Macrophage-derived extracellular vesicles alter cardiac recovery and metabolism in a rat heart model of donation after circulatory death.

Conditions to which the cardiac graft is exposed during transplantation with donation after circulatory death (DCD) can trigger the recruitment of macrophages that are either unpolarized (M0) or pro-inflammatory (M1) as well as the release of extracellular vesicles (EV). We aimed to characterize the effects of M0 and M1 macrophage-derived EV administration on post-ischaemic functional recovery and glucose metabolism using an isolated rat heart model of DCD. Isolated rat hearts were subjected to 20 min aerobic perfusion, followed by 27 min global, warm ischaemia or continued aerobic perfusion and 60 min reperfusion with or without intravascular administration of EV. Four experimental groups were compared: (1) no ischaemia, no EV; (2) ischaemia, no EV; (3) ischaemia with M0-macrophage-dervied EV; (4) ischaemia with M1-macrophage-derived EV. Post-ischaemic ventricular and metabolic recovery were evaluated. During reperfusion, ventricular function was decreased in untreated ischaemic and M1-EV hearts, but not in M0-EV hearts, compared to non-ischaemic hearts (p < 0.05). In parallel with the reduced functional recovery in M1-EV versus M0-EV ischaemic hearts, rates of glycolysis from exogenous glucose and oxidative metabolism tended to be lower, while rates of glycogenolysis and lactate release tended to be higher. EV from M0- and M1-macrophages differentially affect post-ischaemic cardiac recovery, potentially by altering glucose metabolism in a rat model of DCD. Targeted EV therapy may be a useful approach for modulating cardiac energy metabolism and optimizing graft quality in the setting of DCD.

Hypothermic Oxygenated Perfusion Improves Vascular and Contractile Function by Preserving Endothelial Nitric Oxide Production in Cardiac Grafts Obtained With Donation After Circulatory Death.

BACKGROUND: Cardiac donation after circulatory death is a promising option to increase graft availability. Graft preservation with 30 minutes of hypothermic oxygenated perfusion (HOPE) before normothermic machine perfusion may improve cardiac recovery as compared with cold static storage, the current clinical standard. We investigated the role of preserved nitric oxide synthase activity during HOPE on its beneficial effects. METHODS AND RESULTS: Using a rat model of donation after circulatory death, hearts underwent in situ ischemia (21 minutes), were explanted for a cold storage period (30 minutes), and then reperfused under normothermic conditions (60 minutes) with left ventricular loading. Three cold storage conditions were compared: cold static storage, HOPE, and HOPE with Nω-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor). To evaluate potential confounding effects of high coronary flow during early reperfusion in HOPE hearts, bradykinin was administered to normalize coronary flow to HOPE levels in 2 additional groups (cold static storage and HOPE with Nω-nitro-L-arginine methyl ester). Cardiac recovery was significantly improved in HOPE versus cold static storage hearts, as determined by cardiac output, left ventricular work, contraction and relaxation rates, and coronary flow (P<0.05). Furthermore, HOPE attenuated postreperfusion calcium overload. Strikingly, the addition of Nω-nitro-L-arginine methyl ester during HOPE largely abolished its beneficial effects, even when early reperfusion coronary flow was normalized to HOPE levels. CONCLUSIONS: HOPE provides superior preservation of ventricular and vascular function compared with the current clinical standard. Importantly, HOPE's beneficial effects require preservation of nitric oxide synthase activity during the cold storage. Therefore, the application of HOPE before normothermic machine perfusion is a promising approach to optimize graft recovery in donation after circulatory death cardiac grafts.

Simultaneous assessment of mechanical and electrical function in Langendorff-perfused ex-vivo mouse hearts.

