Abstract Body: Background: Myocarditis is defined as an inflammatory process affecting the myocardium, the muscular tissue of the heart. Most common causes are viral including Coxsackievirus B3 (CVB3). Myocarditis has a 39.3% 10-year mortality and up to 30% of cases will develop dilated cardiomyopathy (DCM). We have a very poor understanding of how different immune cell types mediate DCM pathogenesis, particularly fibrosis. While most myocarditis studies are 2D, it is yet to be studied in 3D. Here, using the novel CODA workflow, we present the first of its kind whole-heart 3D reconstruction of acute mouse CVB3 myocarditis at the histological, immunological and transcriptional levels

Research Question: Are immune foci during acute mouse CVB3 myocarditis homogeneous in the 3D space thoughout the heart?

Aim: 3D Profile immune foci in acute mouse CVB3 myocarditis at the histological, immune and transcriptional levels.

Methods: Whole mouse hearts with CVB3 myocarditis were formalin-fixed, paraffin-embedded, and serially sectioned. Serial sections were alternately stained with H&E or IHC. The IHC stain utilized CD3 and CD68 antibodies to identify T cells and macrophages (MΦ). We digitally registered the scanned heart sections and labeled the following at micron-resolution: cardiomyocytes, blood vessels and immune foci. The resulting digital histological annotations and immune cell localization were quantified and incorporated into one 3D model. Visium spatial RNA-Seq was then integrated into the 3D model.

Results: 3D reconstruction of whole CVB3-infected mouse hearts revealed that T cell-dominant and MΦ-dominant immune foci are established in spatially distinct niches during myocarditis, particularly along the sagittal axis (R²=0.5, p-value<0.001). This was validated using large mouse samples via flow cytometry (p-value <0.05) and in human DCM using IHC. We further show that T cell-dominant are more colocalized with fibrosis (p-value<0.05). Intriguingly, it was MΦ within such foci that are in closer proximity to activated fibroblasts (p-value<0.05). These MΦ were not only closer to activated fibroblasts but upregulated Ifngr1 and Cxcl12 (p-value<0.05).

Conclusions: 3D reconstruction is a critical tool to understand the pathology of myocarditis. Here, we show that immune foci formerly thought to be immunologically uniform, are in fact quite spatially heterogeneous. Additionally, this heterogeneity might be critical to better understanding fibrosis development during myocarditis.