Ischaemia-reperfusion injury (IRI) results in myocardial cell death and compromised cardiac function making it a leading risk factor for heart failure.

Background. Hearts from older donors or procured via donation after circulatory death (DCD) can alleviate transplant waitlist; however, these hearts are particularly vulnerable to injury caused by warm ischemic times (WITs) inherent to DCD. This study investigates how the combination of increasing donor age and pharmacologic supplementation affects the ischemic tolerance and functional recovery of DCD hearts and how age impacts cardiac mitochondrial respiratory capacity and oxidative phosphorylation. Methods. Wistar rats (12-, 18-, and 24-mo-old) were subjected to DCD with 20-min fixed WIT. Hearts were procured, instrumented onto a Langendorff perfusion circuit, flushed with Celsior preservation solution with or without supplementation (glyceryl trinitrate [GTN]/erythropoietin [EPO]/zoniporide [Z]) and perfused (Krebs-Henseleit buffer, 37°C Langendorff 30-min, working 30-min). Cardiac functional recovery of aortic flow (AF), coronary flow (CF), cardiac output (CO), and lactate dehydrogenase release were measured. Native heart tissue (3-, 12-, and 24-mo) were assessed for mitochondrial respiratory capacity. Results. Unsupplemented 18- and 24-month DCD hearts showed a 6-fold decrease in AF recovery relative to unsupplemented 12-month DCD hearts. GTN/EPO/Z supplementation significantly increased AF and CO recovery of 18-month DCD hearts to levels comparable to supplemented 12-month hearts; however, GTN/EPO/Z did not improve 24-month DCD heart recovery. Compared to 12-month heart tissue, 24-month hearts exhibited significantly impaired mitochondrial oxygen flux at complex I, II, and uncoupled maximal respiration stage. Conclusions. Reduced ischemic tolerance after DCD was associated with increasing age. Pharmacologic supplementation improves functional recovery of rat DCD hearts but only up to age 18 months, possibly attributed to a decline in mitochondrial respiratory capacity with increasing age.

BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

Introduction: In 2014 the world first DCD heart transplant with distant procurement (DPP) was performed in Sydney, Australia. Programs are now established in the UK and Europe, as well as a recently commenced multi-centre trial in the USA, with >150 performed worldwide. In established centres, DCD donors account for >20% of heart transplants with excellent early- and mid-term outcomes. Despite these encouraging results, there is still much to be learnt about DCD heart transplantation, and with increasing numbers of hospitals wishing to start DCD heart programs this paper will describe the protocols, lessons learnt and current outcomes from our experience. Materials and Methods: Forty-one DCD heart transplants were performed at our institution between July 2014 and February 2020, utilising a DPP and ex-situ perfusion protocol. Male or female DCD donors under the age of 55 years with no known history of cardiac disease were accepted for retrieval. Recipient selection was based on blood group and cross-matching outcomes, heart mass, and clinical urgency. The donor hearts were perfused on the TransMedics OCS device as outlined in Table 1.For analysis, donor details were collected using electronic donor records (Donate Life, Australia) with recipient data collected from clinical notes and electronic databases at our institution. Results: Patients receiving DCD allografts had excellent outcomes with 97.6 and 95.1% 1- and 5-year survival, respectively.In spite of the similar donor and recipient profiles and a similar rate of utilization of allografts reperfused on the OCS device (74% and 72%, early and late cohort, respectively), the rate of delayed graft function requiring ECMO support has dropped dramatically, from 35% to 14%.Interestingly, prolongation of the functional warm ischemic time was not associated with an increased rate of ECMO, however prolongation of the asystolic warm ischemic time was. A longer agonal period from withdrawal to circulatory arrest was in fact associated with a lower rate of ECMO.Discussion: Over our DCD program experience we have made several changes likely to have contributed to the reduced perioperative ECMO requirement. Primarily, we have adopted a more ‘protective’ approach to the management of the heart on the OCS device, targeting in particular lower coronary flows, with a view to reducing endothelial dysfunction and myocardial oedema. In addition, a design change to the OCS module now allows for a controlled cooling of the heart to 16°C prior to delivery of cardioplegia, proving superior protection to the heart during implantation. Conclusion: DCD heart transplantation clearly provides an additional source of donor allografts with excellent recipient outcomes. Our experiences have reinforced the benefit of a ‘protective’ perfusion strategy on the OCS device, and also raise questions about the true tolerable warm ischemic time, which requires further investigation. Curran Foundation. References: 1. Chew, H.C., et al., Outcomes of Donation After Circulatory Death Heart Transplantation in Australia. J Am Coll Cardiol, 2019. 73(12): p. 1447-1459. 2. Messer, S., et al., Outcome after heart transplantation from donation after circulatory-determined death donors. J Heart Lung Transplant, 2017. 36(12): p. 1311-1318.

