Heart Preservation Research Articles

Cold non-ischemic heart preservation with continuous perfusion prevents early graft failure in orthotopic pig-to-baboon xenotransplantation.

Successful preclinical transplantations of porcine hearts into baboon recipients are required before commencing clinical trials. Despite years of research, over half of the orthotopic cardiac xenografts were lost during the first 48hours after transplantation, primarily caused by perioperative cardiac xenograft dysfunction (PCXD). To decrease the rate of PCXD, we adopted a preservation technique of cold non-ischemic perfusion for our ongoing pig-to-baboon cardiac xenotransplantation project. Fourteen orthotopic cardiac xenotransplantation experiments were carried out with genetically modified juvenile pigs (GGTA1- KO/hCD46/hTBM) as donors and captive-bred baboons as recipients. Organ preservation was compared according to the two techniques applied: cold static ischemic cardioplegia (IC; n=5) and cold non-ischemic continuous perfusion (CP; n=9) with an oxygenated albumin-containing hyperoncotic cardioplegic solution containing nutrients, erythrocytes and hormones. Prior to surgery, we measured serum levels of preformed anti-non-Gal-antibodies. During surgery, hemodynamic parameters were monitored with transpulmonary thermodilution. Central venous blood gas analyses were taken at regular intervals to estimate oxygen extraction, as well as lactate production. After surgery, we measured troponine T and serum parameters of the recipient's kidney, liver and coagulation functions. In porcine grafts preserved with IC, we found significantly depressed systolic cardiac function after transplantation which did not recover despite increasing inotropic support. Postoperative oxygen extraction and lactate production were significantly increased. Troponin T, creatinine, aspartate aminotransferase levels were pathologically high, whereas prothrombin ratios were abnormally low. In three of five IC experiments, PCXD developed within 24 hours. By contrast, all nine hearts preserved with CP retained fully preserved systolic function, none showed any signs of PCXD. Oxygen extraction was within normal ranges; serum lactate as well as parameters of organ functions were only mildly elevated. Preformed anti-non-Gal-antibodies were similar in recipients receiving grafts from either IC or CP preservation. While standard ischemic cardioplegia solutions have been used with great success in human allotransplantation over many years, our data indicate that they are insufficient for preservation of porcine hearts transplanted into baboons: Ischemic storage caused severe impairment of cardiac function and decreased tissue oxygen supply, leading to multi-organ failure in more than half of the xenotransplantation experiments. In contrast, cold non-ischemic heart preservation with continuous perfusion reliably prevented early graft failure. Consistent survival in the perioperative phase is a prerequisite for preclinical long-term results after cardiac xenotransplantation.

Abstract 403: Leveraging the Zebrafish to Overcome Barriers in Heart Transplantation

Cardiac transplantation is the only curative therapy for patients with end-stage heart disease; however, there is a severe shortage of viable donor organs. Heart transplantation faces many interwoven challenges, including both biological factors and research limitations. For example, ischemia-reperfusion injury plays a role in early graft dysfunction and is associated with rejection episodes in heart transplantation. Moreover, experimental transplantation relies heavily on animal studies that are laborious and expensive, prohibiting the discovery of novel, bold solutions. We propose that the zebrafish, Danio rerio , would be a valuable tool for the field since it’s amenable to high-throughput screens, captures the complex structure of organs, and offers a suite of tools to monitor the biology of cardiac injury. Here, we develop a new subzero heart preservation method by strategically leveraging animal models from zebrafish to mammalian hearts. Using zebrafish larvae, we screened for agents which preserve hearts at -10°C. As a result of these screens, we identified promising preservative cocktails which restored heartbeat in 82% of larvae immediately post-recovery. Next, we excised adult zebrafish hearts and developed methods to mimic the ex vivo handling practices of hearts destined for transplant using a heart-on-a-plate assay. Using this assay, we carried forward promising agents identified in our initial zebrafish larvae screen to isolated adult zebrafish hearts that were cooled to -10°C and held for up to 24 hours. After rewarming, heart rate was restored and metabolic rate of zebrafish hearts was like time-matched controls (0.213 ± 0.047 and 0.275 ± 0.060, respectively, p = 0.200). Finally, we report our preliminary scale-up efforts whereby rodent hearts are stored for up to 24 hours at -10°C and viability were assessed by the TUNEL assay. The data shows high viability of cardiomyocytes post-preservation, as compared to controls. In summary, we present data to illustrate our efforts in leveraging the zebrafish to aid new discoveries in subzero heart preservation. Similar efforts to model heart transplantation in zebrafish may provide a different vantage point and enable us to make advances faster.

