ABSTRACT: Rationale: Reperfusion therapy is critical to myocardial salvage in the event of a myocardial infarction, but is complicated by ischemia-reperfusion injury (IRI). Limited understanding of the spatial organization of cardiac cells, which governs cellular interaction and function, has hindered the search for targeted intervention minimizing the deleterious effects of IRI. Methods: We used imaging mass cytometry (IMC) to characterize the spatial distribution and dynamics of cell phenotypes and communities in the mouse left ventricle following IRI. Heart sections were collected from 12 cardiac segments (basal, mid-cavity, apical and apex of the anterior, lateral and inferior wall) and 8 time points (prior to ischemia (I-0h, and post reperfusion (R-0h, -2h, -6h, -12h, -1d, -3d, -7d)), and stained with 29 metal-isotope-tagged antibodies. Cell community analysis was performed on reconstructed images, and the most disease-relevant cell type and target protein was selected for intervention of IRI. Results: We obtained a total of 251 multiplexed images, and identified 197,063 single cells, which were grouped into 23 distinct cell communities based on the structure of cellular neighborhoods. The cellular architecture was heterogeneous throughout the ventricular wall, and exhibited swift changes following IRI. Analysis of proteins with post-translational modifications (PTMs) in single cells unveiled 13 PTM intensity clusters, and highlighted increased H3K9me3 as a key regulatory response in endothelial cells during the middle stage of IRI. Erasing H3K9 methylation, by silencing its methyltransferase Suv39h1 or overexpressing its demethylase Kdm4d, in isolated endothelial cells (EC) attenuated cardiac dysfunction and pathological remodeling, compared to PBS and unmodified EC control, when injected into the infarcted area of the myocardium.
