*Purpose: Cardiac function declines in a time-related fashion during ex situ heart perfusion (ESHP) regardless of ventricular load status. We have previously shown that the metabolism of glucose is altered during ESHP, suggesting that the function of the ex situ perfused heart may have a metabolic basis. It is known that extracorporeal perfusion may induce oxidative stress. Our purpose was to evaluate the potential interplay between energy metabolism and oxidative stress in the hearts perfused ex situ for 12 hours.

*Methods: 14 female domestic pigs (47-57 kg) were included. Hearts were perfused on a custom ESHP apparatus in two perfusion modes (non-working mode, NWM n=5 and working mode, WM n=5) with insulin and glucose infusions for metabolic support. Cardiac function was assessed during perfusion. The activation of protein kinase B (AKT), AMP-activated protein kinase (AMPK), activity of glucose-6-phosphate dehydrogenase (G6PD, pentose phosphate pathway), and creatine kinase (CK) enzymes were compared in the left ventricular tissue of the ex situ perfused hearts, and in vivo controls (n=4) using immunoassay methods.

*Results: Cardiac function significantly declined during perfusion in both WM and NWM but the functional parameters were significantly better preserved in the WM (at 12h as a percentage of baseline: cardiac index, WM: 74%, NWM: 39%, p<0.01 and LV stroke work, WM: 67%, NWM: 33%, p=0.01). Glucose utilization did not change over time in either of the perfusion groups but the enzymatic activity of G6PD was significantly higher in ex situ perfused hearts compared to in vivo controls (p=0.03). AKT activation via Thr308 phosphorylation was higher in ex situ perfused hearts compared to in vivo (p<0.001). Ser473 AKT phosphorylation was not different between in vivo and NWM but was higher in WM compared to in vivo and NWM (p<0.01 and p=0.02). AMPK phosphorylation (activation) was significantly higher in WM compare to in vivo and NWM (p<0.01 and p=0.02) but was not different in in vivo and NWM. Creatine kinase activity was significantly lower in NWM compared to both in vivo and WM (p<0.01 and p=0.01) and it was not different in WM and in vivo.

*Conclusions: ESHP in WM is associated with better functional preservation. The simultaneous activation of AMPK and AKT, together with increased activity of G6PD, suggest that the ex situ perfused hearts are exposed to oxidative stress, which affects energy metabolism and thus myocardial function. Higher activity of creatine kinase and AMPK in WM may be associated with the superior functional preservation

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