Non-ischemic Heart Preservation via Hypothermic Cardioplegic Perfusion Induces Immunodepletion of Donor Hearts Resulting in Diminished Graft Infiltration Following Transplantation.

Introduction: Many donor organs contain significant leukocyte reservoirs which upon transplantation activate recipient leukocytes to initiate acute rejection. We aimed to assess whether non-ischemic heart preservation via ex vivo perfusion promotes immunodepletion and alters the inflammatory status of the donor organ prior to transplantation.Methods: Isolated porcine hearts underwent ex vivo hypothermic, cardioplegic perfusion for 8 h. Leukocyte populations were quantified in left ventricle samples by flow cytometry. Cell-free DNA, cytokines, and chemokines were quantified in the perfusate. Tissue integrity was profiled by targeted proteomics and a histological assessment was performed. Heterotopic transplants comparing ex vivo hypothermic preservation and static cold storage were utilized to assess graft infiltration as a solid clinical endpoint.Results: Ex vivo perfusion significantly immunodepleted myocardial tissue. The perfusate displayed a selective, pro-inflammatory cytokine/chemokine pattern dominated by IFN-γ. The tissue molecular profile was improved following perfusion by diminished expression of nine pro-apoptotic and six ischemia-associated proteins. Histologically, no evidence of tissue damage was observed and cardiac troponin I was low throughout perfusion. Cell-free DNA was detected, the source of which may be necrotic/apoptotic leukocytes. Post-transplant graft infiltration was markedly reduced in terms of both leucocyte distribution and intensity of foci.Conclusions: These findings demonstrate that ex vivo perfusion significantly reduced donor heart immunogenicity via loss of resident leukocytes. Despite the pro-inflammatory cytokine pattern observed, a pro-survival and reduced ischemia-related profile was observed, indicating an improvement in graft viability by perfusion. Diminished graft infiltration was observed in perfused hearts compared with those preserved by static cold storage following 48 h of transplantation.

An Addition of U0126 Protecting Heart Grafts From Prolonged Cold Ischemia-Reperfusion Injury in Heart Transplantation: A New Preservation Strategy.

Ischemia-reperfusion injury (IRI) is the major cause of primary graft dysfunction in organ transplantation. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in cell physiological and pathological processes including IRI. This study aims to investigate whether inhibition of ERK signaling with U0126 can prevent prolonged cold IRI in heart transplantation. Rat cardiac cell line H9c2 cells were treated with U0126 before exposure to hypothermic hypoxia/reoxygenation (H/R) conditions. The effect of U0126 on H9c2 cells in response to H/R stress was determined by measuring cell death, reactive oxygen species production, mitochondrial membrane potential, and ERK signaling activation. Mouse syngeneic heterotopic heart transplantation was conducted, where a donor heart was preserved in the University of Wisconsin (UW) solution supplemented with U0126 for 24 hours at 4°C before transplantation. Heart graft function, histopathologic changes, apoptosis, and fibrosis were measured to assess IRI. Phosphorylated ERK was increased in both in vitro H/R-injured H9c2 cells and in vivo heart grafts with IRI. Pretreatment with U0126 inhibited ERK phosphorylation and prevented H9c2 cells from cell death, reactive oxygen species generation, and mitochondrial membrane potential loss in response to H/R. Preservation of donor hearts with U0126-supplemented solution improved graft function and reduced IRI by reductions in cell apoptosis/death, neutrophil infiltration, and fibrosis of the graft. Addition of U0126 to UW solution reduces ERK signal activation and attenuates prolonged cold IRI in a heart transplantation model. ERK inhibition with U0126 may be a useful strategy to minimize IRI in organ transplantation.

Temperature and flow rate limit the optimal ex-vivo perfusion of the heart - an experimental study