Background: The Langendorff-perfused ex-vivo isolated heart model has been extensively used to study cardiac function for many years. However, electrical and mechanical function are often studied separately-despite growing proof of a complex electro-mechanical interaction in cardiac physiology and pathology. Therefore, we developed an isolated mouse heart perfusion system that allows simultaneous recording of electrical and mechanical function. Methods: Isolated mouse hearts were mounted on a Langendorff setup and electrical function was assessed via a pseudo-ECG and an octapolar catheter inserted in the right atrium and ventricle. Mechanical function was simultaneously assessed via a balloon inserted into the left ventricle coupled with pressure determination. Hearts were then submitted to an ischemia-reperfusion protocol. Results: At baseline, heart rate, PR and QT intervals, intra-atrial and intra-ventricular conduction times, as well as ventricular effective refractory period, could be measured as parameters of cardiac electrical function. Left ventricular developed pressure (DP), left ventricular work (DP-heart rate product) and maximal velocities of contraction and relaxation were used to assess cardiac mechanical function. Cardiac arrhythmias were observed with episodes of bigeminy during which DP was significantly increased compared to that of sinus rhythm episodes. In addition, the extrasystole-triggered contraction was only 50% of that of sinus rhythm, recapitulating the "pulse deficit" phenomenon observed in bigeminy patients. After ischemia, the mechanical function significantly decreased and slowly recovered during reperfusion while most of the electrical parameters remained unchanged. Finally, the same electro-mechanical interaction during episodes of bigeminy at baseline was observed during reperfusion. Conclusion: Our modified Langendorff setup allows simultaneous recording of electrical and mechanical function on a beat-to-beat scale and can be used to study electro-mechanical interaction in isolated mouse hearts.

Erratum: Inflammatory extracellular vesicles prompt heart dysfunction via TLR4-dependent NF-κB activation: Erratum.

[This corrects the article DOI: 10.7150/thno.39072.].

A tomographic microscopy-compatible Langendorff system for the dynamic structural characterization of the cardiac cycle.

Introduction: Cardiac architecture has been extensively investigated ex vivo using a broad spectrum of imaging techniques. Nevertheless, the heart is a dynamic system and the structural mechanisms governing the cardiac cycle can only be unveiled when investigating it as such. Methods: This work presents the customization of an isolated, perfused heart system compatible with synchrotron-based X-ray phase contrast imaging (X-PCI). Results: Thanks to the capabilities of the developed setup, it was possible to visualize a beating isolated, perfused rat heart for the very first time in 4D at an unprecedented 2.75 µm pixel size (10.6 µm spatial resolution), and 1 ms temporal resolution. Discussion: The customized setup allows high-spatial resolution studies of heart architecture along the cardiac cycle and has thus the potential to serve as a tool for the characterization of the structural dynamics of the heart, including the effects of drugs and other substances able to modify the cardiac cycle.

Mitochondrial Damage-associated Molecular Patterns as Potential Biomarkers in DCD Heart Transplantation: Lessons From Myocardial Infarction and Cardiac Arrest.

Heart transplantation with donation after circulatory death (DCD) has become a real option to increase graft availability. However, given that DCD organs are exposed to the potentially damaging conditions of warm ischemia before procurement, new strategies for graft evaluation are of particular value for the safe expansion of DCD heart transplantation. Mitochondria-related parameters are very attractive as biomarkers because of their intimate association with cardiac ischemia-reperfusion injury. In this context, a group of mitochondrial components, called mitochondrial damage-associated molecular patterns (mtDAMPs), released by stressed cells, holds great promise. mtDAMPs may be released at different stages of DCD cardiac donation and may act as indicators of graft quality. Because of the lack of information available for DCD grafts, we consider that relevant information can be obtained from other acute cardiac ischemic conditions. Thus, we conducted a systematic review of original research articles in which mtDAMP levels were assessed in the circulation of patients with acute myocardial infarction and cardiac arrest. We conclude that 4 mtDAMPs, ATP, cytochrome c, mitochondrial DNA, and succinate, are rapidly released into the circulation after the onset of ischemia, and their concentrations increase with reperfusion. Importantly, circulating levels of mtDAMPs correlate with cardiac damage and may be used as prognostic markers for patient survival in these conditions. Taken together, these findings support the concept that mtDAMPs may be of use as biomarkers to assess the transplant suitability of procured DCD hearts, and ultimately aid in facilitating the safe, widespread adoption of DCD heart transplantation.

Effects of graft preservation conditions on coronary endothelium and cardiac functional recovery in a rat model of donation after circulatory death.

