Investigating TIP30-Mediated regulation of mTORC1 signaling as a therapeutic strategy for coxsackievirus B3-Induced viral myocarditis.

This study aims to elucidate the role of TIP30 (30 KDa HIV-1 TAT-Interacting Protein) in the progression of coxsackievirus B3 (CVB3)-induced viral myocarditis. TIP30 knockout and wildtype mice were intraperitoneally infected with CVB3 and evaluated at day 7 post-infection. HeLa cells were transfected with TIP30 lentiviral particles and subsequently infected with CVB3 to evaluate viral replication, cellular pathogenesis, and mechanistic target of rapamycin complex 1 (mTORC1) signaling. Deletion of the TIP30 gene heightened heart virus titers and mortality rates in mice with CVB3-induced myocarditis, exacerbating cardiac damage and fibrosis, and elevating pro-inflammatory factors level. In vitro experiments demonstrated the modulation of mTORC1 signaling by TIP30 during CVB3 infection in HeLa cells. TIP30 overexpression mitigated CVB3-induced cellular pathogenesis and VP1 expression, with rapamycin, an mTOR1 inhibitor, reversing these effects. These findings suggest TIP30 plays a critical protective role against CVB3-induced myocarditis by regulating mTORC1 signaling.

Functions and Clinical Significance of Myocardial Cell-Derived Immunoglobulins.

Immunoglobulins (Igs) have been widely accepted to be exclusively expressed by B cells. Nonetheless, this theory is challenged by mounting evidence which suggests that Igs can also be generated by non B cells (non B-Ig), including cardiomyocytes (CM). Non B-Ig exhibits unique physical and chemical characteristics, unique variable region sequences and functions, which diverge from those of B-Ig. For instance, non B-Ig demonstrates hydrophobicity, limited diversity in the variable region, and extracellular matrix protein activity. Likewise, cardiomyocytes can express different classes of Igs, including IgM, IgG, and free Igκ light chains (cardiomyocyte derived-Igs, CM-Igs). In particular, CM-Igs can be secreted into the extracellular space in various cardiovascular diseases, such as myocardial ischaemia and myocardial fibrosis where they might be involved in complement activation and direct damage to cardiomyocytes. Nevertheless, the precise pathological activity of CM-Igs remains unclear. Recently, Zhu et al. focused on studying the sequence characteristics and functions of CM-Igκ; they discovered that the CM-Igκ exhibits a unique VJ recombination pattern, high hydrophobicity, and is principally located on the intercalated discs and cross striations of the cardiomyocytes. Interestingly, loss of Igκ in cardiomyocytes results in structural disorders in intercalated discs and dysfunction in myocardial contraction and conduction. Mechanically, Igκ promotes the stabilisation of plectin, a cytoskeleton cross-linker protein that connects desmin to desomsome, to maintain the normal structure of the intercalated disc. This finding indicates that CM-Igκ plays an integral role in maintaining cytoskeleton structure. Consequently, it is imperative to reveal the physiological functions and mechanisms of pathological injury associated with CM-Igs.

The influence of endurance exercise training on myocardial fibrosis and arrhythmogenesis in a coxsackievirus B3 myocarditis mouse model.