BackgroundEx-vivo heart perfusion can be utilized to study a variety of physiologic and molecular pathways in a controlled system outside of the body. It can also be used in clinical settings such as for organ preservation before transplantation. Myocardial oxygen consumption (MVO2) correlates with energy production in the myocardium and can also be used to determine the balance between the oxygen supply and demand of the perfused heart. This study sought to determine an ex-vivo perfusion rate that matches the metabolic demands of the heart according to different temperatures and solution compositions (with and without the addition of erythrocytes), a flow below which the supply of oxygen is not sufficient to maintain an aerobic state of the perfused heart (“DCRIT”).MethodsUnder general anesthesia, rat hearts were procured and preserved by perfusing with the University of Wisconsin Belzer machine perfusion system (UW Belzer MPS) solution saturated with 100% O2. The key elements of this solution include supraphysiological potassium (to stop the heartbeat and reduce the cellular metabolic demand), starch, gluconate and mannitol (to maintain cell wall integrity), glucose (to sustain basal metabolism), and glutathione (to scavenge free radicals). Three groups of rat hearts (n = 7) were randomly allocated to be perfused at 15 °C, 22 °C or 37 °C, at a varying flow index (FI) starting from a minimum of 380 mL/min/100 g to less than 50 mL/min/100 g, decreasing by 50 mL/min/100 g at 10 min intervals while measuring the MVO2 at each FI. Lactate was measured from coronary sinus samples to determine the onset of tissue hypoxia/anaerobic state.ResultsThe DCRIT at 15 °C was 99.9 ± 4.9 mL/min/100 g; however, at 22 °C and 37 °C we could not reach a DCRIT. The myocardial oxygen demand could not be met at 22 °C and 37 °C with the maximum FI above 380 mL/min/100 g even when erythrocytes (10% V/V) were added to the solution. At 15 °C, the production of lactate was evident only below the DCRIT, while at 22 °C lactate production was present at all flow indices.ConclusionsDetermining the DCRIT for optimal ex-vivo perfusion of the heart is necessary to ensure adequate tissue oxygenation and limit anaerobic state. Temperatures employed above 15 °C limit the efficient ex-vivo perfusion preservation of heart with the UW Belzer MPS solution.

Machine perfusion of donor heart with normothermic blood versus hypothermic HTK in preserving coronary endothelium in a porcine model of DCD.

Both machine perfusion (MP) of donor hearts with autologous blood and crystalloid perfusates have advantages and disadvantages. Currently, which of the aforementioned preservation strategies can better preserve the coronary endothelium has not yet been determined. We aim to compare the impact of hypothermic continuous MP with histidine-tryptophan-ketoglutarate (HTK) solution versus normothermic continuous MP with autologous blood on coronary endothelium in a porcine ex vivo model of donation following circulatory death (DCD). DCD pigs underwent circulatory arrest via asphyxiation followed by 30-minute warm ischemia time. Donor hearts were preserved with either hypothermic MP with HTK solution (MP + HTK group; 4 ℃; n=6), or normothermic MP with blood (MP + blood group; 37 ℃; n=6) for 4 hours. After 2-hour ex vivo reperfusion, the assessment of endothelial-dependent (Edep) and -independent (Eind) relaxation of coronary artery, histopathological analysis, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay were performed. Preservation of DCD hearts with MP + Blood strategy significantly improved both Edep and Eind vasorelaxation of coronary artery compared with MP + HTK strategy (maximum relaxation to bradykinin: MP + HTK 80.9%±2.6% vs. MP + Blood 91.9%±1.9%, P<0.001; maximum relaxation to sodium nitroprusside: MP + HTK 97.1%±1.0% vs. MP + Blood 99.8%±0.2%, P<0.05). MP + Blood strategy significantly decreased nitrotyrosine but increased intercellular adhesion molecule-1 immunoreactivity in the coronary artery. The number of TUNEL-positive cells in MP + Blood group were significantly fewer compared with MP + HTK group. Compared with MP + HTK strategy, MP + Blood strategy significantly alleviates coronary endothelial dysfunction during donor heart preservation. This protective effect is associated with the inhibition of apoptosis and nitro-oxidative stress in coronary artery.

Working on inflammatory injury in heart transplant

Ex vivo perfusion for preservation of donor hearts may be improved by preventing inflammatory tissue injury, including through mechanical work.

A nonrandomized open-label phase 2 trial of nonischemic heart preservation for human heart transplantation

Pre-clinical heart transplantation studies have shown that ex vivo non-ischemic heart preservation (NIHP) can be safely used for 24 h. Here we perform a prospective, open-label, non-randomized phase II study comparing NIHP to static cold preservation (SCS), the current standard for adult heart transplantation. All adult recipients on waiting lists for heart transplantation were included in the study, unless they met any exclusion criteria. The same standard acceptance criteria for donor hearts were used in both study arms. NIHP was scheduled in advance based on availability of device and trained team members. The primary endpoint was a composite of survival free of severe primary graft dysfunction, free of ECMO use within 7 days, and free of acute cellular rejection ≥2R within 180 days. Secondary endpoints were I/R-tissue injury, immediate graft function, and adverse events. Of the 31 eligible patients, six were assigned to NIHP and 25 to SCS. The median preservation time was 223 min (IQR, 202–263) for NIHP and 194 min (IQR, 164–223) for SCS. Over the first six months, all of the patients assigned to NIHP achieved event-free survival, compared with 18 of those assigned to SCS (Kaplan-Meier estimate of event free survival 72.0% [95% CI 50.0–86.0%]). CK-MB assessed 6 ± 2 h after ending perfusion was 76 (IQR, 50–101) ng/mL for NIHP compared with 138 (IQR, 72–198) ng/mL for SCS. Four deaths within six months after transplantation and three cardiac-related adverse events were reported in the SCS group compared with no deaths or cardiac-related adverse events in the NIHP group. This first-in-human study shows the feasibility and safety of NIHP for clinical use in heart transplantation. ClinicalTrial.gov, number NCT03150147

Current status of normothermic ex-vivo perfusion of cardiac allografts.