BACKGROUND: Use of cardiac grafts obtained with donation after circulatory death (DCD) could significantly improve donor heart availability. As DCD hearts undergo potentially deleterious warm ischemia and reperfusion, clinical protocols require optimization to ensure graft quality. Thus, we investigated effects of alternative preservation conditions on endothelial and/or vascular and contractile function in comparison with the current clinical standard. METHODS: Using a rat DCD model, we compared currently used graft preservation conditions, St. Thomas n°2 (St. T) at 4°C, with potentially more suitable conditions for DCD hearts, adenosine-lidocaine preservation solution (A-L) at 4°C or 22°C. Following general anesthesia and diaphragm transection, hearts underwent either 0 or 18 min of in-situ warm ischemia, were explanted, flushed and stored for 15 min with either St. T at 4°C or A-L at 4°C or 22°C, and then reperfused under normothermic, aerobic conditions. Endothelial integrity and contractile function were determined. RESULTS: Compared to 4°C preservation, 22°C A-L significantly increased endothelial nitric oxide synthase (eNOS) dimerization and reduced oxidative tissue damage (p < 0.05 for all). Furthermore, A-L at 22°C better preserved the endothelial glycocalyx and coronary flow compared with St. T, tended to reduce tissue calcium overload, and stimulated pro-survival signaling. No significant differences were observed in cardiac function among ischemic groups. CONCLUSIONS: Twenty-two-degree Celsius A-L solution better preserves the coronary endothelium compared to 4°C St. T, which likely results from greater eNOS dimerization, reduced oxidative stress, and activation of the reperfusion injury salvage kinase (RISK) pathway. Improving heart preservation conditions immediately following warm ischemia constitutes a promising approach for the optimization of clinical protocols in DCD heart transplantation.

Pre-ischemic Lactate Levels Affect Post-ischemic Recovery in an Isolated Rat Heart Model of Donation After Circulatory Death (DCD).

Introduction: Donation after circulatory death (DCD) could substantially improve donor heart availability. In DCD, the heart is not only exposed to a period of warm ischemia, but also to a damaging pre-ischemic phase. We hypothesized that the DCD-relevant pre-ischemic lactate levels negatively affect the post-ischemic functional and mitochondrial recovery in an isolated rat heart model of DCD. Methods: Isolated, working rat hearts underwent 28.5' of global ischemia and 60' of reperfusion. Prior to ischemia, hearts were perfused with one of three pre-ischemic lactate levels: no lactate (0 Lac), physiologic lactate (0.5 mM; 0.5 Lac), or DCD-relevant lactate (1 mM; 1 Lac). In a fourth group, an inhibitor of the mitochondrial calcium uniporter was added in reperfusion to 1 Lac hearts (1 Lac + Ru360). Results: During reperfusion, left ventricular work (heart rate-developed pressure product) was significantly greater in 0.5 Lac hearts compared to 0 Lac or 1 Lac. In 1 vs. 0.5 Lac hearts, in parallel with a decreased function, cellular and mitochondrial damage was greater, tissue calcium content tended to increase, while oxidative stress damage tended to decrease. The addition of Ru360 to 1 Lac hearts partially abrogated the negative effects of the DCD-relevant pre-ischemic lactate levels (greater post-ischemic left ventricular work and less cytochrome c release in 1 Lac+Ru360 vs. 1 Lac). Conclusion: DCD-relevant levels of pre-ischemic lactate (1 mM) reduce contractile, cellular, and mitochondrial recovery during reperfusion compared to physiologic lactate levels. Inhibition of mitochondrial calcium uptake during early reperfusion improves the post-ischemic recovery of 1 Lac hearts, indicating calcium overload as a potential therapeutic reperfusion target for DCD hearts.

The blood oxygen level dependent (BOLD) effect of in-vitro myoglobin and hemoglobin.

The presence of deoxygenated hemoglobin (Hb) results in a drop in T2 and T2* in magnetic resonance imaging (MRI), known as the blood oxygenation level-dependent (BOLD-)effect. The purpose of this study was to investigate if deoxygenated myoglobin (Mb) exerts a BOLD-like effect. Equine Met-Mb powder was dissolved and converted to oxygenated Mb. T1, T2, T2*-maps and BOLD-bSSFP images at 3Tesla were used to scan 22 Mb samples and 12 Hb samples at room air, deoxygenation, reoxygenation and after chemical reduction. In Mb, T2 and T2* mapping showed a significant decrease after deoxygenation (- 25% and - 12%, p < 0.01), increase after subsequent reoxygenation (+ 17% and 0% vs. room air, p < 0.01), and finally a decrease in T2 after chemical reduction (- 28%, p < 0.01). An opposite trend was observed with T1 for each stage, while chemical reduction reduced BOLD-bSSFP signal (- 3%, p < 0.01). Similar deflections were seen at oxygenation changes in Hb. The T1 changes suggests that the oxygen content has been changed in the specimen. The shortening of transverse relaxation times in T2 and T2*-mapping after deoxygenation in Mb specimens are highly indicative of a BOLD-like effect.

