AP39, A Mitochondria-Target Hydrogen Sulfide Donor, Protects Cardiac Allograft From Prolonged Ischemia Reperfusion Injury In Heart Transplantation

Background and objective:Heart transplantation?HTx?has been widely accepted as the standard treatment for end-stage heart diseases.Currently,the preservation method of HTx donor is cold static preservation solution.Due to the tissue specificity and its susceptibility to myocardial ischemia reperfusion injury?MIRI?,therefore,currently the heart donor can only be safely maintained for 4-6 hours in cold preservation solution.Prolonged ischemic preservation time would lead to higher mortality after HTx,primary graft dysfunction,and negatively correlated with long-term survival.Meanwhile,the low preservation time would affect the allocation and utilization of donors.With the increasingly serious organ shortage,it is necessary to constantly optimize the heart donor preservation strategy,which can not only extend the safe storage time of organs and maintain the vitality of organs,but also reduce the reperfusion injury faced by the donor after being transplantated into the receipt.Hydrogen sulfide?H₂S?is an important endogenous gas transmitter,which can play a variety of biological effects in many systems,including the cardiovascular system,and is a new target to reduce MIRI.AP39,a newly synthesized mitochondrial targeted H₂S releasing donor,can specificly increase the concentration of H₂S in the mitochondrial region.AP39 has been reported to be regulatory in many aspects,including ischemia reperfusion injury of kidney transplantation.However,there is no study on its effect on HTx and preservation solution supplementation of heart graft.Therefore,the purpose of this study was to investigate the protective effects of mitochondria-targeted hydrogen sulfide donor AP39 on ischemia-reperfusion injury in HTx.Material and method:An in vitro model of prolonged cold MIRI was used to investigate the role of AP39 in HTx and to investigate the optimal working concentration of AP39 in cold MIRI.HL-1 cells?mouse cardiomyocyte cell line?were exposed to 24 hours of cold ischemia at 10°C,followed by 12 hours of reperfusion at 37°C to induce MIRI in vitro.Cells were treated with different concentrations of AP39?30nM-5uM?dissolved in University of Wisconsin?UW?solution before the induction of ischemia.After ischemia,cells were treated with the same concentration of AP39dissolved in Claycomb medium/UW mixture at the beginning of reperfusion phase.After reperfusion,cell death was dynamically detected using an Incucyte cell-imaging system and by the lactate dehydrogenase method.Cell viability was determined with the MTT assay,to confirm the optimal working concentration.Then cells were treated with the optimal working concentration,using the above stated method.Cell apoptosis was detected with FITC-Annexin V/PI staining followed by flow cytometry.qRT-PCR and Western Blot were used to investigate the role of AP39 on the expression of pro-apoptotic factor Bax,both at the mRNA and protein level.qRT-PCR was used to detect the expression of pro-inflammatory cytokine?IL-6,IL-1?and TNF??,assessing the role of AP39 in inflammation.Mitochondria membrane potential and reactive oxygen species were measured by flow cytometry,to explore the changes of mitochondria function.A primary cardiomyocyte model was also used to investigate the function of AP39 and circular RNA expression was also detected in this model.For in vivo study,a syngeneic heterotopic murine model of HTx was used to mimic the clinical scenario of HTx.Cardiac graft from donor was preserved with the UW solution supplemented with100nM AP39 or alone with UW solution for 24hour at 4°C,followed by being transplanted into the receipt.Cardiac ultrasound was used to measure cardiac function,including the contraction function and flow pattern at postoperative day 7.Tissue injury and tissue fibrosis were used to detect pathological changes at postoperative day 7.Results:AP39 had a regulatory role on cold MIRI in HTx in a dose-depedent manner:5uM and 500nM AP39 increased cell death and decreased cell viability in cold MIRI in HTx;30-300nM AP39 decreased cell death and increased cell viability in cold MIRI in HTx.While 100nM AP39 had the most significant protective effects and thus it was chosen as the optimal working concentration to proceed to further experiments.In further experiments,AP39 significantly decreased percentage of Annexin V/PI double staining cell and significantly decreased expression of pro-apoptotic factor Bax,both at mRNA and protein level,which resulted in reduced cell apoptosis.AP39 reduced expression level of pro-inflammatory cytokines,IL-6?IL-1?and TNF?,leading to reduced inflammatory reaction.AP39 preserved mitochondria function by reducing intracellular reactive oxygen species production and increasing mitochondria membrane potential in cold MIRI in HTx.In the primary cardiomyocyte model,100nM AP39 also reduced cell death.For in vivo study,after 24hours of cold static preservation,transplanted grafts in UW solution barely remained normal function and strong blood flow pattern.For grafts preserved with UW solution+100nM AP39 solution,the transplanted grafts significantly improved contraction function,with both improved ejection fraction and fractional shorterning of left ventricle.Grafts preserved with AP39 also showed significantly higher input blood flow velocity and gradient pressure.Besides,cardiac grafts preserved with AP39 solution retained significantly lower tissue injury and lower areas of cardiac fibrosis.Circular RNA was detected for the mechanism resulting in the protective effects of AP39 in cold MIRI.Circular RNA Aebp2 was significantly improved in group undergoing cold IRI and significantly decreased in group treated with AP39,indicating the potential involvement of circAebp2 in cold MIRI.Discussion and summary:The new mitochondria-target hydrogen sulfide donor,AP39,acted in a dose-depedent manner in cold MIRI in HTx,with 100nM AP39 as the optimal working concentration and most significant protective effects.AP39 provided protective effects in cold MIRI in HTx,by reducing cell deathandapoptosis,decreasinginflammationandpreserving mitochondria function.Grafts treated with AP39 demonstrated improved cardiac function and reduced tissue injury.The protective effects of AP39might be associated with circular RNA Aebp2.The study proves the effectiveness of AP39 in HTx,highlighting its therapeutic potential in preventing MIRI in HTx.