Ex-vivo perfusion has emerged in recent years as an alternative to cold static preservation of organs harvested for transplant. Normothermic ex-vivo perfusion, the subject of this review, maintains the donor heart in a near physiologic state, and allows the transplant team to monitor and control perfusion to the organ prior to implantation. A growing body of evidence has established the safety and viability of this technique, which may improve on current standards of donor management. Following initial single-arm studies over a decade ago, ex-vivo perfusion has been studied in a prospective, randomized fashion in standard donor hearts (PROCEED II trial). The short and intermediate-term results demonstrated similar outcomes compared with cold storage with significantly shorter cold ischemic time. Since then, ex-vivo perfusion has been studied in extended-criteria donor hearts, first in observational studies, and currently in randomized, prospective fashion in the recently completed EXPAND-Heart trial, which is anticipated to be reported in 2020. Normothermic ex-vivo perfusion has an established literature base and holds promise for changing current practices of heart preservation. Results of forthcoming pivotal studies will help determine its role in more widespread clinical adoption.

Cold-inducible RNA binding protein agonist enhances the cardioprotective effect of UW solution during extended heart preservation.

In heart transplantation, time restriction is an unavoidable thorny problem during cardiac transport. Cold storage is an important organ preservation method in donor heart transport. Cold-inducible RNA binding protein (CIRBP) has been proven to play a protective role under cold stress. In this study, we investigated the role of CIRBP in hypothermic cardioprotection during heart preservation in UW solution and explored a new approach to extend the heart preservation time. Cirbp-knockout (Cirbp-/- ), Cirbp-transgenic (Cirbp-Tg), and wild-type rats were, respectively, randomized into two groups based on various heart preservation times (6 or 12-hour group) (n=8 per group). After preservation in UW solution, all hearts were mounted on aLangendorff apparatus andunderwent measurement of cardiac parameters, histological analysis, and molecular study. Within the 6-hour preservation group, no significant difference was found in cardiac functions and histological changes between different rat species. However, after 12 hours of preservation, Cirbp-/- rat hearts showed more apoptosis and worse cardiac function, but less apoptosis and better cardiac function were observed in Cirbp-Tg rat hearts. Furthermore, we found CIRBP-mediated cardiac ubiquinone (CoQ10 ) biosynthesis plays an important role in extending heart preservation, and ubiquinone biosynthesis protein COQ9 was an essential down-stream regulator during this process. Finally, we found that zr17-2, a CIRBP agonist, could enhance the expression of CIRBP, which further enhances the synthesis of CoQ10 and promotes scavenging of reactive oxygen species and ATP production to extend heart preservation. This study demonstrated that CIRBP-enhanced CoQ10 biosynthesis during hypothermic heart preservation and zr17-2-supplemented UW solution could be a promising approach to ameliorate heart damage and extend heart preservation during cardiac transport.

Hypothermic Ex Vivo Perfusion: Protecting the Donor Heart and the Recipient

We congratulate McLeod et al1 on their study demonstrating successful preservation of hearts for 72 hours utilizing normothermic ex vivo perfusion (NEVP) and plasma cross-circulation, using a live para-corporeal donor sheep. This is a great achievement, because heart preservation was well beyond current clinical storage time limits. However, although the authors note future plans to simplify, the preservation method could be considered methodologically challenging. Additionally, the clinical relevance is somewhat difficult to determine, because of the absence of donor brain death (BD) and related sequelae, well reported to impair organ function.

Brain-dead donor heart conservation with a preservation solution supplemented by a conditioned medium from mesenchymal stem cells improves graft contractility after transplantation.