Nonischaemic myocardial fibrosis is associated with cardiac dysfunction, malignant arrhythmias and sudden cardiac death. In the absence of a specific aetiology, its finding as late gadolinium enhancement (LGE) on cardiac magnetic resonance imaging is often attributed to preceding viral myocarditis. Athletes presenting with ventricular arrhythmias often have nonischaemic LGE. Previous studies have demonstrated an adverse effect of exercise on the course of acute viral myocarditis. In this study, we have investigated, for the first time, the impact of endurance training on longer-term outcomes such as myocardial fibrosis and arrhythmogenicity in a murine coxsackievirus B3 (CVB)-induced myocarditis model. Male C57BL/6J mice (n = 72) were randomly assigned to 8 weeks of forced treadmill running (EEX) or no exercise (SED). Myocarditis was induced 2 weeks later by a single intraperitoneal injection with CVB, versus vehicle in the controls (PBS). In a separate study, mice (n = 30) were subjected to pretraining for 13 weeks (preEEX), without continuation of exercise during myocarditis. Overall, continuation of exercise resulted in a milder clinical course of viral disease, with less weight loss and better preserved running capacity. CVB-EEX and preEEX-CVB mice tended to have a lower mortality rate. At sacrifice (i.e. 6 weeks after inoculation), the majority of virus was cleared from the heart. Histological assessment demonstrated prominent myocardial inflammatory infiltration and cardiomyocyte loss in both CVB groups. Inflammatory lesions in the CVB-EEX group contained higher numbers of pro-inflammatory cells (iNOS-reactive macrophages and CD8+ T lymphocytes) compared to these in CVB-SED. Treadmill running during myocarditis increased interstitial fibrosis [82.4% (CVB-EEX) vs. 56.3% (CVB-SED); P = 0.049]. Additionally, perivascular and/or interstitial fibrosis with extensive distribution was more likely to occur with exercise [64.7% and 64.7% (CVB-EEX) vs. 50% and 31.3% (CVB-SED); P = 0.048]. There was a numerical, but not significant, increase in the number of scars per cross-section (1.9 vs. 1.2; P = 0.195), with similar scar distribution and histological appearance in CVB-EEX and CVB-SED. In vivo electrophysiology studies did not induce sustained monomorphic ventricular tachycardia, only nonsustained (usually polymorphic) runs. Their cumulative beat count and duration paralleled the increased fibrosis between CVB-EEX and CVB-SED, but the difference was not significant (P = 0.084 for each). Interestingly, in mice that were subjected to pretraining only without continuation of exercise during myocarditis, no differences between pretrained and sedentary mice were observed at sacrifice (i.e. 6 weeks after inoculation and training cessation) with regard to myocardial inflammation, fibrosis, and ventricular arrhythmogenicity. In conclusion, endurance exercise during viral myocarditis modulates the inflammatory process with more pro-inflammatory cells and enhances perivascular and interstitial fibrosis development. The impact on ventricular arrhythmogenesis requires further exploration.

The natural history of CVB3 myocarditis in C57BL/6J mice: an extended in-depth characterization.

BACKGROUND AND AIMS: Viral infections are the leading cause of myocarditis. Besides acute cardiac complications, late-stage sequelae such as myocardial fibrosis may develop, importantly impacting the prognosis. Coxsackievirus B3 (CVB)-induced myocarditis in mice is the most commonly used translational model to study viral myocarditis and has provided the majority of our current understanding of the disease pathophysiology. Nevertheless, the late stages of disease, encompassing fibrogenesis and arrhythmogenesis, have been underappreciated in viral myocarditis research to date. The present study investigated the natural history of CVB-induced myocarditis in C57BL/6J mice, expanding the focus beyond the acute phase of disease. In addition, we studied the impact of sex and inoculation dose on the disease course. METHODS AND RESULTS: C57BL/6J mice (12 weeks old; n=154) received a single intraperitoneal injection with CVB to induce viral myocarditis, or vehicle (PBS) as control. Male mice (n=92) were injected with 5 × 105 (regular dose) (RD) or 5 × 106 (high dose) (HD) plaque-forming units of CVB, whereas female mice received the RD only. Animals were sacrificed 1, 2, 4, 8, and 11 weeks after CVB or PBS injection. Virally inoculated mice developed viral disease with a temporary decline in general condition and weight loss, which was less pronounced in female animals (P<.001). In male CVB mice, premature mortality occurred between days 8 and 23 after inoculation (RD: 21%, HD: 20%), whereas all female animals survived. Over the course of disease, cardiac inflammation progressively subsided, with faster resolution in female mice. There were no substantial group differences in the composition of the inflammatory cell infiltrates: predominance of cytotoxic T cells at day 7 and 14, and a switch from arginase1-reactive macrophages to iNOS-reactive macrophages from day 7 to 14 were the main findings. There was concomitant development and maturation of different patterns of myocardial fibrosis, with enhanced fibrogenesis in male mice. Virus was almost completely cleared from the heart by day 14. Serum biomarkers of cardiac damage and cardiac expression of remodeling genes were temporarily elevated during the acute phase of disease. Cardiac CTGF gene upregulation was less prolonged in female CVB animals. In vivo electrophysiology studies at weeks 8 and 11 demonstrated that under baseline conditions (i.e. in the absence of proarrhythmogenic drugs), ventricular arrhythmias could only be induced in CVB animals. The cumulative arrhythmia burden throughout the entire stimulation protocol was not significantly different between CVB and control groups. CONCLUSION: CVB inoculation in C57BL/6J mice represents a model of acute self-limiting viral myocarditis, with progression to different patterns of myocardial fibrosis. Sex, but not inoculation dose, seems to modulate the course of disease.