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.

The definition of WIT in DCD heart transplantation has proved controversial, however with more in-depth analysis of the patterns associated with WLS and performance of the donor allograft it does not appear that a prolonged WIT, as defined by any of the current or proposed definitions, is in fact associated with sPGD. At present the only clear negative predictor is a prolonged aWIT. This ought to provoke renewed discussions regarding these definitions, and an increased focus on the minimisation of aWIT during DCD allograft retrievals.

A major obstacle in heart transplantation is the limited number of donor hearts available to meet recipient demand. This has forced consideration of marginal donor hearts, those from older donors, with pre-existing heart disease, after prolonged periods (>6 hours) of cold storage, or obligatory warm ischemic times during donation after circulatory death (DCD) including mandatory stand-off times. However, marginal donor hearts are more susceptible to ischemia-reperfusion injury (IRI) in response to dysregulated ion homeostasis caused by the inhibition of the plasma membrane sodium/potassium ATPase and activation of ion exchangers such as the sodium-hydrogen exchanger (NHE) and the sodium-calcium exchanger. The detrimental consequences of IRI can be counteracted through pharmacological supplementation of donor heart preservation solutions with NHE inhibitors (NHEIs). The addition of the NHEI cariporide or zoniporide to the heart preservation solutions, either as a single supplement or combined with erythropoietin and glyceryl trinitrate, dramatically improves the recovery of donor hearts in animal models of heart transplantation. 1 Iyer A Gao L Doyle A et al. Increasing the tolerance of DCD hearts to warm ischemia by pharmacological postconditioning. Am J Transplant. 2014; 14: 1744-1752 Crossref PubMed Scopus (85) Google Scholar Although potent cardioprotection was observed in a Phase III clinical trial of the NHEI cariporide in patients undergoing high-risk coronary artery bypass, repeated intravenous bolus administration of cariporide was unfortunately associated with stroke-like syndrome neurotoxicity and increased mortality, 2 Mentzer Jr, RM Bartels C Bolli R et al. Sodium-hydrogen exchange inhibition by cariporide to reduce the risk of ischemic cardiac events in patients undergoing coronary artery bypass grafting: results of the EXPEDITION study. Ann Thorac Surg. 2008; 85: 1261-1270 Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar preventing any further clinical development of NHEIs for this indication. Current clinical marginal brain death and DCD donor heart preservation at our institution incorporates supplementation using erythropoietin and glyceryl trinitrate. 3 Chew HC Iyer A Connellan M et al. Outcomes of donation after circulatory death heart transplantation in Australia. J Am Coll Cardiol. 2019; 73: 1447-1459 Crossref PubMed Scopus (119) Google Scholar Given the strong cardioprotective benefits of NHEIs in pre-clinical studies of donor heart preservation, identification of a safe alternative to cariporide with the cardioprotective benefits of an NHEI is warranted.