Hearts are usually procured from brain-dead (BD) donors. However, brain death may induce hemodynamic instability, which may contribute to posttransplant graft dysfunction. We hypothesized that BD-donor heart preservation with a conditioned medium (CM) from mesenchymal stem cells (MSCs) would improve graft function after transplantation. Additionally, we explored the PI3K pathway's potential role. Rat MSCs-derived CM was used for conservation purposes. Donor rats were either exposed to sham operation or brain death by inflation of a subdural balloon-catheter for 5.5hours. Then, the hearts were explanted, stored in cardioplegic solution-supplemented with either a medium vehicle (BD and sham), CM (BD+CM), or LY294002, an inhibitor of PI3K (BD+CM+LY), and finally transplanted. Systolic performance and relaxation parameters were significantly reduced in BD-donors compared to sham. After transplantation, systolic and diastolic functions were significantly decreased, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells and endonuclease G positive cells were increased in the BD-group compared to sham. Preservation of BD-donor hearts with CM resulted in a recovery of systolic graft function (dP/dtmax : BD+CM: 3148±178 vs BD: 2192±94mmHg/s at 110µL, P<.05) and reduced apoptosis. LY294002 partially lowered graft protection afforded by CM in the BD group. Our data suggest that PI3K/Akt pathway is not the primary mechanism of action of CM in improving posttransplant cardiac contractility and preventing caspase-independent apoptosis.

Heart Preservation with the Organ Care System in Extended Criteria Donor Hearts: A Three-Center Experience

The Organ Care System (OCS) Heart® is non-inferior to standard cold storage for preservation of donor hearts. We hypothesized that its properties for prolonged heart preservation might be especially beneficial for extended criteria donor (ECD) hearts. Patients transplanted with ECD hearts preserved using the OCS Heart® at two German and one Italian centers between 11/2011 and 10/2019 were prospectively followed and included into the study. ECD were defined as fulfilling at least one of the following criteria: age between 45 and 55 years without coronary angiography; age >55 years; echocardiographic evidence of left ventricular hypertrophy (LVH); need for cardiopulmonary resuscitation (CPR) longer than 20 minutes; left ventricular ejection fraction (LVEF)<40%; evidence of coronary artery disease (CAD); carbon monoxide intoxication with preserved LVEF; moderate-severe alcohol consumption; diabetes mellitus without CAD. During the study period, the OCS Heart® was employed in 122 patients transplanted at the three institutions. Among these 122 patients, 82 (67%) patients were transplanted with an ECD heart, as defined by the aforementioned criteria (median donor age, 48 years; LVH, n=17; CPR, n=28; mild CAD, n=5; LVEF<40%, n=2; alcohol consumption, n=36; diabetes mellitus, n=1), and thus considered in the study. Five further ECD hearts were considered non-transplantable while on OCS and were discarded. Before transplantation, 64 (78%) recipients had undergone at least one cardiac operation (LVAD/BVAD, n=47, 57%), 12 (15%) were on ECLS, and 65 (79%) were on the high-urgency waiting list. Median OCS perfusion and out-of-body times were 4.5 and 6.2 hours, respectively. Postoperatively, 20 (24%) and 38 (46%) patients required re-thoracotomy for bleeding and dialysis (temporary, n=32), respectively. Sixteen (19%) patients required postoperative ECLS or intraaortic balloon pump (IABP), due to PGD in 11 cases. Eleven (13%) patients died in-hospital. At discharge, all surviving patients showed a LVEF>60% in echocardiography. At 2-year follow-up, survival and freedom from biopsy-confirmed rejection (ISHLT grade >1R) were 78% and 86%, respectively. OCS Heart® allowed safe transplantation of extended criteria donor hearts. Despite out-of-body time was longer than 6 hours, patient and graft outcomes were favorable.

Utilization of New Heart Preservation Technology in the Era of New Organ Allocation and Extended Ischemic Times

Current ice bag heart preservation technique is associated with wide fluctuations of organ temperature, resulting in sub-optimal preservation and freeze injury. These shortcomings may be particularly impactful with increased ischemic times given increasing retrieval distances associated with new organ allocation. In comparison, the SherpaPakTM Cardiac Transport System is designed for the static hypothermic preservation of donor hearts within a controlled temperature range. This method avoids excess cooling and optimizes preservation during transportation. The purpose of this study is to assess the outcomes of heart transplantation using the static hypothermic preservation (SHP) system as compared with conventional ice bag preservation (IP). Retrospective cohort analysis was performed on adult patients undergoing heart transplantation between 6/30/2018 and 09/02/2019 at a single institution. Donor and recipient baseline characteristics and post-transplant outcomes were compared between patients receiving organs with SHP versus IP for donor heart transport. Patients who underwent multiple organ transplantation were excluded. Of 48 consecutive patients undergoing heart transplantation, 20 (41.7%) organs were transported using SHP and 28 (58.3%) using IP. SHP was used for older recipients (61.5 vs 57.5 years, p=0.02), and longer donor heart cold ischemic times (4.35 vs 3.49 hours, Figure1, p=0.018). There were no 30-day mortalities. Despite longer ischemic times, there was no difference in the incidence of any primary graft dysfunction (PGD) (14.3% vs 15.0%, p=1) or severe PGD requiring extracorporeal membrane oxygenation (10.7% vs 5.0%, Table1, p=0.63) between SHP and IP. There was no worsening in clinical outcomes with SHP despite longer ischemic times and older recipient ages. With new heart allocation changes, SHP may be beneficial in cases with further transport distances, by mitigating the effect of prolonged ischemic times.

