Fibrotic remodelling after coxsackievirus-induced myocarditis in mice
Authors: Favere K , Van Hecke M , Eens S , Bosman M , Stobbelaar K , Delputte P , De Sutter J , Roskams T , Heidbuchel H , Guns P 
Published: June 2022

Abstract

Funding Acknowledgements
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Research Foundation Flanders

Introduction
Myocarditis is an inflammatory disease of the heart with viral infections being the most common aetiology. Often, myocardial fibrosis (MF) is observed during the resolution of myocarditis. MF is a strong independent predictor for outcome in patients with myocarditis. Nevertheless, the late stages of disease, encompassing fibrogenesis and arrhythmogenesis, have been underappreciated in viral myocarditis research to date.

Purpose
The primary aim of this study was to characterise the natural history of coxsackievirus B3 (CVB3)-induced myocarditis in C57BL/6J mice, with emphasis on fibrogenesis and its electrical impact. Secondary aims were 1) to study the impact of inoculation dose and sex on the course of disease, and 2) to develop an in-house histopathological scoring system for inflammation and fibrosis.

Methods
C57BL/6J mice (12 weeks old; n=154) received intraperitoneal injection with CVB3 (Nancy strain). Male mice received 5 x 10^5 (regular dose)(RD) or 5 x 10^6 (high dose) plaque-forming units, whereas female mice received the RD only. Mice injected with PBS served as control groups. Animals were sacrificed 7, 14, 28, 56 and 77 days after CVB3 or PBS injection. Inflammation (haematoxylin-eosin) and fibrosis (Picrosirius red (PSR)) were evaluated histologically. Arrhythmogenicity was assessed through right ventricular programmed stimulation.

Results
CVB3 animals sacrificed or with spontaneous death within 14 days after inoculation showed cardiac infiltration in all (100%) and necrosis in 88% (35/40). In male CVB3-infected mice, premature mortality was ±20% in both regular and high dose groups, occurring between 9 and 23 days post inoculation. No mortality was observed in the female group (p&lt;0.025 for comparison male and female RD). Cardiac inflammation had resolved in the majority of animals at day 77. Perivascular fibrosis was seen in both infected and control mice. Pericellular fibrosis and organised scarring were seen in CVB3-infected animals only, and mainly became evident 56 and 77 days after inoculation (51% of infected animals). Also total cross-sectional PSR-positive area was 2-3-fold higher in CVB-infected mice compared to sex-matched controls, independent of inoculation dose (p&lt;0.05). At the 56- and 77-day timepoint, nonsustained ventricular tachycardia was induced in 17% (5/29) of the intervention animals (all with pericellular fibrosis or scarring) and none (0/14) of the control animals, but this did not reach statistical significance (p=0.098).

Conclusion
We report on the characterisation of fibrotic remodelling in a CVB3-induced myocarditis mouse model. Fibrotic remodelling was seen in a majority of mice after 8 to 11 weeks. It was associated with a numerical (but not significant) increased arrhythmogenicity, requiring further exploration given the clinical significance of fibrotic remodelling. Further research on the molecular pathways and cellular interactions triggered by myocarditis is ongoing.