https://doi.org/10.1007/s00392-025-02625-4
1Universitäts-Herzzentrum Freiburg - Bad Krozingen Klinik für Kardiologie und Angiologie Freiburg im Breisgau, Deutschland; 2Deutsches Herzzentrum der Charite (DHZC) Klinik für Kardiologie, Angiologie und Intensivmedizin | CBF Berlin, Deutschland; 3Universitätsklinikum Tübingen Kardiopathologie Tübingen, Deutschland; 4Universitäts-Herzzentrum Freiburg - Bad Krozingen Innere Medizin III, Kardiologie und Angiologie Freiburg im Breisgau, Deutschland
Background:
The clinical course of myocarditis presents a wide range of disease trajectories, from complete recovery to heart failure. In mice, Coxsackievirus B3 (CVB3) injection is used as a well-established model for viral myocarditis. In the resolution of myocarditis—in both humans and mice—the complete elimination of the virus is a crucial factor in reducing long-term damage. At the same time, an excessive immune response must be avoided, as it could also cause lasting damage to heart tissue. Therefore, a precisely timed interplay of pro- and anti-inflammatory immune signals is required for myocarditis to heal completely without sequelae.
Objectives and experimental design:
To better understand the course of immune responses during the disease progression, this project focus on a detailed time-course analysis of CVB3-induced myocarditis (5, 7 and 9 days post-infection). The time-course of immune response, virus load, and viral replication in the heart and in other organs involved in the immune response are examined via RNA sequencing and gene expression analysis. Furthermore, in vitro infection with CVB3 sheet light on the contribution of different cardiac and immune cell types to the disease progression.
Results:
In our study, we observed that the virus load in the left ventricle (LV) was highest on day 5 post-infection, and was significantly declined at day 9. Notably, one animal had completely eliminated the virus from the LV by day 9. Analysis of blood, lymph nodes, and spleen revealed the highest virus load in the spleen, though this also decreased between day 5 and day 9.
We performed RNA sequencing on LV tissue samples collected at days 5, 7, and 9. The largest number of differentially expressed genes (DEGs) was found on day 5, with 861 DEGs (460 upregulated and 401 downregulated). On day 7, we identified 657 DEGs, while day 9 showed only 192 DEGs when comparing CVB3-infected animals to sham controls. These findings were reflected with the results of GO term analysis. On day 5, 44 cytokine-related GO terms were upregulated, whereas on day 7, this number decreased to 36. By day 9, only 19 cytokine-related GO terms showed significant enrichment. Interestingly, GO terms associated with apoptosis were most significantly enriched at day 7, suggesting possible apoptosis of immune cells at this time point.
To further explore dynamics of virus infection and replication, we infected different cardiac cell types with CVB3 for 24 hours. Cardiomyocytes showed a substantial increase in virus load, with a 300-fold increase after 24 hours. In contrast, T cells showed only a modest 1.5-fold increase, B cells increased virus load 3-fold, and macrophages actually reduced their virus load within 24 hours. The results were confirmed in vivo: In situ hybridization of LV tissue samples showed that viral RNA was mainly detected in cardiomyocytes but also in immune cell infiltrates, which cell composition is currently analysed in more detail.
Summary:
The RNA sequencing revealed a decreased inflammatory response from day 5 to day 9, which is in line with the decreased virus load in LV tissue in the same timeframe. In vitro experiments as well as in situ hybridization of viral RNA in LV tissue samples showed virus replication mainly in cardiomyocytes and to a lesser extend in B- and T cells. These findings provide insight into the dynamics of viral replication, immune response, and cellular susceptibility in the heart during CVB3-induced myocarditis.