Minimizing Cardiac Oedema during Ex Vivo Perfusion in a Juvenile Porcine Model - How Much Does Coronary Flow Matter?

Transmedic Organ Care System (OCS) allows preservation of the donor heart by perfusing the organ at 34°C in a beating state and providing additional assessment options. The perfusion protocols as established by the manufacturer is valid for adult human hearts, using coronary flows in the 8-900ml/min range guided by lactate measurements. Higher flow can facilitate higher degrees of cardiac oedema which can be associated with diastolic dysfunction, and generel impairment of contractility of the heart. The aim of the study is to establish the optimal coronary flow in a juvenile porcine heart, similar in size to the adult human heart, that minimizes oedema and still meets the metabolic requirements. 18 pigs in the range of 60-70kg was used for the trial and randomized into three groups with a coronary flow of 6-, 7- or 800ml/min for 180 minuts. After intubation and propofol/fentanyl anesthesia was induced, median sternotomy was performed and the heart prepared for harvest. Immediately after crossclamp 1000ml of HTK Custodiol cardioplegia was administered, and the heart prepared as per protocol for the human heart. The heart was connected a OCS-based system and randomized to one of three regimens: a coronary flow of 6-, 7- or 800ml/min for 180 minutes at 34 degree celsius, with the pressure autoregulated to 65mmHg. The pump flow was adjusted to give the desired coronary flow. Haemodynamic parameteres were continously recorded, arterial and venous lactates sampled every 30 minutes. Biopsies taken form the apex of the left ventricle and the heart was weighed after 180 minuts of perfusion. Our primary endpoint was met, as the weight gain in the 800ml/min group was significantly higher than the 600ml/min group (103g +/-20g vs 72g +/- 15g, p<0.05). There were no significant difference in the final lactate (relative to the initial levels) after three hours of perfusion. All hearts had end arterial lactate under 5.0 mmol/l. At the end of the perfusion, Troponin T levels in the blood and pathohistological examination of the biopsies showed no significant differences between the groups. We found a lower degree of oedema formation in the 600ml/min perfusion group, but similar metabolic response as the group with higher flows. . We suggest using this slightly reduced flow when investigating juvenile porcine hearts in an ex-vivo perfusion model.

First Experience with a New Storage Device for Cold Preservation

The standard technology for heart preservation for transplantation is cold static storange on ice. No temperature control is performed routinley and therefore lower or higher temperatures can occur leading to injury of the graft. The Paragonix SherpaPak™Cardiac Transportation System (CTS) (Paragonix Technologies, MA, USA) has been approved in Europe and the USA for clinical use. This single-use disposable device is designed for cold preservation of donor hearts. We report our first clinical experience with the SherpaPak™ METHODS: Since November 2018 SherpaPak™ has been used in 6 non consecutive cases in our insitutition. Decision to use the device was done in procurements with either high risk donors, long ischemic times or both. Donor risk was calculated with both the Eurotransplant donor heart risk score and the donor heart risk index (both JHLT 2012). Recipient risk was calculated via the IMPACT score (Ann Thor Surg 2011). Median recipient age was 64,5 years. All patients were male and 50% had previous sternotomies (2 VAD patients). Median impact score was 9,5 (17% expected 1 year mortality). All donors were male with a median age of 45,5 years. Median LVEF was 55%. Median norepinephrine support was 0,11mcg/kg/min. Median ET donor risk score was 18 (40% risk of non-acceptance) and median Donor risk score was 7 (18% expected 1-year mortality). Median ischemic time was 290 minutes. Donor hearts were preserved at a median of 5,5C temperature. 4 Patients were successfully weaned from bypass at the first attempt with low inotropic support. 2 Patients developed primary graft dysfunction ISHLT Grade 2 and were weaned from bypass via ECMO. Both heart recovered within 72 hours and ECMO could be explanted. All patients could be extubated within 7 days post transplant and are alive at a median of 6 months post transplant with normal graft function. The Paragonix SherpaPak™ provides consistent temperature during transportation of grafts and could be successfully used with long ischemic times and high risk donor hearts.