miR-29b-3p regulates cardiomyocytes pyroptosis in CVB3-induced myocarditis through targeting DNMT3A.

BACKGROUND: Viral myocarditis (VMC) is a disease resulting from viral infection, which manifests as inflammation of myocardial cells. Until now, the treatment of VMC is still a great challenge for clinicians. Increasing studies indicate the participation of miR-29b-3p in various diseases. According to the transcriptome sequencing analysis, miR-29b-3p was markedly upregulated in the viral myocarditis model. The purpose of this study was to investigate the role of miR-29b-3p in the progression of VMC. METHODS: We used CVB3 to induce primary cardiomyocytes and mice to establish a model of viral myocarditis. The purity of primary cardiomyocytes was identified by immunofluorescence. The cardiac function of mice was detected by Vevo770 imaging system. The area of inflammatory infiltration in heart tissue was shown by hematoxylin and eosin (H&E) staining. The expression of miR-29b-3p and DNMT3A was detected by quantitative real time polymerase chain reaction (qRT-PCR). The expression of a series of pyroptosis-related proteins was detected by western blot. The role of miR-29b-3p/DNMT3A in CVB3-induced pyroptosis of cardiomyocytes was studied in this research. RESULTS: Our data showed that the expression of miR-29b-3p was upregulated in CVB3-induced cardiomyocytes and heart tissues in mice. To explore the function of miR-29b-3p in CVB3-induced VMC, we conducted in vivo experiments by knocking down the expression of miR-29b-3p using antagomir. We then assessed the effects on mice body weight, histopathology changes, myocardial function, and cell pyroptosis in heart tissues. Additionally, we performed gain/loss-of-function experiments in vitro to measure the levels of pyroptosis in primary cardiomyocytes. Through bioinformatic analysis, we identified DNA methyltransferases 3A (DNMT3A) as a potential target gene of miR-29b-3p. Furthermore, we found that the expression of DNMT3A can be modulated by miR-29b-3p during CVB3 infection. CONCLUSIONS: Our results demonstrate a correlation between the expression of DNMT3A and CVB3-induced pyroptosis in cardiomyocytes. These findings unveil a previously unidentified mechanism by which CVB3 induces cardiac injury through the regulation of miR-29b-3p/DNMT3A-mediated pyroptosis.

CXCL4/CXCR3 axis regulates cardiac fibrosis by activating TGF-ß1/Smad2/3 signaling in mouse viral myocarditis.

BACKGROUND: Severe myocarditis is often accompanied by cardiac fibrosis, but the underlying mechanism has not been fully elucidated. CXCL4 is a chemokine that has been reported to have pro-inflammatory and profibrotic functions. The exact role of CXCL4 in cardiac fibrosis remains unclear. METHODS: Viral myocarditis (VMC) models were induced by intraperitoneal injection of Coxsackie B Type 3 (CVB3). In vivo, CVB3 (100 TCID50) and CVB3-AMG487 (CVB3: 100 TCID50; AMG487: 5 mg/kg) combination were administered in the VMC and VMC+AMG487 groups, respectively. Hematoxylin and eosin staining, severity score, Masson staining, and immunofluorescence staining were performed to measure myocardial morphology in VMC. Enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed to quantify inflammatory factors (IL-1ß, IL-6, TNF-α, and CXCL4). Aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase-myocardial band (CK-MB) levels were analyzed by commercial kits. CXCL4, CXCR3B, α-SMA, TGF-ß1, Collagen I, and Collagen III were determined by Western blot and immunofluorescence staining. RESULTS: In vivo, CVB3-AMG487 reduced cardiac injury, α-SMA, Collagen I and Collagen III levels, and collagen deposition in VMC+AMG487 group. Additionally, compared with VMC group, VMC+AMG group decreased the levels of inflammatory factors (IL-1ß, IL-6, and TNF-α). In vitro, CXCL4/CXCR3B axis activation TGF-ß1/Smad2/3 pathway promote mice cardiac fibroblasts differentiation. CONCLUSION: CXCL4 acts as a profibrotic factor in TGF-ß1/Smad2/3 pathway-induced cardiac fibroblast activation and ECM synthesis, and eventually progresses to cardiac fibrosis. Therefore, our findings revealed the role of CXCL4 in VMC and unveiled its underlying mechanism. CXCL4 appears to be a potential target for the treatment of VMC.