Introduction: Many donor hearts are unsuitable for transplantation due to ischemia-reperfusion injury during retrieval. Improved donor heart recovery is observed after preservation solution supplementation with sodium-hydrogen exchange inhibitors (NHEI) cariporide/zoniporide however these NHEI are no longer approved for clinical use due to neurotoxicity observed in cariporide clinical trials. One promising alternative is the sodium-glucose cotransporter 2 inhibitor Empagliflozin (Empa) – the first anti-diabetic drug to yield positive cardiovascular outcomes, reduce heart failure hospitalisations and mortality. In vitro studies have shown Empa mimics NHEI and slows pH recovery in cardiomyocytes following an acidic load. Whether Empa is beneficial in the context of donor heart preservation is unknown. Aim: To test Empa-supplementation during prolonged cold storage of donor hearts. Materials and Methods: Wistar rat (320–420g; n=5-7) hearts were perfused ex vivo with Krebs-Henseleit buffer (KHB) and baseline hemodynamic measurements acquired. Hearts were arrested and stored in Celsior±0.3–10µM Empa, or Celsior+zoniporide (1µM) (6h, 4°C) then reperfused (KHB, 37°C, Langendorff 15min, working 30min). Post-reperfusion aortic flow (AF), coronary flow (CF), and cardiac output (CO=AF+CF) 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. Statistical significance was determined using one-way ANOVA (functional studies), or two-way ANOVA (LDH studies) with Tukey’s multiple comparisons test. Results and Discussion: Donor hearts supplemented with Empa during cold storage showed a dose-dependent improvement in cardiac functional recovery. Compared to unsupplemented control hearts, 10µM Empa-supplementation significantly improved AF (54±11% vs 10±7%, p=0.006; Fig 1), showed a trend for increased CF (70±14% vs 36±12%, Fig 2), and significantly improved CO (55±12 vs 16±8, p=0.03; Fig 3) recovery. The improved functional hemodynamics observed after 10µM Empa-supplementation were comparable to hearts supplemented with 1µM zoniporide which showed significantly improved AF (63±7%, p=0.002 vs control, Fig 1), CF (90±6%, p=0.039, Fig 2) and CO (65±9%, p=0.01, Fig 3) recovery. A trend for reduced LDH efflux, a marker of cellular injury, was observed at 30 min reperfusion in hearts supplemented with 1-10µM Empa or 1µM zoniporide when compared to control hearts. Conclusion: Supplementation of the cardiac preservation solution with 10µM Empa yields significantly improved functional recovery after prolonged cold storage and reduced cellular injury, with similar cardioprotective efficacy to the NHEI zoniporide. The safety profile and approved clinical use of Empa highlights its potential as a suitable alternative for traditional NHEI (e.g., zoniporide and cariporide) in donor heart preservation.St Vincent’s Clinic Foundation. National Health and Medical Research Council.

Background: DCD hearts are now an important source for transplantation [1]. Among unknowns during DCD organ retrieval is the need for ante-mortem heparin which is currently not permitted in some jurisdictions due to ethical concerns. While administration of ante-mortem heparin has been shown experimentally to improve perfusion of abdominal organs during regional perfusion after withdraw of life support (WLS) [2], the impact of ante-mortem heparin on the recovery of cardiac function following retrieval from DCD donor has not been reported. In a recent analysis of clinical outcomes of DCD heart transplantation in our program, we found that the asystolic warm ischemic time (aWIT) between circulatory arrest and the commencement of cold flush of the heart was critical to the early outcome of the donor heart after implantation [3]. Here, in a DCD model designed to mimic this clinical scenario, we aimed to investigate: 1) the susceptibility of DCD hearts to increasing aWIT; 2) the impact of ante-mortem heparin administration on recovery of DCD heart during normothermic ex situ reperfusion. Methods: Male Wistar rats (340-420g) were anesthetized and carotid artery was cannulated for blood pressure monitoring. WLS was initiated by tracheal ligation with or without ante-mortem injection of 500 IU heparin. Circulatory arrest was declared when pulse pressure trace disappeared. Animals were randomized into experimental groups exposed to increasing aWIT of 10, 15 and 20 min. After each aWIT period, hearts were excised then flushed with 100 ml ice-cold Celsior followed by 1 hr reperfusion (30 min Langendorff, 30 min working mode) with Krebs solution at 37oC. Sham hearts were removed after heparin injection without tracheal ligation. Recovery of cardiac function after reperfusion was assessed by measurement of aortic flow (AF), pulse pressure (PP), heart rate (HR) and coronary flow (CF). Timed collection of coronary effluents during reperfusion were assessed for lactate dehydrogenase (LDH) and troponin-I (cTnI). Results: All DCD hearts had significantly lower functional recoveries after reperfusion compared to sham controls. The recovery of DCD heart function was inversely proportional to the duration of aWIT (Fig1). LDH levels were significantly higher in 15 and 20 aWIT hearts (Fig2). When exposed to aWIT of 10min, hearts retrieved after ante-mortem heparin had significantly better functional recovery (Fig3) and reduced LDH (Fig4) and cTnI release compared to those without ante-mortem heparin. Conclusions: This is the first study specifically investigating the impact of ante-mortem heparin on the recovery of DCD hearts after retrieval. We show ante-mortem heparin administration significantly enhanced recovery of DCD heart function, associated with reduced LDH and cTnI release. We believe that administration of ante-mortem heparin in DCD heart retrieval is beneficial and may improve donor heart quality with the potential to reduce risk of PGD post-implantation.St Vincent’s Clinical Foundation of Australia (SVCF-PM). National Health & Medical Research Council of Australia (program grant ID 1074386). References: 1. Chew HC, Iyer A, Connellan M, et al. Outcome of Donation after Circulatory Death Heart Transplantation in Australia. J Am Coll Cardio 2019; 73:1447– 59. 2. Rojas-Pena A, et al. Timing of Heparin and Perfusion Temperature During Procurement of Organs with Extracorporeal Support in Donors After Circulatory Determination of Death. ASAIO J 2011; 57: 368-74. 3. Scheuer SE, et al. Getting the time right: what matters, what doesn’t, and how should we really be defining ischemic times in DCD withdrawals? Journal of Heart & Lung Transplantation; 2020, in press.