Comparison of Continuous Hypothermic Oxygenated Crystalloid Perfusion with the Novel Solution “Custodiol N ” and Warm Blood Perfusion in a Porcine DCD Donation Model

Transplantation programs are worldwide burdened by the limited availability of suitable donor hearts, despite extending the donation criteria. Organ donation after circulatory death (DCD) became a reliable alternative over the past few years. Nevertheless, the grafts fetched as DCDs are submitted to a further ischemic insult due to the progressive hypoxia and circulatory arrest occurring between withdrawal of care and declaration of death. Although diverse storage and perfusion strategies exist to preserve DCD hearts, no direct comparison has yet been performed. The aim of the present study is to examine the effects of a novel cold crystalloid preservation solution (Custodiol-N) and warm blood perfusion protocols compared to simple cold storage in a porcine model. In 39 pigs, DCD was induced by asphyxia and the hearts underwent 30 min initial warm ischemia in the donor. The hearts were than explanted and divided in 4 groups: 1) control group (n=8) was placed directly in a Langendorff model. In group 2 (CSG, n=8) the hearts were flushed with 2L of 4°C Custodiol N solution and then stored in the same solution for 4h before evaluation. In group 3 (CCP, n=8), the hearts underwent a 4h continuous oxygenated 4°C cold machine perfusion cycle with Custodiol N. In group 4 (WBP, n=7) the hearts were placed on warm blood reperfusion for 4h. Left ventricular function was defined by dP/dt min & max at different ventricle filling volumes. Coronary blood flow (CBF) was measured at 100 mmHg perfusion pressure and Troponin T was collected up to 2 hours. Both dP/dt min & max were significantly higher in the CPP group compared to the WBP group [-1038±182 vs -344±38 mmHg/s; p=0.0356 and 1441±194 vs 498±53 mmHg/s; p=0.0123 respectively]. CBF was also significantly higher in the CCP group [1145±57vs 726±72ml/min; p=0.0032]. Moreover, Troponin T was significantly elevated in the WBP compared to the CCP group [27477±11777 vs 251034±90247 pg/ml, p=0.0239]. There were no significant differences among the WBP, CSG and control groups. Continuous hypothermic oxygenated crystalloid perfusion with the novel preservation solution Custodiol N offers superior preservation and resuscitation of DCD hearts in terms of functional recovery and myocardial enzyme release.

Normothermic Ex-Vivo Perfusion for Donor Heart Preservation in Transplantation of Patients Bridged with Ventricular Assist Devices

Recently, it has been shown that survival after heart transplantation is impaired in patients bridged with left ventricular assist devices (LVAD), mostly due to increased rates of primary graft dysfunction (PGD). Ex vivo perfusion (EVP) is a graft preservation technique non-inferior to standard cold storage (CS). It allows to maintain the heart in a beating and perfused status during challenging surgical dissection in LVAD patients undergoing heart transplantation, and to reduce ischemic time potentially lowering the risk of PGD. Patients bridged on LVAD and transplanted in two European centers (Udine, Italy and Hanover, Germany) since 2016 were included in the study and prospectively followed. Grafts were transported with EVP using the Organ Care System (Transmedics, Andover, MA) in 44 cases and 21 with CS. The underlying demographics were not different, except for urgency status (86% vs. 57%, p=0.01) and chronic renal failure (59% vs. 29%, p=0.02) in EVP and CS respectively. Median age was 56(21-71) vs. 54(25-65), p=0.73; male sex was 77% vs. 81%, p=0.50 and most frequent ethiology was dilated cardiomyopathy in 73% vs. 52%, p=0.11. No differences were found in donors characteristics, with the employment of extended criteria donors in many cases (41% vs. 43%, p=0.55).Five extended criteria donor hearts were assessed as non-transplantable while on EVP.The remaining grafts were transported for a mean time of 296±64 min in EVP.Ischemic times were significantly shorter in the EVP group (132±29 vs. 223±48 min, p<0.01). The early outcome showed no significant statistical differences between the two groups, but a favourable trend in 30-day mortality after HTx in the EVP group (5% vs. 14%, p=0.19). Severe PGD (11% vs. 19% p=0.32), need of mechanical ventilation >96h (25% vs. 33%, p=0.48), of dialysis (66% vs. 52%, p=0.29), revision for bleeding (16% vs. 19%, p=0.50) and ICU stay (6.1-123 vs. 5.1-195 days, p=0.48) were reported. Furthermore, a trend to better survival at mid-term was found (p=0.25) with 88% vs. 76% survival at 3 years. VAD patients have a high risk of morbidity during HTx. EVP warrants a trend towards improved outcomes in this difficult recipient cohort. The longer available time to perform surgery and the reduced ischemia permit a safer procedure including cases of extended criteria donor employment.