Prevalence and impact of viral myocarditis in patients with severe fever with thrombocytopenia syndrome.

To explore the association and impact between viral myocarditis and mortality in patients with severe fever with thrombocytopenia syndrome. A dynamic analysis was conducted between fatal group and nonfatal group regarding the daily epidemiology data, clinical symptoms, and electrocardiogram (ECG), echocardiogram, and laboratory findings. Outcomes of patients with and without viral myocarditis were compared. The association between viral myocarditis and mortality was analyzed. Among 183 severe fever with thrombocytopenia syndrome patients, 32 were in the fatal group and 151 in the nonfatal group; there were 26 (81.25%) with viral myocarditis in the fatal group, 66 (43.70%) with viral myocarditis in the nonfatal group (p < 0.001), 79.35% of patients had abnormal ECG results. The abnormal rate of ECG in the fatal group was 100%, and in the nonfatal group was 74.83%. Univariate analysis found that the number of risk factors gradually increased on Day 7 of the disease course and reached the peak on Day 10. Combined with the dynamic analysis of the disease course, alanine aminotransferase, aspartate aminotransferase, creatine kinase, creatine kinase fraction, lactate dehydrogenase, hydroxybutyrate dehydrogenase, neutrophil count, serum creatinine, Na, Ca, carbon dioxide combining power, amylase, lipase, activated partial thromboplastin time and thrombin time had statistically significant impact on prognosis. The incidence of fever with thrombocytopenia syndrome combined with viral myocarditis is high, especially in the fatal group of patients. Viral myocarditis is closely related to prognosis and is an early risk factor. The time point for changes in myocarditis is Day 7 of the course of the disease.

CXCL4:NLRP3-mediated pyroptosis product that regulates cardiac fibrosis.

Severe myocarditis is often accompanied by cardiac fibrosis, but the underlying mechanism has not been fully elucidated. NOD-like receptor protein 3 (NLRP3) inflammation is involved in the development of myocarditis and is closely related to the form of cell death. Inhibiting pyroptosis mediated by NLRP3 inflammasome can reduce cardiac fibrosis, although its exact mechanism remains unknown. In this study, we induced Viral myocarditis (VMC) via infection of CVB3 to explore the relationship between pyroptosis and fibrosis. Our results showed that intraperitoneal injection of an NLRP3 inhibitor MCC950 or use of NLRP3-/- mice inhibited cardiac pyroptosis mediated by NLRP3 inflammasome in VMC. CXCL4 is a chemokine that has been reported to have pro-inflammatory and pro-fibrotic functions. In VMC, we further found that pyroptosis of Mouse myocardial fibroblasts (MCF) promoted the secretion of CXCL4 by activating Wnt/ß-Catenin signaling. Subsequently, the transcriptome sequencing data showed that CXCL4 could promote cardiac fibrosis by activating PI3K/AKT pathway. In summary, infection of CVB3 induced host oxidative stress to further activate the NLRP3 inflammasome and ultimately lead to heart pyroptosis, in which MCF secreted CXCL4 by activating Wnt/ß-Catenin signaling and CXCL4 participated in cardiac fibrosis by activating PI3K/AKT pathway. Therefore, our findings revealed the role of CXCL4 in VMC and unveiled its underlying mechanism. CXCL4 appears to be a potential target for the treatment of VMC.

Effect of Yangxin Huoxue Jiedu recipe on inflammatory factors and oxidative stress on viral myocarditis in children.