Cardiac Graft Assessment in the Era of Machine Perfusion: Current and Future Biomarkers.

Heart transplantation remains the treatment of reference for patients experiencing end-stage heart failure; unfortunately, graft availability through conventional donation after brain death is insufficient to meet the demand. Use of extended-criteria donors or donation after circulatory death has emerged to increase organ availability; however, clinical protocols require optimization to limit or prevent damage in hearts possessing greater susceptibility to injury than conventional grafts. The emergence of cardiac ex situ machine perfusion not only facilitates the use of extended-criteria donor and donation after circulatory death hearts through the avoidance of potentially damaging ischemia during graft storage and transport, it also opens the door to multiple opportunities for more sensitive monitoring of graft quality. With this review, we aim to bring together the current knowledge of biomarkers that hold particular promise for cardiac graft evaluation to improve precision and reliability in the identification of hearts for transplantation, thereby facilitating the safe increase in graft availability. Information about the utility of potential biomarkers was categorized into 5 themes: (1) functional, (2) metabolic, (3) hormone/prohormone, (4) cellular damage/death, and (5) inflammatory markers. Several promising biomarkers are identified, and recommendations for potential improvements to current clinical protocols are provided.

Hypothermic, oxygenated perfusion (HOPE) provides cardioprotection via succinate oxidation prior to normothermic perfusion in a rat model of donation after circulatory death (DCD).

In donation after circulatory death (DCD), cardiac grafts are subjected to warm ischemia in situ, prior to a brief period of cold, static storage (CSS) at procurement, and ex situ, normothermic, machine perfusion (NMP) for transport and graft evaluation. Cold ischemia and normothermic reoxygenation during NMP could aggravate graft injury through continued accumulation and oxidation, respectively, of mitochondrial succinate, and the resultant oxidative stress. We hypothesized that replacing CSS with hypothermic, oxygenated perfusion (HOPE) could provide cardioprotection by reducing cardiac succinate levels before NMP. DCD was simulated in male Wistar rats. Following 21 minutes in situ ischemia, explanted hearts underwent 30 minutes hypothermic storage with 1 of the following: (1) CSS, (2) HOPE, (3) hypothermic deoxygenated perfusion (HNPE), or (4) HOPE + AA5 (succinate dehydrogenase inhibitor) followed by normothermic reperfusion to measure cardiac and metabolic recovery. After hypothermic storage, tissue ATP/ADP levels were higher and succinate concentration was lower in HOPE vs CSS, HNPE, and HOPE + AA5 hearts. After 60 minutes reperfusion, cardiac function was increased and cellular injury was decreased in HOPE compared with CSS, HNPE, and HOPE + AA5 hearts. HOPE provides improved cardioprotection via succinate oxidation prior to normothermic reperfusion compared with CSS, and therefore is a promising strategy for preservation of cardiac grafts obtained with DCD.

Circulating extracellular vesicles as non-invasive biomarker of rejection in heart transplant.

BACKGROUND: Circulating extracellular vesicles (EVs) are raising considerable interest as a non-invasive diagnostic tool, as they are easily detectable in biologic fluids and contain a specific set of nucleic acids, proteins, and lipids reflecting pathophysiologic conditions. We aimed to investigate differences in plasma-derived EV surface protein profiles as a biomarker to be used in combination with endomyocardial biopsies (EMBs) for the diagnosis of allograft rejection. METHODS: Plasma was collected from 90 patients (53 training cohort, 37 validation cohort) before EMB. EV concentration was assessed by nanoparticle tracking analysis. EV surface antigens were measured using a multiplex flow cytometry assay composed of 37 fluorescently labeled capture bead populations coated with specific antibodies directed against respective EV surface epitopes. RESULTS: The concentration of EVs was significantly increased and their diameter decreased in patients undergoing rejection as compared with negative ones. The trend was highly significant for both antibody-mediated rejection and acute cellular rejection (p < 0.001). Among EV surface markers, CD3, CD2, ROR1, SSEA-4, human leukocyte antigen (HLA)-I, and CD41b were identified as discriminants between controls and acute cellular rejection, whereas HLA-II, CD326, CD19, CD25, CD20, ROR1, SSEA-4, HLA-I, and CD41b discriminated controls from patients with antibody-mediated rejection. Receiver operating characteristics curves confirmed a reliable diagnostic performance for each single marker (area under the curve range, 0.727-0.939). According to differential EV-marker expression, a diagnostic model was built and validated in an external cohort of patients. Our model was able to distinguish patients undergoing rejection from those without rejection. The accuracy at validation in an independent external cohort reached 86.5%. Its application for patient management has the potential to reduce the number of EMBs. Further studies in a higher number of patients are required to validate this approach for clinical purposes. CONCLUSIONS: Circulating EVs are highly promising as a new tool to characterize cardiac allograft rejection and to be complementary to EMB monitoring.