The proton-gated acid-sensing ion channel 1a (ASIC1a) is implicated in the injury response to cerebral ischemia but little is known about its role in cardiac ischemia. We provide genetic evidence that ASIC1a is involved in myocardial ischemia-reperfusion injury (IRI) and show that pharmacological inhibition of ASIC1a yields robust cardioprotection in rodent and human models of cardiac ischemia, resulting in improved post-IRI cardiac viability and function. Consistent with a key role for ASIC1a in cardiac ischemia, we show that polymorphisms in the ASIC1 genetic locus are strongly associated with myocardial infarction. Collectively, our data provide compelling evidence that ASIC1a is a key target for cardioprotective drugs to reduce the burden of disease associated with myocardial ischemia.

Aim: Acid-sensing ion channel 1a (ASIC1a) has been found to play a critical role in acidosis-induced neuronal injury during cerebral ischaemia. This project sought to investigate the potential cardioprotective effects of Hi1a, a novel ASIC1a inhibitor, derived from funnel-web spider venom, in donor allograft preservation.

Background. Storage of donor hearts in cardioplegic solutions supplemented with conditioning agents activating endogenous mitochondrial protective signaling enhanced their postreperfusion recovery. The present study investigates the role of timing and duration of cardiac exposure to cyclosporine A (CsA), another putative mitochondrial protectant, on cardiac functional recovery and potential mechanisms of CsA action in an isolated working rat heart model of donor heart retrieval and storage. Methods. After measurement of baseline function, hearts were arrested and stored for 6 hours at 4°C in either Celsior alone or Celsior + CsA (0.2 µM), then reperfused for 45 minutes in Krebs solution, when functional recovery was assessed. Two additional groups of Celsior-alone stored hearts were exposed to 0.2 µM CsA for the initial 15 minutes (nonworking period) or the full 45-minute period of reperfusion. Coronary effluent was collected pre- and poststorage for assessment of lactate dehydrogenase release. Tissue samples were collected at the end of each study for immunoblotting and histological studies. Results. CsA supplementation during cold storage or the first 15-minute reperfusion significantly improved functional recovery and significantly increased phospho-AMPKα Thr172 and phospho-ULK-1 Ser757 . Hearts exposed to CsA for 45 minutes at reperfusion recovered poorly with no phospho–AMP-activated protein kinase α activation, decreased phospho-eNOS Ser633 , and decreased mitochondrial cytochrome c content with increased lactate dehydrogenase release. Conclusions. Inclusion of CsA during cold storage is cardioprotective. Effects of CsA addition to the perfusate during reperfusion were time dependent, with benefits at 15 minutes but not 45 minutes of reperfusion. The toxic effect with the presence of CsA for the full 45-minute reperfusion is associated with impaired mitochondrial integrity and decreased eNOS phosphorylation.