OBJECTIVE: This observation purposed to investigate the effect of the Yangxin Huoxue Jiedu formula on children with viral myocarditis and its effect on inflammatory factors and oxidative response. MATERIALS AND METHODS: A total of 121 children with viral myocarditis were randomly divided into two groups, namely the control group (N = 60) and the traditional Chinese medicine group (N = 61). The control group was mainly treated with routine therapy, while the traditional Chinese medicine group was treated with Yangxin Huoxue Jiedu recipes based on the control group. The creatine kinase, creatine kinase myocardial isoenzyme, aspartate aminotransferase, lactic dehydrogenase, hydroxybutyrate dehydrogenase, cardiac troponin I, brain natriuretic peptide, interleukin-6, interleukin-8, and tumour necrosis factor-alpha, superoxide dismutase and malondialdehyde in viral myocarditis patients were tested to estimate the myocardial function, inflammation, and oxidative situation. RESULTS: After Yangxin Huoxue Jiedu treatment, 15 cases were recovered, 20 were excellent, and 21 were effective, which had a significant difference from the control group. The concentration of creatine kinase, creatine kinase myocardial isoenzyme, aspartate aminotransferase, lactic dehydrogenase, hydroxybutyrate dehydrogenase, cardiac troponin I and brain natriuretic peptide was decreased in the traditional Chinese medicine group. The levels of interleukin-6, interleukin-8, and tumour necrosis factor-alpha in the traditional Chinese medicine group were significantly lower than those in the control group. Superoxide dismutase was higher and malondialdehyde was lower than those in the control group. CONCLUSION: The use of Yangxin Huoxue Jiedu in the treatment of viral myocarditis has a definite clinical effect, which could improve myocardial function, reduce body inflammation, and promote oxidative recovery.

MG53 protects against Coxsackievirus B3-induced acute viral myocarditis in mice by inhibiting NLRP3 inflammasome-mediated pyroptosis via the NF-κB signaling pathway.

Pyroptosis, a novel programmed cell death mediated by NOD-like receptor protein 3 (NLRP3) inflammasome, is a critical pathogenic process in acute viral myocarditis (AVMC). Mitsugumin 53 (MG53) is predominantly expressed in myocardial tissues and has been reported to exert cardioprotective effects through multiple pathways. Herein, we aimed to investigate the biological function of MG53 in AVMC and its underlying regulatory mechanism in pyroptosis. BALB/c mice and HL-1 cells were infected with Coxsackievirus B3 (CVB3) to establish animal and cellular models of AVMC. As inflammation progressed in the myocardium, we found a progressive decrease in myocardial MG53 expression, accompanied by a significant enhancement of cardiomyocyte pyroptosis. MG53 overexpression significantly alleviated myocardial inflammation, apoptosis, fibrosis, and mitochondrial damage, thereby improving cardiac dysfunction in AVMC mice. Moreover, MG53 overexpression inhibited NLRP3 inflammasome-mediated pyroptosis, reduced pro-inflammatory cytokines (IL-1ß/18) release, and suppressed NF-κB signaling pathway activation both in vivo and in vitro. Conversely, MG53 knockdown reduced cell viability, facilitated cell pyroptosis, and increased pro-inflammatory cytokines release in CVB3-infected HL-1 cells by promoting NF-κB activation. These effects were partially reversed by applying the NF-κB inhibitor BAY 11-7082. In conclusion, our results suggest that MG53 acts as a negative regulator of NLRP3 inflammasome-mediated pyroptosis in CVB3-induced AVMC, partially by inhibiting the NF-κB signaling pathway. MG53 is a promising candidate for clinical applications in AVMC treatment.

Clinical Effect Analysis of Different Doses of Creatine Phosphate Sodium Combined with Immunoglobulin in the Treatment of Pediatric Viral Myocarditis.

