Whole-heart 3D reconstruction of mouse CVB3 myocarditis reveals spatial and transcriptomic heterogeneity of immune foci.

aimsMyocarditis is an inflammation of the myocardium and is characterized by poor prognosis in symptomatic patients and significant correlation with dilated cardiomyopathy (DCM). We investigated spatial, morphological, immunological, and transcriptomic heterogeneity of myocarditis inflammatory foci in the 3D space. METHODS AND

resultsUtilizing a novel large tissue histological reconstruction workflow, CODA (not an acronym), we generated whole-heart 3D reconstructions of acute mouse Coxsackievirus B3 (CVB3) myocarditis at the single-cell resolution. Furthermore, we integrated immunohistochemical staining and spatial RNA-sequencing into the CODA workflow. This approach allowed the simultaneous morphological, immunological and transcriptional 3D analysis of acute mouse CVB3 myocarditis. In addition, flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses were used to validate our 3D findings. We show that acute mouse CVB3 myocarditis foci are multi-branched, elongated, and found mostly within myocardial regions of the left ventricular wall. We further show that significant T-cell- and macrophage-specific niches exist within the same focus. At the organ level, we show that T-cell-rich foci are more likely to be found anteriorly while macrophage-rich foci are more likely to be found posteriorly. We further demonstrate that T-cell hotspots are well-vascularized spatial niches. Moreover, using spatial transcriptomics and qRT-PCR, we show that T-cell-rich regions of anterior immune foci uniquely upregulate the expression of collagen I and T-cell-specific chemokines. Conversely, surrounding homogeneous foci regions upregulate the expression of broad-target chemokines and T-cell retentive molecules. We show the translatability of our work since 3D immunological heterogeneity mouse results were similar to human DCM explanted hearts with similar immune cell heterogeneity along the sagittal axis.

conclusionOur multimodal findings highlight that acute mouse CVB3 myocarditis exhibits heterogeneous immune phenotype with distinct anterior T-cell hotspots that are well-vascularized and upregulate the expression of T-cell-specific chemokines and collagen I.