SARS-CoV-2 pathogenesis in an angiotensin II-induced heart-on-a-chip disease model and extracellular vesicle screening.

Adverse cardiac outcomes in COVID-19 patients, particularly those with preexisting cardiac disease, motivate the development of human cell-based organ-on-a-chip models to recapitulate cardiac injury and dysfunction and for screening of cardioprotective therapeutics. Here, we developed a heart-on-a-chip model to study the pathogenesis of SARS-CoV-2 in healthy myocardium established from human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and a cardiac dysfunction model, mimicking aspects of preexisting hypertensive disease induced by angiotensin II (Ang II). We recapitulated cytopathic features of SARS-CoV-2-induced cardiac damage, including progressively impaired contractile function and calcium handling, apoptosis, and sarcomere disarray. SARS-CoV-2 presence in Ang II-treated hearts-on-a-chip decreased contractile force with earlier onset of contractile dysfunction and profoundly enhanced inflammatory cytokines compared to SARS-CoV-2 alone. Toward the development of potential therapeutics, we evaluated the cardioprotective effects of extracellular vesicles (EVs) from human iPSC which alleviated the impairment of contractile force, decreased apoptosis, reduced the disruption of sarcomeric proteins, and enhanced beta-oxidation gene expression. Viral load was not affected by either Ang II or EV treatment. We identified MicroRNAs miR-20a-5p and miR-19a-3p as potential mediators of cardioprotective effects of these EVs.

Major Group-B Enterovirus populations deleted in the noncoding 5' region of genomic RNA modulate activation of the type I interferon pathway in cardiomyocytes and induce myocarditis.

Major 5'-terminally deleted (5'TD) RNA forms of group-B coxsackievirus (CVB-5'TD) has been associated with myocarditis in both mice and humans. Although it is known that interferon-ß (IFN-ß) signaling is critical for an efficient innate immune response against CVB-induced myocarditis, the link between CVB-5'TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored. In a mouse model of CVB3/28-induced myocarditis, major early-emerging forms of CVB-5'TD RNA have been characterized as replicative viral populations that impair IFN-ß production in the heart. Synthetic CVB3/28 RNA forms mimicking each of these major 5'TD virus populations were transfected in mice and have been shown to modulate innate immune responses in the heart and to induce myocarditis in mice. Remarkably, transfection of synthetic viral RNA with deletions in the secondary structures of the 5'-terminal CVB3 RNA domain I, modifying stem-loops "b", "c" or "d", were found to impair IFN-ß production in human cardiomyocytes. In addition, the activation of innate immune response by Poly(I:C), was found to restore IFN-ß production and to reduce the burden of CVB-5'TD RNA-forms in cardiac tissues, thereby reducing the mortality rate of infected mice. Overall, our results indicate that major early-emerging CVB3 populations deleted in the domain I of genomic RNA, in the 5' noncoding region, modulate the activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in mice. These findings shed new light on the role of replicative CVB-5'TD RNA forms as key pathophysiological factors in CVB-induced human myocarditis.

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.

Loss of TRIM29 mitigates viral myocarditis by attenuating PERK-driven ER stress response in male mice.

Viral myocarditis, an inflammatory disease of the myocardium, is a significant cause of sudden death in children and young adults. The current coronavirus disease 19 pandemic emphasizes the need to understand the pathogenesis mechanisms and potential treatment strategies for viral myocarditis. Here, we found that TRIM29 was highly induced by cardiotropic viruses and promoted protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated endoplasmic reticulum (ER) stress, apoptosis, and reactive oxygen species (ROS) responses that promote viral replication in cardiomyocytes in vitro. TRIM29 deficiency protected mice from viral myocarditis by promoting cardiac antiviral functions and reducing PERK-mediated inflammation and immunosuppressive monocytic myeloid-derived suppressor cells (mMDSC) in vivo. Mechanistically, TRIM29 interacted with PERK to promote SUMOylation of PERK to maintain its stability, thereby promoting PERK-mediated signaling pathways. Finally, we demonstrated that the PERK inhibitor GSK2656157 mitigated viral myocarditis by disrupting the TRIM29-PERK connection, thereby bolstering cardiac function, enhancing cardiac antiviral responses, and curbing inflammation and immunosuppressive mMDSC in vivo. Our findings offer insight into how cardiotropic viruses exploit TRIM29-regulated PERK signaling pathways to instigate viral myocarditis, suggesting that targeting the TRIM29-PERK axis could mitigate disease severity.

ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection.

AIMS: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. METHODS AND RESULTS: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. CONCLUSION: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.

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.