Immunosuppression modulates post-ischemic myocardial scar formation and remodelling in LEA29Y pigs
H Heymer, N Hornaschewitz, M Corsten, X Li, E Wolf, A Parzefall, N Klymiuk, C Kupatt, A BaehrAbstract
Background
Scar formation during myocardial remodelling after ischemia/reperfusion (I/R) is tightly regulated by the immune system and its effector cells. Genetically modified immunocompromised pigs (LEA29Y) allow investigation of remodelling mechanisms. To account for strong regional immune responses, we performed a detailed whole-heart examination of I/R injury and remodelling, combining functional and molecular data with comprehensive immunohistochemical assessment of differently affected regions.
Methods
Using a catheter-based approach, 45–60 minutes of ischemia were induced in the left anterior descending artery (LAD) of LEA29Y pigs and WT controls, followed by permanent reperfusion. I/R-treated and control animals were functionally assessed and sacrificed after 3, 9, 14, & 26 days. Each heart was sectioned into 36 locally assigned left ventricular samples, classified as remote zone, area at risk, or infarct zone, and separated for fixation and cryoconservation. All fixed samples were stained with HE and Sirius Red. Immunohistochemistry for IBA1, CD3 T-cells, and neutrophils was performed on every fourth systematically selected sample per region. Whole-slide scans were analysed using QuPath for global high-resolution quantification by creating pixel classifiers and complemented by cluster analysis in ImageJ. Statistical comparisons were performed using t tests.
Results
LEA29Y and WT animals displayed similar infarct sizes. LV angiography revealed a tendency of greater early loss of ejection fraction in transgenic pigs up to day 14, but less aggravated impairment after 26 days (-18.96%) compared to WT (-27.34%, p=0.0016). Collagen analysis demonstrated an earlier, lower peak at day 9 in LEA29Y versus day 14 in WT animals. Global IBA1-stained macrophages were significantly reduced in transgenic pigs (p=0.000751) with rapid post-MI recovery to WT-comparable levels after 3 days. In infarct areas, macrophage levels were initially higher in LEA29Y pigs but declined rapidly, whereas WT levels peaked at day 9, resulting in higher WT levels at this timepoint and similar levels at day 26. Cluster analysis of IBA1-stained infarcted areas showed homogeneous macrophage clustering at day 3, followed by increased clustering in LEA29Y and decreased clustering in WT, which later reversed; by day 26 WT animals exhibited greater clustering. Quantitative measurements of stained CD3 T-cells showed significantly lower levels in LEA29Y pigs at baseline and they remained reduced in infarct areas through day 26.
Conclusion
Histological findings in post-I/R hearts align with functional data indicating slower but more sustained recovery of ejection fraction in LEA29Y pigs. Proteomics and snRNAseq support the suggestion of a delayed and reduced remodelling under immunosuppression, co-inciding with a more pronounced clustering of macrophages in infarcted areas. Early cardiac remodelling thus depends on precise immune orchestration.