Inflammatory extracellular vesicles prompt heart dysfunction via TRL4-dependent NF-κB activation.

Background: After myocardial infarction, necrotic cardiomyocytes release damage-associated proteins that stimulate innate immune pathways and macrophage tissue infiltration, which drives inflammation and myocardial remodeling. Circulating inflammatory extracellular vesicles play a crucial role in the acute and chronic phases of ischemia, in terms of inflammatory progression. In this study, we hypothesize that the paracrine effect mediated by these vesicles induces direct cytotoxicity in cardiomyocytes. Thus, we examined whether reducing the generation of inflammatory vesicles within the first few hours after the ischemic event ameliorates cardiac outcome at short and long time points. Methods: Myocardial infarction was induced in rats that were previously injected intraperitoneally with a chemical inhibitor of extracellular-vesicle biogenesis. Heart global function was assessed by echocardiography performed at 7, 14 and 28 days after MI. Cardiac outcome was also evaluated by hemodynamic analysis at sacrifice. Cytotoxic effects of circulating EV were evaluated ex-vivo in a Langendorff, system by measuring the level of cardiac troponin I (cTnI) in the perfusate. Mechanisms undergoing cytotoxic effects of EV derived from pro-inflammatory macrophages (M1) were studied in-vitro in primary rat neonatal cardiomyocytes. Results: Inflammatory response following myocardial infarction dramatically increased the number of circulating extracellular vesicles carrying alarmins such as IL-1α, IL-1ß and Rantes. Reducing the boost in inflammatory vesicles during the acute phase of ischemia resulted in preserved left ventricular ejection fraction in vivo. Hemodynamic analysis confirmed functional recovery by displaying higher velocity of left ventricular relaxation and improved contractility. When added to the perfusate of isolated hearts, post-infarction circulating vesicles induced significantly more cell death in adult cardiomyocytes, as assessed by cTnI release, comparing to circulating vesicles isolated from healthy (non-infarcted) rats. In vitro inflammatory extracellular vesicles induce cell death by driving nuclear translocation of NF-κB into nuclei of cardiomyocytes. Conclusion: Our data suggest that targeting circulating extracellular vesicles during the acute phase of myocardial infarction may offer an effective therapeutic approach to preserve function of ischemic heart.

Mechanical Postconditioning Promotes Glucose Metabolism and AMPK Activity in Parallel with Improved Post-Ischemic Recovery in an Isolated Rat Heart Model of Donation after Circulatory Death.

Donation after circulatory death (DCD) could improve donor heart availability; however, warm ischemia-reperfusion injury raises concerns about graft quality. Mechanical postconditioning (MPC) may limit injury, but mechanisms remain incompletely characterized. Therefore, we investigated the roles of glucose metabolism and key signaling molecules in MPC using an isolated rat heart model of DCD. Hearts underwent 20 minutes perfusion, 30 minutes global ischemia, and 60 minutes reperfusion with or without MPC (two cycles: 30 seconds reperfusion-30 seconds ischemia). Despite identical perfusion conditions, MPC either significantly decreased (low recovery = LoR; 32 ± 5%; p < 0.05), or increased (high recovery = HiR; 59 ± 7%; p < 0.05) the recovery of left ventricular work compared with no MPC (47 ± 9%). Glucose uptake and glycolysis were increased in HiR vs. LoR hearts (p < 0.05), but glucose oxidation was unchanged. Furthermore, in HiR vs. LoR hearts, phosphorylation of raptor, a downstream target of AMPK, increased (p < 0.05), cytochrome c release (p < 0.05) decreased, and TNFα content tended to decrease. Increased glucose uptake and glycolysis, lower mitochondrial damage, and a trend towards decreased pro-inflammatory cytokines occurred specifically in HiR vs. LoR MPC hearts, which may result from greater AMPK activation. Thus, we identify endogenous cellular mechanisms that occur specifically with cardioprotective MPC, which could be elicited in the development of effective reperfusion strategies for DCD cardiac grafts.

