Aquaporin 4 (AQP4) belongs to the family of small integrin transmembrane proteins that are highly permeable to water. It was recently shown that human and mouse cardiomyocytes also express AQP4 and that AQP4-deficient mice are protected from acute myocardial ischemia/reperfusion injury. The goal of this study was to test whether AQP4 inhibition reduces ischemia reperfusion injury following transplantation and improves the survival of cardiac allografts.C57BL/6 (H-2^b) mice were transplanted with BALB/c (H-2^d) heart allografts subjected to 8 h of cold ischemia (in contrast to 0.5 h storage typical for most mouse heart transplantation models). To inhibit AQP4, donors were injected with AQP4 inhibitor, AER 270, 0.5 h prior to the heart harvest; excised hearts were perfused with Ringers solution supplemented with AER270 and stored for 8 h on ice in Ringers solution plus AER270. After transplantation, recipients were injected with AER271 (converted to AER 270 in vivo) every 6 h for 5 days. Control donor hearts were perfused and stored in Ringers solution without the AQP4 inhibitor, and then transplanted into recipients treated with PBS. In the absence of additional immunosuppression, treatment with AQP4 inhibitor significantly prolonged heart allograft survival compared to control recipients (MST of 25 d, n=24 vs. 6 d, n=5). We have previously reported that prolonged cold ischemia storage (CIS) results in increased allograft infiltration by endogenous memory T cells within 24 h post-transplant. AQP4 inhibition decreased early T cell infiltration and the intragraft expression of molecules either expressed or induced by T cells such as IFNγ, granzyme B, perforin, FASL, CXCR3, ICOS, and CXCL10 compared to untreated controls. Recall ELISPOT assays at the time of rejection showed that the frequencies of donor-reactive spleen cells secreting IFNγ in AQP4 inhibitor treated recipients were four fold lower than those in untreated controls. Blocking AQP4 during allograft storage and early after transplantation prolongs the survival of fully MHC-mismatched heart allografts subjected to 8 h CIS. The graft prolongation is associated with the reduced early memory T cell infiltration into the graft and with decreased de novo donor-specific T cell activation. Our data identify AQP4 as a promising therapeutic target to diminish the detrimental effects of prolonged CIS on transplant outcome.

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