The purpose of this paper was to unravel the clinical effect analysis of different doses of creatine phosphate sodium (CPS) combined with immunoglobulin in the treatment of pediatric viral myocarditis (VMC). One hundred and twenty children with VMC were recruited and randomized into three groups (40 patients each). Group I received 1.0 g of CPS dissolved in 100 mL of 5% glucose injection intravenously 1 time/day; group II received 1.25 g of CPS dissolved in 125 mL of 5% glucose injection intravenously 1 time/day; group III received 1.5 g of CPS dissolved in 150 mL of 5% glucose injection intravenously 1 time/day; then all three groups were treated with combined use of immunoglobulin (300-400 mg/day) intravenously once a day; and all three groups were treated for 14 days. The clinical efficacy, cardiac function, serum inflammatory factor levels, immune function, and the occurrence of drug toxicity and adverse effects of the children in the three groups were compared after 14 days of treatment. All three groups achieved better therapeutic effects after treatment, in which the effective rate of the Group II and Group III was notably higher versus the Group I. Lower levels of cTnI, CK-MB, LDH, AST, IL-18, IL-6, IFN-γ, and LVEDD and higher CD3+, CD4+, and CD4+/CD8+, FS, and LVEF values were noted in the Group II and Group III versus the Group I, and the results were more pronounced in the high-dose group. The liver and kidney functions of the children in the three groups before and after treatment did not show any significant changes and the incidence of adverse reactions during the treatment period was low in all three groups. Children with VMC can be treated with high-dose CPS in combination with immunoglobulin, which can improve their cardiac function and immune function and reduce the inflammatory response with good overall therapeutic efficacy and fewer adverse effects.

Clinical characteristics and influencing factors of severe fever with thrombocytopenia syndrome complicated by viral myocarditis: a retrospective study.

OBJECTIVE: This study aimed to investigate the clinical characteristics of severe fever with thrombocytopenia syndrome complicated by viral myocarditis (SFTS-VM) and analyze relevant influencing factors. METHODS: Retrospective analysis was conducted on clinical data from 79 SFTS-VM patients, categorized into common (SFTS-CVM, n = 40) and severe groups (SFTS-SVM, n = 39). Clinical manifestations, laboratory results, cardiac ultrasonography, and electrocardiogram features were analyzed. Univariate and multivariate analyses identified significant indicators, which were further assessed using ROC curves to predict SFTS-SVM. RESULTS: SFTS-SVM group exhibited higher rates of hypotension, shock, abdominal pain, cough with sputum, and consciousness disorders compared to SFTS-CVM group. Laboratory findings showed elevated platelet count, ALT, AST, amylase, lipase, LDH, D-dimer, procalcitonin, TNI, and NT-proBNP in SFTS-SVM. Abnormal electrocardiograms, especially atrial fibrillation, were more prevalent in SFTS-SVM (P < 0.05). Multivariate analysis identified elevated LDH upon admission (OR = 1.004, 95% CI: 1-1.008, P = 0.050), elevated NT-proBNP (OR = 1.005, 95% CI: 1.001-1.008, P = 0.007), and consciousness disorders (OR = 112.852, 95% CI: 3.676 ~ 3464.292, P = 0.007) as independent risk factors for SFTS-SVM. LDH and NT-proBNP had AUCs of 0.728 and 0.744, respectively, in predicting SFTS-SVM. Critical values of LDH (> 978.5U/L) and NT-proBNP (> 857.5pg/ml)) indicated increased likelihood of SFTS progression into SVM. CONCLUSION: Elevated LDH, NT-proBNP, and consciousness disorders independently correlate with SFTS-SVM. LDH and NT-proBNP can aid in early identification of SFTS-SVM development when above specified thresholds.

Multitargeted Immunomodulatory Therapy for Viral Myocarditis by Engineered Extracellular Vesicles.

