A20 (TNFAIP3) alleviates viral myocarditis through ADAR1/miR-1a-3p-dependent regulation

Abstract

Objective To investigate the effect of A20 and how A20 is regulated in viral myocarditis (VMC). Methods BABL/C mice, primary neonatal rat cardiomyocytes and H9c2 cells were infected with Coxsackie virus B3 (CVB3) to establish animal and cellular models of VMC. H&E staining revealed the pathologic condition of myocardium. ELISA measured the serum levels of creatine kinase, creatine kinase isoenzyme and cardiac troponin I. The effects of A20, miR-1a-3p and ADAR1 were investigated using gain and loss of function approaches. ELISA measured the levels of IL-6, IL-18 and TNF-α in serum or cell culture supernatant. TUNEL staining and flow cytometry assessed the apoptosis of myocardium and cardiomyocytes, respectively. RNA-binding protein immunoprecipitation and dual-luciferase reporter assays verified the binding between A20 and miR-1a-3p. Co-immunoprecipitation assay verified the binding between ADAR1 and Dicer. Results A20 was underexpressed and miR-1a-3p was overexpressed in the myocardium of VMC mice as well as in CVB3-infected cardiomyocytes. Overexpression of A20 suppressed cardiomyocyte inflammation and apoptosis in vivo and in vitro. miR-1a-3p promoted CVB3-induced inflammation and apoptosis in cardiomyocytes by binding to A20. The expression of miR-1a-3p was regulated by ADAR1. ADAR1 promoted the slicing of miR-1a-3p precursor by binding to Dicer. Conclusion A20, regulated by ADAR1/miR-1a-3p, suppresses inflammation and cardiomyocyte apoptosis in VMC.

A Model of iPSC-Derived Macrophages with TNFAIP3 Overexpression Reveals the Peculiarities of TNFAIP3 Protein Expression and Function in Human Macrophages

“…The effects of A20 overexpression were explored in fewer studies and largely in cells other than macrophages. The reported effects include: an inhibition of the phosphorylation of p38 and c-JUN and an augmentation of the levels of IkB in intestinal epithelioid cells IEC-6 bearing an ectopic A20 expression and stimulated with LPS or CpG [ 43 ]; a repression of retinoic acid-inducible gene I (RIG-1) and RIG-1-dependent antiviral activity and an inhibition of TIR-Domain-Containing-Adaptor-Molecule 1 (TRIF)-mediated activation of interferon-stimulated response element (ISRE), NF-κB and IFN-β promoter in HEK293 cells co-transfected with A20 and its molecular targets [ 19 , 41 ]; an inhibition of IL-8 secretion by intestinal epithelial CaCo cells transfected with an A20-expressing plasmid and stimulated with Pam3CSK4 [ 42 ]; a downregulation of the expression of proinflammatory chemokines Ccl2, Cxcl1 and Cxcl10 in A20 -transfected Stat3 -knockout mouse embryo fibroblasts triggered with TNF-α [ 45 ]; a decrease in the expression of IL-6, IL-18 and TNF-α in primary neonatal rat cardiomyocytes transduced with an A20 -expressing lentivirus [ 46 ]; a decreased production of IFN-γ and IL-17 by CD4 + T cells derived from patients with SLE and transfected with an A20 -expressing plasmid [ 44 ]; and a decreased TNF-α-induced apoptosis in A20 -overexpressing Jurkat cells [ 40 ]. Although the studies are consistent in terms of anti-inflammatory A20 effects, they do not concern macrophages, and it cannot be excluded that in different cells the nuances of A20 inhibitory activity may be different.…”

“…However, most A20 studies were conducted in conditions wherein there was a deficit of A20. Whether the overexpression of the protein can efficiently restrict inflammatory reactions has been examined in a limited number of studies which did not involve macrophages [ 19 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ] (this is considered in more detail in the Discussion section).…”

Adenosine deaminase acting on RNA 1 (ADAR1) as crucial regulators in cardiovascular diseases: structures, pathogenesis, and potential therapeutic approach

“…ADAR1 has a certain effect on virus replication and can inhibit or promote virus replication ( Samuel, 2011 ). ADAR1 is overexpressed in the myocardium of viral myocarditis (VMC) mice as well as in Coxsackie virus B3 (CVB3)-induced cardiomyocytes ( Li and Xie, 2022 ). ADAR1 promotes the cleavage of pre-miR-1a-3p by binding to Dicer, thereby regulating the functional activity of A20 which inhibits of cardiomyocyte inflammation and apoptosis in CVB3-induced myocarditis ( Li and Xie, 2022 ).…”

Coxsackievirus B3 infection induces glycolysis to facilitate viral replication

“…To determine whether CVB3 induces glycolysis in myocardial cells, H9c2 and HL-1 cells were infected with CVB3 to establish cellular models of viral myocarditis (Tabor-Godwin et al, 2012;Li et al, 2014Li et al, , 2019Wang et al, 2018;Li and Xie, 2022;Pappritz et al, 2022). We measured glucose consumption and lactate production, which approximatelyreflect fluctuations in glycolysis (Sun et al, 2011;Mazzon et al, 2018;Lee et al, 2020).…”