LEA29Y expression affects myocardial ischemia/reperfusion outcome in pigs

Healing and scar formation in myocardial remodeling after ischemia/reperfusion (I/R) are complex processes strictly regulated by the immune system and its effector cells. The fusion protein LEA29Y is a commonly used immunosuppressant that blocks co-stimulation of T cells. Pigs systematically expressing LEA29Y have been established as a model for general immunosuppression that was used to study I/R remodeling under immuno-compromised conditions including a focus on the amount of collagen as an indicator of scar formation as well as quantification of immune cells.

LEA29Y transgenic and wildtype pigs were subjected to a catheter-based procedure of I/R in which the LAD was temporarily occluded by a PCTA catheter. A total of 28 animals were assigned to follow-up periods of 3, 9, 14 and 26 days. Results were compared to transgenic and control animals without intervention to determine baseline conditions. Infarct size was calculated and the left ventricle (LV) was systematically randomly sampled. Functional measurements by left ventricular angiography as well as ECG and echocardiography are supplemented by molecular and histological assessments including immunohistochemical assessment of immune cell infiltration. Scar formation is determined by global, whole slide analysis after picrosirius red staining using “QuPath” software. QuPath is an open-source bioimage analysis program that allows for the analysis of wholly scanned slides, thus enabling a global, total tissue determination of collagen content also in the hearts of large animal models.  For this,  two QuPath-filters were created – one for tissue detection and one for detection of collagen in Sirius red. For the detection of tissue a pixel threshold was used, for collagen-detection an artificial neural network was trained to classify and measure the area within the boundary of the detected tissue.

LEA29Y and WT animals display similar infarct sizes. However, LV angiography demonstrated a significantly higher loss of ejection fraction in transgenic pigs after 3 days (-15.48±1.069 LEA29Y vs. -7.554±1.873 WT), which was not detectable for longer follow-up periods. High resolution QuPath analysis of stained tissue and subsequently of stained collagen areas in per-cent as well as in micrometers square detects scar formation over time. Initial calculations point to a slower increase in collagen in LEA29Y transgenic pigs compared to WT, indicating slower healing under immunosuppression (day 3: 13,33% of LEA29Y LV vs. 12,6% of WT LV; day 9: 18,36% of LV vs. 16,57% of LV; day 14: 18,4% of LV vs. 20,75% of LV; day 26: 16,49% of LV vs. 15,04% of LV). At 3 days post I/R, immunohistochemistry reveals a peak in the volume of neutrophils in both groups (0.29% of LEA29Y LV vs. 0.36% of WT LV,) whereas T cells numbers are markedly reduced in LEA29Y animals (0.15% of LEA29Y LV vs. 0.36% of WT LV). Macrophages show a delayed and not as prominent accumulation in transgenic pigs (day 3: 1.5% of LEA29Y LV vs. 4.12% of WT LV; day 9: 3.22% of LEA29Y LV vs. 4.23% of WT LV).

The lower recruitment to the ischemic area with concomitant higher proportion of circulating cells suggests LEA29Y mediated T cell anergy. This includes a reduced ability to proliferate, differentiate and migrate into injured tissue, which also affects macrophages and collagen formation and leads to an initial higher loss of function after I/R, but seems to be compensated through alternative mechanisms of the immune system.