Simvastatin pretreatment reduces caspase-9 and RIPK1 protein activity in rat cardiac allograft ischemia-reperfusion.

BACKGROUND

In transplantation-associated ischemia/reperfusion injury (Tx-IRI), tumor necrosis factor alpha and damage-associated molecular patterns promote caspase-8 and -9 apoptotic and receptor-interacting protein kinase-1 and -3 (RIPK1/3) necroptotic pathway activation. The extent of cell death and the counterbalance between apoptosis and regulated necrosis eventually determine the immune response of the allograft. Although simvastatin prevents Tx-IRI, its role in apoptotic and necroptotic activity remains unsolved.

METHODS

Rat allograft donors and recipients were treated with a single-dose of simvastatin 2h prior to allograft procurement and reperfusion, respectively. Intragraft caspase-3, -8, and -9 and RIPK1 and -3 mRNA expression was analysed by quantitative RT-PCR and protein activity measured by immunohistochemistry and luminescent assays 6h after reperfusion. Lactate and lactate dehydrogenase (LDH) levels were analysed from allograft recipient and from hypoxic endothelial cell cultures having treated with activated simvastatin.

RESULTS

When compared to without cold ischemia, prolonged 4-hour cold ischemia significantly enhanced intragraft mRNA expression of caspase-3 and -9, and RIPK1 and -3, and elevated protein activity of caspase-9 and RIPK1 in the allografts. Simvastatin pretreatment decreased mRNA expression of caspase-3 and -9, and RIPK1 and -3 and protein activity of caspase-9 and RIPK1 in the allografts. Intragraft caspase-8 mRNA expression remained constant regardless of cold ischemia or simvastatin pretreatment. Simvastatin pretreatment attenuated lactate and LDH levels, both in the allograft recipients and in hypoxic endothelial cell cultures.

CONCLUSIONS

The beneficial effects of simvastatin pretreatment in cardiac allograft IRI may involve prevention of apoptosis and necroptosis.