Purpose ASIC1a has been demonstrated to play a critical role in acidosis-induced neuronal injury following cerebral ischaemia, however its role in the heart is poorly understood. Given similar pathophysiology, this project sought to investigate the cardioprotective effects of Hi1a, a novel ASIC1a inhibitor, derived from funnel web spider venom, in the context of donor allograft preservation. Methods Studies utilized a working rodent model of DCD heart preservation. Following asphyxia, hearts were retrieved from male Wistar rats (350-425g) after a 10 min warm ischaemic time (WIT) followed by 2 min stand-off, mimicking a clinical DCD withdrawal. Hearts were then flushed with either Celsior alone, Celsior + Hi1a (CHi1a), or Celsior + Hi1a, GTN and EPO (n=7/group). Allografts were reperfused ex-vivo in resting Langendorff mode for 30 min, prior to assessment of cardiac function in working mode for 30 min. Results Recovery of aortic flow (AF), pulse pressure (PP), and cardiac output (CO) was significantly improved in the CHi1a group and CHi1a + GTN, EPO group when compared to Celsior alone (p<0.006 and p<0.0001 respectively, for all parameters). Furthermore, LDH measurements from coronary effluent during working reperfusion demonstrated that allografts treated with CHi1a + GTN, EPO had a significantly reduced LDH level when compared to Celsior alone (p=0.008). In fact, the LDH level at 30 mins of working reperfusion in this group was comparable to a heart at baseline, prior to any ischaemic insult. Conclusion Supplementation of Celsior with either Hi1a as a single supplement, or in combination with GTN and EPO significantly improved cardiac allograft function following warm ischaemia. Both the functional and biochemical results demonstrate profound cardioprotective effects of Hi1a. These results suggest the potential of Hi1a, or other ASIC1a inhibitors to increase the tolerable WIT during DCD retrieval, leading to further increases in heart transplant volume.

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated channel that is activated by tissue acidosis. We utilized genetic loss of function to demonstrate that ASIC1a mediates the injury response to myocardial ischemia-reperfusion injury (IRI). We show that pharmacological inhibition of ASIC1a affords cardioprotection across a range of rodent and human models of cardiac ischemia, resulting in improved cardiac viability and functional recovery post-IRI. Consistent with this data, we show that polymorphisms in the ASIC1 gene locus are associated with myocardial infarction and small-vessel stroke in humans. This study provides compelling evidence that ASIC1a is a novel target for cardioprotective drugs that could reduce the burden of disease associated with myocardial ischemia.

Aims Metformin may have clinical benefits in dialysis patients; however, its safety in this population is unknown. This systematic review evaluated the safety of metformin in dialysis patients. Methods MEDLINE, Embase, CENTRAL, PsycINFO and the Cochrane Library were searched for randomised controlled trials and observational studies evaluating metformin use in dialysis patients. Three authors reviewed the studies and extracted data. The primary outcomes were mortality, occurrence of lactic acidosis and myocardial infarction (MI) in patients taking metformin during dialysis treatment for ≥12 months ( long term ). Risk of bias was assessed using Risk Of Bias In Nonrandomised Studies of Interventions (ROBINS‐1). Overall quality of evidence was assessed using Grading of Recommendations Assessment, Development and Evaluation (GRADE). Results Fifteen observational studies were eligible; 7 were prospective observational studies and 8 were case reports/case series. No randomised controlled trials were identified. The 7 prospective observational studies ( n = 194) reported on cautious metformin use in patients undergoing maintenance dialysis. Only 3 provided long‐term follow‐up data. In 2 long‐term studies of metformin therapy (≤1000 mg/d) in patients undergoing peritoneal dialysis (PD), 1 reported 6 deaths (6/83; 7%) due to major cardiovascular events (3 MI) and the other reported no deaths (0/35). One long‐term study of metformin therapy (250 mg to 500 mg thrice weekly) in patients undergoing haemodialysis reported 4 deaths (4/61; 7%) due to major cardiovascular events (2 MI). These findings provide very low‐quality evidence as they come from small observational studies. Conclusion The evidence regarding the safety of metformin in people undergoing dialysis is inconclusive. Appropriately designed randomised controlled trials are needed to resolve this uncertainty.

