Abstract Body (Do not enter title and authors here): Introduction: Cellular senescence often involves a p16-pathway, and p16 overexpression is a hallmark of senescent cells. The role of cellular senescence in myocardial infarction (MI) and any mediating mechanisms remain unclear.

Aims: To investigate the effect of p16⁺ cell clearance on survival post MI and elucidate underlying mechanisms.

Methods: We utilized INK-ATTAC transgenic mice, in which p16⁺ cells undergo targeted apoptosis upon exposure to AP20187 (AP). Sham and MI mice were treated with AP or vehicle (V) twice-weekly for one month, starting 3-4 hours post-MI. Survival rate improvement post MI in the AP group (Fig A, P<0.001) prompted us to evaluate effects of senescent cell clearance at the cardiac cellular level and its involvement in MI-associated pathways at 3 days post-MI. The infarct and peri-infarct areas were collected and snap-frozen. Nuclei were extracted with modified gentleMACS methods; libraries were constructed using 10X Genomics. Standard software (CellRanger, Seurat) were employed for single-nuclei-RNA-seq data analysis.

Results: Cardiomyocytes (CMs) showed 5 distinct subsets. CM subclusters 1 and 2 showed enrichments of genes characteristic of infarct boarder zones, while most sham CMs were found in subcluster 0, reflecting healthy-CMs. AP-treated mice exhibited a higher proportion of healthy CMs (Fig B). Differentially expressed genes (DEGs) between V and AP group were enriched in the healthy CMs, suggesting that clearance of p16⁺ cells improves the status of CMs post MI (Fig C). Immune-cell profiling showed that by far the largest number of DEG for AP vs vehicle were expressed in the (arginase-1) Arg1 macrophage subgroup (Fig D). The percentage of Arg1 macrophages decreased in the AP group (FDR=0.059) (Fig E). We noted significant enrichment in the hypoxia and cardiac rupture pathways in Arg1 macrophages (Fig F), correlating with the high death rate from myocardial rupture in V-mice.

Conclusion: These results suggest a significant role of p16⁺ senescent cells in post-MI mortality, potentially through effects on cardiomyocytes and a specific macrophage subpopulation, Arg1 macrophages.