Background:
Myocarditis exhibits a highly variable clinical course, ranging from full recovery to chronic heart failure. In mice, infection with Coxsackievirus B3 (CVB3) represents a well-established model for viral myocarditis. Complete viral clearance is a key determinant of recovery, while persistent infection is associated with enhanced fibrosis and long-term functional decline. Previous studies have linked immune cell infiltration to fibrotic remodelling and demonstrated fibrosis resolution following viral clearance. However, the temporal dynamics of immune and fibrotic responses during disease resolution remain incompletely understood.
Objectives and Experimental Design:
To elucidate the temporal immune response during disease progression, we performed a detailed time-course analysis of CVB3-induced myocarditis at 5-, 7-, 9-, and 16-days post-infection (dpi). Left ventricular (LV) tissue samples were subjected to bulk RNA sequencing at each time point to assess transcriptional changes, complemented by functional and histological analyses at 16 dpi.
Results:
Virus load in the LV peaked at 5 dpi and markedly declined by 9 dpi, with one mouse showing complete virus clearance. By 16 dpi, only 3 of 11 animals still exhibited detectable viral RNA in the myocardium, despite initial comparable virus load in the blood at 4 dpi. Gene Ontology (GO) analysis of differentially expressed genes (DEGs) revealed a progressive decrease in immune system activation and defence response to virus between 5 and 9 dpi, while apoptosis-related pathways such as “apoptotic signalling pathway” were most enriched at 7 dpi, suggesting immune cell apoptosis. Ongoing TUNEL staining of corresponding tissue sections aims to validate this observation.
At 16 dpi, animals that had cleared the virus from the LV displayed no significant DEGs compared with sham controls, whereas those with persistent infection exhibited 813 DEGs. Additionally, animals with persistent infection showed reduced body weight, impaired cardiac function, and increased fibrosis compared to sham controls. In contrast, animals with successful virus clearance did not differ significantly from sham controls. Remarkably, GO terms related to extracellular matrix organization such as “extracellular matrix structural constituent” or “collagen trimer” were markedly downregulated, including downregulated DEGs such as Col1a1, Col1a2, Col3a1 but also the collagenase Mmp13, as confirmed by TaqMan gene expression analysis. Correlation analysis further revealed a significant negative association between Mmp13 expression and fibrotic tissue area, which might reflect a delayed resolution of fibrosis compared to animals with successful virus clearance.
Conclusion:
Our data highlight distinct temporal phases of immune activation, apoptosis, and cardiac tissue remodelling during CVB3-induced myocarditis. Complete viral clearance was associated with transcriptional normalization and preserved cardiac structure, while persistent infection led to functional impairment and fibrosis despite downregulation of collagen genes. These findings underscore the importance of timely viral elimination to prevent maladaptive remodelling and may provide insight into mechanisms driving chronic myocarditis and heart failure
