CXCL4/CXCR3 axis regulates cardiac fibrosis by activating TGF-β1/Smad2/3 signaling in mouse viral myocarditis

Background: Severe myocarditis is often associated with cardiac fibrosis, although the precise mechanisms remain unclear. CXCL4, a chemokine known for its pro-inflammatory and profibrotic roles, may play a key role in this process, but its exact contribution to cardiac fibrosis is not fully understood.

Methods: Viral myocarditis (VMC) models were developed through intraperitoneal injections of Coxsackievirus B3 (CVB3). The VMC group received CVB3 at 100 TCID50, while the VMC+AMG487 group received a combination of CVB3 (100 TCID50) and AMG487 (5 mg/kg). Myocardial morphology was assessed through hematoxylin-eosin (H&E) staining, severity scoring, Masson staining, and immunofluorescence. Inflammatory markers (IL-1β, IL-6, TNF-α, and CXCL4) were quantified using enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription PCR (qRT-PCR). Serum levels of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB) were measured using commercial kits. Western blotting and immunofluorescence were performed to evaluate CXCL4, CXCR3B, α-SMA, TGF-β1, and Collagen I/III levels.

Results: Treatment with AMG487 reduced cardiac damage, α-SMA expression, collagen deposition, and levels of Collagen I and III in the VMC+AMG487 group compared to the VMC group. Furthermore, inflammatory markers (IL-1β, IL-6, and TNF-α) were significantly lower in the VMC+AMG487 group. In vitro studies demonstrated that activation of the CXCL4/CXCR3B axis promotes cardiac fibroblast differentiation via the TGF-β1/Smad2/3 signaling pathway.

Conclusion: CXCL4 functions as a profibrotic mediator by driving TGF-β1/Smad2/3 pathway-dependent cardiac fibroblast activation AMG 487 and extracellular matrix (ECM) synthesis, contributing to cardiac fibrosis. These findings highlight the role of CXCL4 in VMC and suggest that targeting CXCL4 may offer a potential therapeutic strategy for treating viral myocarditis.