Abstract Body (Do not enter title and authors here): CD8⁺ T-cells are adverse regulators post myocardial infarction (MI), leading to increased mortality and impaired cardiac function. We hypothesize that CD8⁺ T-cells impair cardiac function by altering scar composition.

MI was induced by ligating the left anterior descending coronary artery in C57BL6/J (WT; 3-7 months of age, n≥2/sex) and CD8a^tm1mak mice (CD8^-/-; 3-7 months of age, n≥2/sex/treatment). CD8^-/- mice were injected with either vehicle or naïve splenic CD8⁺ T-cells (2x10⁶ cells/injection) via tail vein, 4 hours post-MI. Infarct tissue was collected post-MI Day 7 and underwent biomechanical, histological, and biochemical analyses. Effects of granzyme (Gzm) A, B, and K on collagen cleavage were tested using a fluorogenic collagen cleavage assay to examine possible mechanisms of scar alteration.

Mice lacking CD8⁺ T-cells had improved ejection fraction and decreased dilation (p<0.05 for both) compared to WT mice at post-MI Day 7. This protective effect was lost in CD8^-/- mice that received splenic CD8⁺ T-cells (p=0.09 vs WT). Biomechanical testing revealed CD8^-/- mice had a 2-fold increase in regional scar stiffness compared to WT mice (p<0.05). Re-supplementation with splenic CD8⁺ T-cells decreased scar tissue stiffness compared to vehicle treated mice (p<0.05) mimicking biomechanics of WT mice. Picrosirius red staining showed no significant differences in total collagen levels (p=0.51) across all groups. However, further investigation using collagen hybridization peptide staining showed increased collagen cleavage in WT mice in comparison to CD8^-/-mice (p<0.05) as demonstrated by overall fluorescence intensity. Evaluation of percent area of CHP staining showed WT mice had increased cleavage distribution within the infarct compared to vehicle-treated CD8^-/- mice (p<0.05). This effect was lost with CD8⁺ T-cell supplementation (p=0.13 vs WT). Ex-vivo cleavage assay demonstrated GzmA and K cleaved collagen in a concentration (0-50 AU) and temporal-dependent manner (0-24 hrs). Both GzmA and K demonstrated a 10-fold greater capacity in collagen cleavage compared to GzmB at 25 AU, implicating distinctly separate roles for the different granzymes during post-MI scar formation and maturation.

In conclusion, our data demonstrates that CD8⁺ T-cells regulate cardiac fibrosis in part through the release of granzymes, leading to impairments of biomechanical capability of the left ventricle and increased LV dilation.