It is well known that heart graft deteriorates rapidly during the hypothermic preservation phase before transplantation. Many preservative solutions are used in clinical practice, but there is no consensus on any of them. A new preservation solution named LYon Preservative Solution (LYPS) was developed in our laboratory to improve heart survival during preservation to allow a better functional recovery after transplantation.

Our objective is to understand cell death mechanisms that occur during cold ischemia at different temperatures and to test the LYPS compared to the other solutions currently used in clinic (UW, Celsior, and Plégisol).

We performed 20hours of cold to mild-hypothermic (5, 10, 15 and 20°C) static preservation, followed by 2hours of warm “reoxygenation” (37°C) on H9C2 cells. PI, Annexin V and DILC1 flow cytometry staining were used to determined cell necrosis, cell apoptosis and mitochondrial membrane potential respectively at the end of the preservation, and at the end of the reoxygenation. Apoptosis was also quantified by caspase 3 cleavage by Western-Blot. Evaluation of autophagy was made using immunofluorescent labelling of LC3B protein.

It appears that LYPS solution protects from necrotic cell death compared to Celsior, UW and Plegisol (8.8% compared to 72.8%; 66.2%; 95% respectively at 5°C). In addition, LYPS allows a better maintenance of the mitochondrial membrane potential (76% compared to 4.6%; 26%; 3.3%, respectively). This trend is observed at all temperatures studied. Furthermore, we observed that autophagic marker LC3B was stimulated in LYPS compared to other studied solutions.

All of these data show that the LYPS solution allows better activation of survival signals, preservation of mitochondrial membrane potential and protection against necrosis. The next step is now to study other cell death pathways such as necroptosis, pyroptosis, and ferroptosis to better understand the mechanisms involved in graft deterioration during preservation in order to find the best preservation conditions and expand the number of potential grafts.