Melatonin Protects Donation After Cardiac Death Hearts Preserved With Normothermic Machine Perfusion Via Inhibiting Pyroptosis

Objective:The adoption of hearts from donation after cardiac death(DCD)is a promising approach for the shortage of suitable organs in heart transplantation.However,DCD hearts suffer from serious ischemia reperfusion injury(IRI).Recent studies have shown that NLRP3 inflammasome-mediated pyroptosis is a new target to ameliorate myocardial IRI,and melatonin has been shown to inhibit pyroptosis.This study is designed to verify the hypothesis that melatonin combined with Normothermic machine perfusion(NMP),as an effective DCD preservation strategy,may alleviate myocardial IRI in DCD hearts and protect donor hearts by inhibiting NLRP3 inflammasome mediated pyroptosis.Methods:Donor heart rats were randomly divided into three groups:(1)blank control group(n=8):non-DCD heart was taken from beating heart rats,and allogeneic blood based perfusion solution was continuously injected for 105 min during NMP system;(2)DCD-vehicle group(n=8);And(3)DCD-melatonin group(n=8):The rats were asphyxiated by clamping the trachea.DCD was defined as systolic blood pressure lower than 30 mmHg or cardiac arrest.After 25 minutes,cardiac arrest fluid was injected into the DCD heart through the right common carotid artery,and the DCD heart was preserved for 105 minutes during the NMP system.Melatonin(DCD-melatonin group,with a final concentration of 200 μmol/L in cardiac arrest fluid or NMP Perfusion fluid)or absolute ethyl alcohol(DCD-control group)were added during cardioplegia infusion and throughout NMP.During NMP,cardiac function was evaluated every 30 min(T0,T30,T60,and T90).Cardiac function parameters included left ventricular Developed pressure(DP),Heart rate(Heart rate,HR),the maximum rate of increase of left ventricular pressure(dP/dtmax)and the maximum rate of decrease of left ventricular pressure(dP/dtmin).After NMP,oxidative stress(SOD,MDA,HNE),inflammatory response(IL-6,TNF-α,NF-κB P65),apoptosis(TUNEL staining,Cleaved Caspase-3,and NLRP3 inflammasome-mediated pyroptosis(NLRP3,ASC,Cleaved caspase-1,IL-1β,IL-18,Cleaved GSDMD)of donor hearts were evaluated.Results:After twenty five-minute warm ischemia injury,DP,dP/dtmax,and dP/dtmin of the DCD heart preserved with NMP significantly decreased compared with blank control group,while DP and dP/dtmax of DCD heart in DCD-melatonin group significantly increased compared with DCD-vehicle group.In addition,compared with blank control group,oxidative stress(SOD,MDA,HNE),inflammatory response(IL-6,TNF-α,NF-κB P65),apoptosis(TUNEL staining,Cleaved Caspase-3),NLRP3 inflammasome-mediated pyroptosis(NLRP3,ASC,Cleaved caspase-1,IL-1 β,IL-18,and Cleaved GSDMD)induced by warm ischemia injury in donor heart have significantly increased.However,melatonin supplementation throughout the perfusion of cardioplegia and NMP phase significantly reduced the levels of oxidative stress,inflammatory response,apoptosis,and NLRP3 inflammasome-mediated pyroptosis in NMP preserved donor hearts compared with the DCD-vehicle group.Conclusion:NMP combined with melatonin is a promising strategy for DCD heart preservation,which can alleviate IRI of DCD heart,effectively repair DCD heart and expand the source of donor heart by inhibiting NLRP3 inflammasome mediated pyroptosis.