Nitric oxide pathway after long-term cold storage and reperfusion in a heterotopic rat heart transplantation model

Recent studies have suggested the involvement of the nitric oxide (NO) pathway in ischemia-reperfusion injury related to cardiac transplantation. Herein, we assessed the NO pathway by quantifying endothelial (e) and inducible (i) nitric oxide synthase (NOS) expression and total NOS activity in a rat heart transplant model during cold ischemia with Celsior cardioplegia and reperfusion. Experiments were performed using a modified Lewis-Lewis heterotopic abdominal heart transplantation with 3 or 6 hours of ischemia with or without 1 hour of blood reperfusion. NOS expression and activity were determined using Western blotting and colorimetric assays, respectively, on freeze-clamped hearts after ischemia without (n = 10) or with reflow (n = 12) compared with basal values. Hearts submitted to 3 hours of ischemia and 1 hour of reperfusion showed a postischemic rate pressure product of 5190 +/- 3047 mm Hg/min (reversible ischemia), but no contractility was observed after 6 hours of ischemia. eNOS protein levels were lower after 3 hours of ischemia compared with the basal value (P = .0005) and were further decreased after 6 hours of ischemia (P < .0001 versus basal value and P = .0018 versus 3 hours of ischemia). Reperfusion did not further decrease eNOS protein levels. iNOS protein was not detected in any condition. NOS activity was increased after 3 hours of ischemia versus basal value (P = .0065) but not after 6 hours of ischemia without any effect of reperfusion. We concluded that eNOS expression was altered during ischemia and the amplitude of the alteration depended on the duration of ischemia. Reversible ischemia was associated with increased NOS activity at the end of ischemia with no variation at reperfusion.