus in switching the usage of a template between these processes. These proteins inhibit translation either by displacing a stimulator of translation or by binding to an alternative site. Both mechanisms lead to ribosome clearance and availability of the genomic strand for replication. We have shown that HuR also helps in maintaining this balance by inhibiting replication and subsequently promoting translation and packaging.

The Role of B Cells in Regulation of Th Cell Differentiation in Coxsackievirus B3-Induced Acute Myocarditis.

Viral myocarditis (VMC) is the major cause of sudden death in adolescents. To date, no effective treatment has been identified for VMC. Studies have shown that T helper (Th) cells such as Th1, Th2, Th17, and Th22 cells are involved in the pathogenesis of VMC. However, the role of B cells and their impact on Th cells in VMC is unclear. In this study, we investigated the role of B cells in Th cell differentiation in myocardial damage in an animal model of VMC. C57BL/6 mice were infected with Coxsackievirus B3 (CVB3) intraperitoneally or injected with phosphate-buffered saline as a control condition. At day 7, samples from these mice were analyzed by histology, ELISA, flow cytometry, and gene expression assays. We found that TNF-α-, IL-6-, and IL-17-producing B cell numbers were significantly increased, while IL-4-producing B cell population was significantly reduced in acute VMC. Furthermore, we performed B cell knockout (BKO), SCID, and SCID+B cells reconstitution experiments. We found that BKO alleviated the cardiac damage following CVB3 infection, may hamper the differentiation of Th1 and Th17 cells, may promote the differentiation of Th2 cells, and proved ineffective for the differentiation of Th22 cells. In contrast, SCID+B cells reconstitution experiment exacerbated the cardiac damage. Ex vivo studies further revealed that B cells promote the differentiation of Th1 and Th17 cells and inhibit the differentiation of Th2 cells. Our study shows that B cells are activated and have strong abilities of antigen presentation and producing cytokines in VMC; B cells not only play a pathogenic role in VMC independent of T cells but also promote Th1 and Th17 cell differentiation, and hamper Th2 cell differentiation in VMC.

A Crucial Role of ACBD3 Required for Coxsackievirus Infection in Animal Model Developed by AAV-Mediated CRISPR Genome Editing Technique.

Genetic screens using CRISPR/Cas9 have been exploited to discover host-virus interactions. These screens have identified viral dependencies on host proteins during their life cycle and potential antiviral strategies. The acyl-CoA binding domain containing 3 (ACBD3) was identified as an essential host factor for the Coxsackievirus B3 (CVB3) infection. Other groups have also investigated the role of ACBD3 as a host factor for diverse enteroviruses in cultured cells. However, it has not been tested if ACBD3 is required in the animal model of CVB3 infection. Owing to embryonic lethality, conventional knockout mice were not available for in vivo study. As an alternative approach, we used adeno-associated virus (AAV)-mediated CRISPR genome editing to generate mice that lacked ACBD3 within the pancreas, the major target organ for CVB3. Delivery of sgRNAs using self-complementary (sc) AAV8 efficiently induced a loss-of-function mutation in the pancreas of the Cas9 knock-in mice. Loss of ACBD3 in the pancreas resulted in a 100-fold reduction in the CVB3 titer within the pancreas and a noticeable reduction in viral protein expression. These results indicate a crucial function of ACBD3 in CVB3 infection in vivo. AAV-mediated CRISPR genome editing may be applicable to many in vivo studies on the virus-host interaction and identify a novel target for antiviral therapeutics.

MicroRNA-30a-5p silencing polarizes macrophages toward M2 phenotype to alleviate cardiac injury following viral myocarditis by targeting SOCS1.

Viral myocarditis (VMC) is a life-threatening disease characterized by severe cardiac inflammation generally caused by coxsackievirus B3 (CVB3) infection. Several microRNAs (miRNAs or miRs) are known to play crucial roles in the pathogenesis of VMC. The study aimed to decipher the role of miR-30a-5p in the underlying mechanisms of VMC pathogenesis. We first quantified miR-30a-5p expression in a CVB3-induced mouse VMC model. The physiological characteristics of mouse cardiac tissues were then detected by hematoxylin and eosin (HE) and Picrosirius red staining. We established the correlation between miR-30a-5p and SOCS1, using dual-luciferase gene assay and Pearson's correlation coefficient. The expression of inflammatory factors (IFN-γ, IL-6, IL-10, and IL-13), M1 polarization markers [TNF-α, inducible nitric oxide synthase (iNOS)], M2 polarization markers (Arg-1, IL-10), and myocardial hypertrophy markers [atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)] was detected by RT-qPCR and Western blot analysis. miR-30a-5p was found to be highly expressed in VMC mice. Silencing of miR-30a-5p improved the cardiac function index and reduced heart weight-to-body weight ratio, myocardial tissue pathological changes and fibrosis degree, serological indexes, as well as proinflammatory factor levels, while enhancing anti-inflammatory factor levels in VMC mice. Furthermore, silencing of miR-30a-5p inhibited M1 polarization of macrophages while promoting M2 polarization in vivo and in vitro. SOCS1 was a target gene of miR-30a-5p, and the aforementioned cardioprotective effects of miR-30a-5p silencing were reversed upon silencing of SOCS1. Overall, this study shows that silencing of miR-30a-5p may promote M2 polarization of macrophages and improve cardiac injury following VMC via SOCS1 upregulation, constituting a potential therapeutic target for VMC treatment.NEW & NOTEWORTHY We found in this study that microRNA (miR)-30a-5p inhibition might improve cardiac injury following viral myocarditis (VMC) by accelerating M2 polarization of macrophages via SOCS1 upregulation. Furthermore, the anti-inflammatory mechanisms of miR-30a-5p inhibition may contribute to the development of new therapeutic strategies for VMC.

Coxsackievirus B3 Infection of Human iPSC Lines and Derived Primary Germ-Layer Cells Regarding Receptor Expression.

The association of members of the enterovirus family with pregnancy complications up to miscarriages is under discussion. Here, infection of two different human induced pluripotent stem cell (iPSC) lines and iPSC-derived primary germ-layer cells with coxsackievirus B3 (CVB3) was characterized as an in vitro cell culture model for very early human development. Transcriptomic analysis of iPSC lines infected with recombinant CVB3 expressing enhanced green fluorescent protein (EGFP) revealed a reduction in the expression of pluripotency genes besides an enhancement of genes involved in RNA metabolism. The initial distribution of CVB3-EGFP-positive cells within iPSC colonies correlated with the distribution of its receptor coxsackie- and adenovirus receptor (CAR). Application of anti-CAR blocking antibodies supported the requirement of CAR, but not of the co-receptor decay-accelerating factor (DAF) for infection of iPSC lines. Among iPSC-derived germ-layer cells, mesodermal cells were especially vulnerable to CVB3-EGFP infection. Our data implicate further consideration of members of the enterovirus family in the screening program of human pregnancies. Furthermore, iPSCs with their differentiation capacity into cell populations of relevant viral target organs could offer a reliable screening approach for therapeutic intervention and for assessment of organ-specific enterovirus virulence.