Background: Normothermic machine perfusion (NMP) utilises 1.2-1.5L of donor blood (DB) with a target perfusate haematocrit of 25% to reanimate the donor heart. Limitations to current practise include small donor size and donor anaemia which can impact on the recovery of these hearts. Furthermore, in DCD, blood collection delays delivery of cardio-protective solution resulting in longer warm ischaemic times. Banked blood (BB) is a potential alternative to donor blood. We investigate the effect of banked blood for the reanimation and perfusion of donor hearts in a DCD porcine model. Methods: Series 1: Landrace pigs (n=12) underwent DCD withdrawal with subsequent procurement of the heart; 6 were reperfused with autologous blood; 6 were reperfused with BB (collected 24 hours prior). Hearts were maintained on NMP for 5 hours with continuous pressure and flow measurement. Serial venous and arterial lactate measurements were performed as a marker of organ health. Series 2: Ten further studies in the BB group with NO pre-treatment (n=5), and Sodium Nitroprusside (SNP) infusion (n=5). Results: BB demonstrates severe metabolic and electrolyte disturbances requiring correction before organ perfusion. Both group were successful in cardiac reanimation and demonstrated favourable lactate trend during perfusion. BB had higher perfusion pressure which continues to rise overtime with resultant deterioration in cardiac function. The introduction of NO and SNP only provided temporary relief to the observed hypertension. Conclusion: BB in the reperfusion of donor hearts on NMP results in significantly higher perfusion pressures leading to progressive deterioration of cardiac recovery. Treatment with vasodilators produced short-lived benefits.

Aims: This project sought to investigate the cardioprotective effects of Hi1a, an acid-sensing ion channel 1a (ASIC1a) inhibitor derived from funnel-web spider venom, in the context of donor heart preservation.

To investigate the effect of ageing on donor heart recovery after exposure to DCD withdrawal in a rodent model.

The ryanodine receptor antagonist dantrolene inhibits calcium release from the sarcoplasmic reticulum and reduces cardiac ischaemia-reperfusion injury (IRI) in global warm ischaemia models however the cardioprotective potential of dantrolene under hypothermic conditions is unknown. This study addresses whether the addition of dantrolene during cardioplegia and hypothermic storage of the donor heart can improve functional recovery and reduce IRI. Using an ex vivo isolated working heart model, Wistar rat (3 month and 12 month) hearts were perfused to acquire baseline haemodynamic measurements of aortic flow, coronary flow, cardiac output, pulse pressure and heart rate. Hearts were arrested and stored in Celsior preservation solution supplemented with 0.2–40 μM dantrolene for 6 hours at 4°C, then reperfused (15 min Langendorff, 30 min working mode). In 3-month hearts, supplementation with 1 μM dantrolene significantly improved aortic flow and cardiac output compared to unsupplemented controls however lactate dehydrogenase (LDH) release and contraction bands were comparable. In contrast, 40 μM dantrolene-supplementation yielded poor cardiac recovery, increased post-reperfusion LDH but reduced contraction bands. All 3-month hearts stored in dantrolene displayed significantly reduced cleaved-caspase 3 intensities compared to controls. Analysis of cardioprotective signalling pathways showed no changes in AMPKα however dantrolene increased STAT3 and ERK1/2 signaling in a manner unrelated to functional recovery and AKT activity was reduced in 1 μM dantrolene-stored hearts. In contrast to 3-month hearts, no significant improvements were observed in the functional recovery of 12-month hearts following prolonged storage in 1 μM dantrolene. Conclusions: Dantrolene supplementation at 1 μM during hypothermic heart preservation improved functional recovery of young, but not older (12 month) hearts. Although the molecular mechanisms responsible for dantrolene-mediated cardioprotection are unclear, our studies show no correlation between improved functional recovery and SAFE and RISK pathway activation.