Immune regulation therapies are considered promising for treating classically activated macrophage (M1)-driven viral myocarditis (VM). Alternatively, activated macrophage (M2)-derived extracellular vesicles (M2 EVs) have great immunomodulatory potential owing to their ability to reprogram macrophages, but their therapeutic efficacy is hampered by insufficient targeting capacity in vivo. Therefore, we developed cardiac-targeting peptide (CTP) and platelet membrane (PM)-engineered M2 EVs enriched with viral macrophage inflammatory protein-II (vMIP-II), termed CTP/PM-M2 EVsvMIP-II-Lamp2b, to improve the delivery of EVs "cargo" to the heart tissues. In a mouse model of VM, the intravenously injected CTP/PM-M2 EVsvMIP-II-Lamp2b could be carried into the myocardium via CTP, PM, and vMIP-II. In the inflammatory microenvironment, macrophages differentiated from circulating monocytes and macrophages residing in the heart showed enhanced endocytosis rates for CTP/PM-M2 EVsvMIP-II-Lamp2b. Subsequently, CTP/PM-M2 EVsvMIP-II-Lamp2b successfully released functional M2 EVsvMIP-II-Lamp2b into the cytosol, which facilitated the reprogramming of inflammatory M1 macrophages to reparative M2 macrophages. vMIP-II not only helps to increase the targeting ability of M2 EVs but also collaborates with M2 EVs to regulate M1 macrophages in the inflammatory microenvironment and downregulate the levels of multiple chemokine receptors. Finally, the cardiac immune microenvironment was protectively regulated to achieve cardiac repair. Taken together, our findings suggest that CTP-and-PM-engineered M2 EVsvMIP-II-Lamp2b represent an effective means for treating VM and show promise for clinical applications.

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.

Transcriptional and functional analysis of plasma exosomal microRNAs in acute viral myocarditis.

AIM: To assess the differential expression profiles of exosome-derived microRNA (miRNA) and reveal their potential functions in patients with acute viral myocarditis (AVMC). MATERIALS & METHODS: Peripheral blood samples were collected from 9 patients diagnosed with AVMC and 9 healthy controls (HC) in the Affiliated Hospital of Qingdao University from July 2021 to September 2022. The exosomal miRNA expression were tested using RNA high-throughput sequencing. We conducted the GO and KEGG functional analysis to predict the potential molecular, biological functions and related signaling pathways of miRNAs in exosomes. Target genes of exosomal miRNAs were predicted and miRNA-target gene network was mapped using gene databases. Differentially expressed exosomal miRNAs were selected and their expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) to verify the sequencing results. RESULTS: P < 0.05 and Fold Change>2 were considered as cut-off value to screen miRNAs that were differently expressed. This study identified 14 upregulated and 14 downregulated exosome-derived miRNAs. GO and KEGG analysis showed that differentially expressed miRNAs may be related to ß-catenin binding, DNA transcription activities, ubiquitin ligase, PI3K-Akt, FoxO, P53, MAPK, and etc.. The target genes of differentially expressed miRNAs were predicted using gene databases. Real-time PCR confirmed the upregulation of hsa-miR-548a-3p and downregulation of hsa-miR-500b-5p in AVMC. CONCLUSIONS: Hsa-miR-548a-3p and hsa-miR-500b-5p could serve as a promising biomarker of AVMC. Exosomal miRNAs may have substantial roles in the mechanisms of AVMC.

High risk and low prevalence diseases: Myocarditis.

INTRODUCTION: Myocarditis is a serious condition that carries with it a high rate of morbidity and mortality. OBJECTIVE: This review highlights the pearls and pitfalls of myocarditis, including presentation, diagnosis, and management in the emergency department (ED) based on current evidence. DISCUSSION: Myocarditis is an inflammatory syndrome of myocardium, most often resulting from a viral infection, that can cause life-threatening cardiovascular collapse. It has a highly variable presentation and no widely available specific diagnostic test, making it a challenging diagnosis. Emergency clinicians should obtain an electrocardiogram and perform bedside ultrasound to assess cardiac function. Treatment in the ED is largely supportive, focusing on resuscitation, cardiovascular support, cardiology specialist consultation, and appropriate disposition. CONCLUSIONS: An understanding of myocarditis can assist emergency clinicians in diagnosing and managing this potentially deadly disease.

CD4+TEM cells drive the progression from acute myocarditis to dilated cardiomyopathy in CVB3-induced BALB/c mice.