Non-Ischemic Heart Preservation versus Static Cold Storage in Human Heart Transplantation

Pre-clinical studies have shown that ex vivo non-ischemic heart preservation (NIHP) method can be safely used for 24 hours. This state-of-the-art method has never been applied on humans. ໿໿The primary objective of the study was to evaluate the efficacy and safety of the NIHP method on early and late human heart allograft function compared with static cold storage (SCS). We performed a prospective, open-label, non-randomised phase II study. All adult recipients listed for heart transplantation were included, unless they met any exclusion criteria. The primary endpoint was a composite of survival free of severe primary graft dysfunction, free of ECMO use within 7 days, and free of acute cellular rejection ≥2R within 180 days. Secondary endpoints were I/R-tissue injury, immediate graft function, and adverse event. Of the 37 eligible patients, nine were assigned to the NIHP method and 28 to SCS. The median age was 51 years (interquartile range (IQR), 37-58) for the donors and 56 years (IQR, 46 - 63) for the recipients. The median preservation time was significant longer for the NIHP group, 251 min (IQR, 219-269) compared with the SCS group, 199 min (IQR, 164 - 227), P=0.008. Over the first three months, all of the patients assigned to the NIHP group achieved event-free survival, compared with 21 (75%) of those assigned to the SCS group (Kaplan-Meier estimate of event free survival 75% (95% CI 55-87%); P=0.124). CK-MB assessed 6±2 h after ending perfusion was 77 (IQR, 54-101) ng/mL for the NIHP group compared with 137 (IQR, 73-196) ng/mL for the SCS group, P=0.030. Four (16%) death within six months after transplantation and three (12%) cardiac-related adverse events were reported in the SCS group compared with no deaths or cardiac-related adverse events in the NIHP group. This first-in-human study shows the NIHP method's feasibility and safety for use in the clinic of heart transplantation.

Empagliflozin Improves Cardiac Functional Recovery after Prolonged Cold Storage of Donor Hearts in an Isolated Working Rat Heart Model

Many donor hearts are unsuitable for transplantation after prolonged cold storage or donation after circulatory death. Improved donor heart recovery is observed after supplementation of preservation solutions with sodium-hydrogen exchange inhibitors (NHEI) cariporide/zoniporide (Gao et al, 2010, BJP; Cropper et al, 2003, JHLT). NHEI are not clinically used due to neurotoxicity observed in cariporide clinical trials however the search for suitable NHEI alternatives continues. One promising alternative is the sodium-glucose cotransporter 2 inhibitor Empagliflozin (Empa) - the first anti-diabetic drug to yield positive cardiovascular outcomes. Studies have shown Empa mimics NHEI and slows pH recovery in cardiomyocytes following an acidic load. This study tests the cardioprotective efficacy of Empa-supplementation in cardiac preservation solution during prolonged cold storage of donor hearts. Wistar rat (320-420g; n=5-7) hearts were perfused ex-vivo and baseline hemodynamic measurements acquired. Hearts were arrested and stored in Celsior±0.3-10µM Empa, or zoniporide (1µM) (6h, 4°C) then reperfused (37°C, Langendorff 15min, working 30min). Post-reperfusion aortic flow (AF) and cardiac output (CO) were expressed as a percentage of baseline measurements. Coronary effluent (pre-storage, 15, 30, and 45 min reperfusion) was assessed for lactate dehydrogenase (LDH) release. Donor hearts supplemented with Empa during cold storage showed a dose-dependent improvement in cardiac functional recovery. Compared to unsupplemented control hearts, 10µM Empa-supplemented hearts showed significantly improved AF (54±11% vs 10±7%, p=0.006) and CO (55±12 vs 16±8, p=0.03) recovery. Hearts supplemented with 1µM zoniporide also showed significantly improved AF (63±7%, p=0.002 vs control) and CO (65±9%, p=0.01) recovery. A trend for reduced LDH efflux was observed in hearts supplemented with 1-10µM Empa or zoniporide compared to control hearts. Supplementation of cardiac preservation solution with 10µM Empa yields significantly improved functional recovery after prolonged cold storage, with similar cardioprotective efficacy to the NHEI zoniporide. Given the approved clinical use of Empa, this study highlights its potential as a suitable alternative for NHEI in donor heart preservation.