Refining a preclinical model of viral myocarditis in accordance with biotech standards

Background: Viral myocarditis is a clinically relevant inflammatory heart disease that can progress to dilated cardiomyopathy and heart failure. However, preclinical models often suffer from limited translational relevance and high animal burden. Methods: This study aimed to refine a murine model of Coxsackievirus B3 (CVB3)-induced myocarditis by integrating dietary and imaging innovations to improve animal welfare, data quality, and clinical applicability. BALB/cJ mice were infected with heart-passaged CVB3 and monitored for up to 56 days. A hydrolyzed amino acid diet was introduced to mitigate CVB3-induced pancreatitis and associated weight loss. Cardiac function was assessed using 1D, 2D, and 4D echocardiography, including speckle-tracking strain analysis. Results: The refined diet significantly reduced animal health burden, reduced weight loss, and stabilized blood glucose, during development of cardiac inflammation and fibrosis. 4D echocardiography provided a highly sensitive and reproducible detection of systolic dysfunction, while strain analysis revealed early and regional myocardial impairment, closely mirroring clinical myocarditis phenotypes. Histopathological and AI-based analyses confirmed dose- and time-dependent inflammation and fibrosis, with peak inflammation at day 7 and persistent abnormalities through day 56. Conclusions: In summary, this model offers a robust and ethically optimized platform for translational research and therapeutic development in inflammatory cardiomyopathies, aligned with the principles of the 3Rs.