Olanzapine-mediated cardiotoxicity is associated with altered energy metabolism in isolated rat hearts.

Olanzapine is an antipsychotic drug routinely used for the treatment of schizophrenia. Although the olanzapine treatment is associated with disturbed electrical heart activity, the exact mechanism underlying this severe adverse effect remains unclear. Recently, olanzapine administration was demonstrated to be associated with elevation of blood glucose and lower levels of free fatty acids. Therefore, we investigated the effect of acute olanzapine administration on pathways regulating the cardiac energy metabolism in an isolated heart. Electrical activity and contractile parameters were recorded in isolated, spontaneously beating, adult male rat hearts, perfused with either olanzapine (100 nmol/l) or the vehicle for 10 min. Regulation of key signalling molecules was evaluated by immunoblotting and ATP levels were measured spectrophotometrically. Olanzapine prolonged the QTc intervals and induced a higher number of premature ventricular beats. Furthermore, olanzapine significantly decreased the coronary flow, the rate-pressure product and the contractility (+dP/dt and -dP/dt). These changes were associated with an increased acetyl-CoA carboxylase phosphorylation and tissue ATP levels. We also found a trend for lower phosphorylation levels of Akt and its downstream products AS160, a key regulator of GLUT4 trafficking and glycogen synthase kinase­3ß in olanzapine­treated hearts when compared to vehicle-treated controls. These data should contribute to the elucidation of mechanisms that underlie the adverse cardiac effects of olanzapine.

Comparison of Experimental Rat Models in Donation After Circulatory Death (DCD): in-situ vs. ex-situ Ischemia.

Introduction: Donation after circulatory death (DCD) could substantially improve donor heart availability. However, warm ischemia prior to procurement is of particular concern for cardiac graft quality. We describe a rat model of DCD with in-situ ischemia in order to characterize the physiologic changes during the withdrawal period before graft procurement, to determine effects of cardioplegic graft storage, and to evaluate the post-ischemic cardiac recovery in comparison with an established ex-situ ischemia model. Methods: Following general anesthesia in male, Wistar rats (404 ± 24 g, n = 25), withdrawal of life-sustaining therapy was simulated by diaphragm transection. Hearts underwent no ischemia or 27 min in-situ ischemia and were explanted. Ex situ, hearts were subjected to a cardioplegic flush and 15 min cold storage or not, and 60 min reperfusion. Cardiac recovery was determined and compared to published results of an entirely ex-situ ischemia model (n = 18). Results: In donors, hearts were subjected to hypoxia and hemodynamic changes, as well as increased levels of circulating catecholamines and free fatty acids prior to circulatory arrest. Post-ischemic contractile recovery was significantly lower in the in-situ ischemia model compared to the ex-situ model, and the addition of cardioplegic storage improved developed pressure-heart rate product, but not cardiac output. Conclusion: The in-situ model provides insight into conditions to which the heart is exposed before procurement. Compared to an entirely ex-situ ischemia model, hearts of the in-situ model demonstrated a lower post-ischemic functional recovery, potentially due to systemic changes prior to ischemia, which are partially abrogated by cardioplegic graft storage.

Differential effects of ischemia/reperfusion on endothelial function and contractility in donation after circulatory death.

