Background: Pigs are an indispensable model in translational cardiovascular research, amongst others for the investigation of novel therapeutic strategies against myocardial infarction (MI) and its sequelae. Given the notable immunological proximity of these animals to humans, porcine models seem to be especially suitable to investigate therapeutic approaches targeting the immune response to cardiac injury. However, leukocytes in pig hearts have not yet been systematically studied, partly because of the rather limited immunological toolbox for this species. Therefore, we aimed to develop a workflow for further examination of these cells with basic immunological methods.

Methods and Results: In 2- to 3-month-old female German Landrace pigs MI was induced by a percutaneous coronary balloon occlusion of the left anterior descending artery over 90 minutes. Some animals underwent a corresponding sham procedure. After 3, 7 or 14 days of recovery the pigs were sacrificed. Their hearts were explanted and the coronary vessels were flushed with saline. The dissected organs from the MI group consistently showed extensive transmural infarction of the left ventricular myocardium at different maturation stages and with clear macroscopic delineation. Tissue samples were taken from the infarct core, the adjacent “border region” and the non-infarcted “remote myocardium” of the left ventricle. For immunophenotyping, the tissue was enzymatically digested. Leukocytes were enriched from the cell suspension using anti-pig CD45 antibodies and magnetic cell separation. Eventually, flow cytometry was performed after staining with a viability dye and several antibodies against porcine immune cell markers (CD3e, CD4a, CD8a, CD79a, CD163, CD172a, CD203a, Foxp3) described in the literature and validated in our hands. Also, tissue cryosections were stained with a selection of these as well as other antibodies and subjected to immunofluorescence microscopy.  

With our protocol we could isolate CD45+ leukocytes from all myocardial tissue samples and purify them in sufficient numbers and quality for further analysis. Flow cytometry enabled us to identify and to relatively quantify the major myeloid/lymphoid immune cell populations. Moreover, several subsets were differentiated. In addition, immunofluorescence microscopy allowed us to detect and localize different leukocytes in the tissue. Compared to sham animals or to non-infarcted regions, we found a massive accumulation of leukocytes in the infarct core with a marked predominance of myeloid cells (mostly monocytes/macrophages), particularly in the early phase (90.8% CD172a+ cells 3d post MI vs. 45.9% in pooled sham), showing a decreasing trend over time. Inversely, the local lymphocyte fraction increased up to 19.7% on day 14 after MI, with an early peak in CD79a+ B cells and a growing population of CD4+ Foxp3+ T cells (2.1% 7d post MI vs. 0.6% in pooled sham). These results were in line with our expectations based on data from mice and humans.

Summary: We were able to establish a workflow for the identification and phenotypic characterization of immune cells in the pig heart, particularly after MI. Next, we will investigate in depth the temporospatial dynamics of immune cell infiltration and activation in this context. Since our protocols use standard methods and commercially available antibodies, they might serve other researchers as a starting point for own cardio-immunological studies in this large animal model.