Characterizing immune-fibroblast distribution in pigs after myocardial infarction

Background: Myocardial infarction (MI) and its complications, such as heart failure, are still a major burden of disease despite improved treatment options. After MI, the interplay of immune cells and fibroblasts is indispensable for healing and scarring of the injured myocardium. Although pigs exhibit close anatomical and immunological similarities to humans, they have not been used extensively for research on this topic. In a closed-chest porcine model that resembles the pathophysiology of reperfused MI in humans, our group recently described key aspects of the MI induced immune response in more detail. Based on this, we aimed to characterize the spatiotemporal distribution of immune cell and fibroblast subtypes following experimental MI in the pig. In addition, we investigated the transcriptional features and interactions of the immune cells involved.

Methods and Results: 2.5-month-old Landrace pigs underwent MI induction by a 90-minute interventional balloon occlusion of the proximal to mid left anterior descending artery, followed by a 3 to 14-day reperfusion period. Tissue cryosections from three distinct regions of the myocardium (infarct core, border zone, remote myocardium) at three different time points (days 3, 7, 14) after MI were analyzed by immunofluorescence staining of immune and stromal cells. On day 3, we identified macrophages and activated (periostin⁺) fibroblasts in the border zone, which have eventually advanced into the infarct core on day 7 after MI. At this time point, a marked expansion of (α-smooth muscle actin⁺) myofibroblasts can also be observed. On day 14, the macrophages have become less abundant in the border zone.
Noteworthy is the infiltration pattern of these cell types: The macrophages closest to the infarct core are followed by activated fibroblasts and myofibroblasts, with each cell type creating its distinct zone.

To further analyze the infiltrating immune cells at day 7 post MI, scRNA-seq was performed. Using the Seurat R package, we identified several myeloid populations that are mainly found in the infarct core. By using recently published gene signatures, we can further delineate these cells as pro-inflammatory and pro-repair populations, indicating different functions regarding the expansion of activated (periostin⁺) fibroblasts and (α-smooth muscle actin⁺) myofibroblasts.
The CellChat package enabled us to describe cell-cell communication, showing cell type-dependent signaling in the myeloid cells for inflammatory and fibrosis-regulating pathways, complementing the above-mentioned findings.

Summary and Implications: We present insights into the spatiotemporal infiltration pattern and the current cell states of immune cells and fibroblasts after experimental MI. In combination with examining the communication of the participating myeloid cells, our analysis can serve as a resource for studying cardiac repair mechanisms post MI in this translational large animal model.