Acute viral myocarditis can progress to chronic myocarditis leading to dilated cardiomyopathy (DCM). Persistent CD4+ T-cell-mediated autoimmunity triggered by infection plays a critical role in this progression. Increasing evidence demonstrates that effector memory CD4+T (CD4+TEM) cells, a subset of memory CD4+ T cells, are crucial pathogenic mediators of many autoimmune diseases. However, the role of CD4+TEM cells during the progression from acute viral myocarditis to DCM remains unknown. In this study, we observed an increase in CD4+TEM cells both in the periphery and the heart, and memory CD4+ T cells were the predominant sources of IL-17A and IFN-γ among inflamed heart-infiltrating CD4+ T cells during the progression from acute myocarditis to chronic myocarditis and DCM in CVB3-induced BALB/c mice. Moreover, splenic CD4+TEM cells sorted from DCM mice induced by CVB3 were found to respond to cardiac self-antigens ex vivo. Additionally, adoptive transfer experiments substantiated their pathogenic impact, inducing sustained myocardial inflammation, tissue fibrosis, cardiac injury, and impairment of cardiac systolic function in vivo. Our findings illustrate that long-lived CD4+TEM cells are important contributors to the progression from acute viral myocarditis into DCM.

Identification of the communal pathogenesis and immune landscape between viral myocarditis and dilated cardiomyopathy.

AIMS: Studies have confirmed that viral myocarditis (VMC) is one of the risk factors for dilated cardiomyopathy (DCM). The molecular mechanisms underlying the progression from VMC to DCM remain unclear and require further investigation. METHODS AND RESULTS: The mRNA microarray datasets GSE57338 (DCM) and GSE1145 (VMC) were obtained from the Gene Expression Omnibus database. The candidate key genes were further screened using weighted correlation network analysis (WGCNA), protein-protein interaction and external dataset validation, and the correlation between the candidate key genes and immune cells and the signalling pathways of the candidate key genes were observed by enrichment analysis and immune infiltration analysis. The expression of key genes was validated in the external dataset GSE35182. The crosstalk genes between DCM and VMC were mainly enriched in 'transcriptional misregulation in cancer', 'FoxO signalling pathway', 'AGE-RAGE signalling pathway in diabetic complications', 'thyroid hormone signalling pathway', 'AMPK signalling pathway', and other signalling pathways. The immune infiltration analysis indicated that VMC was mainly associated with resting dendritic cells and M0 macrophages, while DCM was mainly associated with monocytes, M0 macrophages, CD8+ T cells, resting CD4 memory T cells, naive CD4+ T cells, and resting mast cells. In DCM-related dataset GSE57338 and VMC-related dataset GSE1145, a total of 18 candidate key genes were differentially expressed. BLC6, FOXO1, and UBE2M were identified as the key genes that lead to the progression from VMC to DCM by GSE35182. CONCLUSIONS: Three key genes (BLC6, FOXO1, and UBE2M) were identified and provided new insights into the diagnosis and treatment of VMC with DCM.

M2 macrophage exosome-derived lncRNA AK083884 protects mice from CVB3-induced viral myocarditis through regulating PKM2/HIF-1α axis mediated metabolic reprogramming of macrophages.

Viral myocarditis (VM) is a clinically common inflammatory disease. Accumulating literature has indicated that M2 macrophages protect mice from Coxsackievirus B3 (CVB3)-induced VM. However, mechanisms that underlie M2 macrophages alleviating myocardial inflammation remain largely undefined. We found that M2 macrophage-derived exosomes (M2-Exo) can effectively attenuate VM. The long non-coding RNA (lncRNA) AK083884 in M2-Exo was found to be involved in the regulation of macrophage polarization by exosome lncRNA sequencing combined with in vitro functional assays. M2-Exo-derived AK083884 promotes macrophage M2 polarization and protects mice from CVB3-induced VM. Furthermore, we identified pyruvate kinase M2 (PKM2) as a protein target binding to AK083884 and found that PKM2 knockdown could promote macrophages to polarize to M2 phenotype. Intriguingly, functional assay revealed that downregulation of AK083884 promotes metabolic reprogramming in macrophages. In addition, co-immunoprecipitation was performed to reveal AK083884 could interact with PKM2 and inhibition of AK083884 can facilitate the binding of PKM2 and HIF-1α. Collectively, our findings uncovered an important role of M2-Exo-derived AK083884 in the regulation of macrophage polarization through metabolic reprogramming, identified a new participant in the development of VM and provided a potential clinically important therapeutic target.