Cold Preservation Of Donor Hearts And Establishment Of Evaluation System In A Porcine Heart Transplantation Model

For patients with end-stage heart failure,cardiac transplantation is the only effective treatment.However,several hurdles need to be overcome for the successful transplantation such as ischemia-reperfusion injury(IRI)and the limited cold preservation time.IRI causes tissue damage due to the loss of blood supply and resultant ischemia as well as the inflammation and reoxygenation during the reperfusion.IRI is associated with the delayed donor graft function and organ failure as well as chronic organ dysfunction and mortality.In clinic,utilizing cold preservation solution to perfuse and ex vivo store donor hearts is popular due to its simplicity and low cost.However,the metabolism cannot be completely shut off,resulting in an accumulation of harmful cellular metabolites and consequently inflammation and organ injury during reperfusion.The current safe myocardial preservation time is 4-6 hours,the limited storage time window intensifies the conflict between shortage of donor hearts and the increasing need for cardiac transplantation.Therefore,developing new solutions that can attenuate IRI and extend organ preservation time is critically needed.IRI induces activation of multiple signaling pathways and regulates expression of various genes,subsequently causing cell apoptosis and necrosis that promote organ damage.Apoptosis,complement activation,and inflammation pathways are the best-defined and studied causative factors associated with the IRI.Recent studies suggest that add the synthetic small interfering RNA(siRNA)into solutions has a potential to effectively inhibit IRI-caused damage in organ preservation,however,most studies are conducted in murine models,and small animals are often unable to model human disease.Swine provides an excellent model for human condition given their similarities in anatomy,physiology and immunology.The large heart/body weight and high degree of similarities in cardiovascular system also make the swine a valuable and highly relevant model for human cardiovascular research.Therefore,in this study,we developed a new siRNA contained solution which could reduce IRI and prolong cold preservation time in a porcine model.siRNA delivery is a current hurdle for the clinical application.In previous studies,a large quantity of naked siRNA is added to the preservation solution,which appears to work in the small animals such as rats.However,high dose of siRNA may not be feasible for clinic use or porcine cardiac grafts.Transfection reagents(TR)have been extensively used either in vitro or in vivo for siRNA delivery,the reagents also allow to minimize the siRNA dosage,which is more suitable for clinical application.siRNAs targeting C3(complement activation),p65(inflammation),Caspase 8 and Caspase 3(apoptosis)were designed and synthesized,the most effective siRNAs for each individual gene were selected by testing in PK15 and ST cells and used in subsequent experiments.siRNA contained Celsior solution with or without TR were used to perfuse and cold preserve donor hearts from minipigs,and we found that solution with low dosage of siRNA and TR(4 OD siRNA-TR)showed more effective in genes knock down and apoptosis reduction than the solution with high dosage of siRNA and non-TR(16 OD siRNA).The cold preservation and subsequent reperfusion often cause cardiac cell apoptosis.Although TR-contained siRNA solution appeared to reduce apoptosis in the cold-preserved hearts,it is unclear if the solution has the same effect on heart tissue after reperfusion.To test this,we designed and established the evaluation system of large animal donor hearts.Taking the leading in assembling large animal hearts ex vivo perfusion system,which can hemoperfused isolated hearts in body temperature.This perfusion system and cardiac transplantation model can be used to re-perfuse donor hearts ex vivo and in vivo respectively,and during and after the re-perfusion,series of parameters as left ventrical hemodynamics,myocardial survival,structure alteration,myocardial damage and necrocytosis,oxidative stress,innate immunity triggering and tissue inflammation will be measured for quality evalution of donor hearts.Indeed,this evaluation system has laid a solid foundation and technology platform for the clinical research of cardiac function and cardiovascular diseases.To test the effect of the siRNA-TR solution in re-perfused hearts,we incubated the porcine hearts in 500 ml Celsior solution(regular preservation group,Celsior,CEL),or TR-containing Celsior solution with 4 OD scramble siRNA(Control group,Ctrl)or 2,4,6 OD siRNAs(siRNA groups).After 12 h of the cold preservation,the heart grafts were connected to the perfusion system and allowed to work 3 h.Comparing to the hearts from CEL and Ctrl,We found the reduction of cell apoptosis,structure alteration,myocardial damage and tissue inflammation by reduced biochemistry and markers and protein expression of proinflammatory cytokines,no triggering innate immunity by lower protein expression of TLR-7 and MyD88,and improvement of cardiac function as shown by the improved hemodynamic indices in siRNA treated hearts.In the 3 siRNA groups,the solution with 4 OD siRNA produced the best outcome compared to the other treatments,consistanted with the result of the cold storage hearts.Based on the results of the isolated working hearts,we hypothesized that 4 OD siRNA with TR would also provide better protection to the porcine hearts undergoing cardiac orthotopic transplantation than the conventional method.To prove this hypothesis,6 grafts of adult male minipigs were divided into 2 groups,the hearts without siRNA treatment were preserved for the standard 6 h(CEL),and the hearts with siRNA treatment were prolonged to 12 h preservation(siRNA).After old preservation,all donor hearts were transplanted to recipients and allowed to beat for 3 h and the hemodynamic indices were recorded.After the 3 h work,all hearts were sampled and accepted the same detections as the isolated hearts.Comparing to the 6 h preserved CEL hearts,we found the siRNA solution(4 OD-TR)protects the 12 h preserved transplanted hearts from IRI-induced apoptosis,structure alteration,tissue damage,inflammation and innate immunity,and enhanced the cardiac function.These findings demonstrate blockade of inflammation and apoptosis pathways using siRNA in the preservation solution can prolong cold preservation time and better protect donor heart function in cardiac transplantation of large animals,which may be beneficial for human heart preservation.