BACKGROUND: Donation after circulatory death (DCD) could significantly improve cardiac graft availability. However, DCD hearts undergo potentially deleterious warm ischemia/reperfusion (I/R). As endothelial damage is a key factor in cardiac I/R injury, we aimed to investigate the tolerance of cardiac and endothelial function after various durations of warm ischemia to improve the timing and choice of cardioprotective therapies. METHODS: Isolated, working rat hearts were perfused for 20 minutes aerobically, then underwent various periods of warm global ischemia and either 30 or 60 minutes of reperfusion. RESULTS: Compared with non-ischemic hearts, recovery of left ventricular work (heart rate-developed pressure product) was significantly reduced at 60 minutes of reperfusion with ≥27 minutes of ischemia (p <0.05 for all), but was unchanged after 21 or 24 minutes of ischemia. Markers of cell death and edema significantly increased with ≥27-minute ischemia compared with non-ischemic hearts (p <0.05 for all). Endothelial-dependent vasodilation was significantly impaired compared with non-ischemic hearts with ≥24 minutes of ischemia, whereas endothelial-independent vasodilation was impaired with ≥27 minutes of ischemia (p <0.05 for all). Furthermore, with ≥24 minutes of ischemia, superoxide production by nitric oxide synthase and peroxynitrite levels were significantly increased compared with non-ischemic hearts, suggesting endothelial nitric oxide synthase (eNOS) uncoupling (p <0.05 for both). CONCLUSIONS: The first signs of endothelial dysfunction after cardiac ischemia occur with less ischemia than cardiac functional alterations, and may result from increased eNOS uncoupling. Strategies aimed at improving eNOS coupling may thus help to optimize both endothelial and myocardial recovery, ultimately facilitating DCD heart transplantation.

Heart Transplantation With Donation After Circulatory Death.

Heart transplantation remains the preferred option for improving quality of life and survival for patients suffering from end-stage heart failure. Unfortunately, insufficient supply of cardiac grafts has become an obstacle. Increasing organ availability with donation after circulatory death (DCD) may be a promising option to overcome the organ shortage. Unlike conventional donation after brain death, DCD organs undergo a period of warm, global ischemia between circulatory arrest and graft procurement, which raises concerns for graft quality. Nonetheless, the potential of DCD heart transplantation is being reconsidered, after reports of more than 70 cases in Australia and the United Kingdom over the past 3 years. Ensuring optimal patient outcomes and generalized adoption of DCD in heart transplantation, however, requires further development of clinical protocols, which in turn require a better understanding of cardiac ischemia-reperfusion injury and the various possibilities to limit its adverse effects. Thus, we aim to provide an overview of the knowledge obtained with preclinical studies in animal models of DCD heart transplantation, to facilitate and promote the most effective and efficient advancement in preclinical research. A literature search of the PubMed database was performed to identify all relevant preclinical studies in DCD heart transplantation. Specific aspects relevant for DCD heart transplantation were analyzed, including animal models, graft procurement and storage conditions, cardioprotective approaches, and graft evaluation strategies. Several potential therapeutic strategies for optimizing graft quality are identified, and recommendations for further preclinical research are provided.

Mitochondrial integrity during early reperfusion in an isolated rat heart model of donation after circulatory death-consequences of ischemic duration.

BACKGROUND: Cardioprotection and graft evaluation after ischemia-reperfusion (IR) are essential in facilitating heart transplantation with donation after circulatory death. Given the key role of mitochondria in IR, we aimed to investigate the tolerance of cardiac mitochondria to warm, global ischemia and to determine the predictive value of early reperfusion mitochondria-related parameters for post-ischemic cardiac recovery. METHODS: Isolated, working rat hearts underwent 0, 21, 24, 27, 30, or 33 minutes of warm, global ischemia, followed by 60 minutes of reperfusion. Functional recovery (developed pressure × heart rate) was determined at 60 minutes of reperfusion, whereas mitochondrial integrity was measured at 10 minutes of reperfusion. RESULTS: Functional recovery at 60 minutes of reperfusion decreased with ≥ 27 minutes of ischemia vs no ischemia (n = 7-8/group; p < 0.01). Cytochrome c, succinate release, and mitochondrial Ca2+ content increased with ≥ 27 minutes of ischemia vs no ischemia (p < 0.05). Ischemia at ≥ 21 minutes decreased mitochondrial coupling, adenosine 5'-triphosphate content, mitochondrial Ca2+ retention capacity, and increased oxidative damage vs no ischemia (p < 0.05). Reactive oxygen species (ROS) from reverse electron transfer increased with 21 and 27 minutes of ischemia vs no ischemia and 33 minutes of ischemia (p < 0.05), whereas ROS from forward electron transfer increased only with 33 minutes of ischemia vs no ischemia (p < 0.05). Mitochondrial coupling and adenosine 5'-triphosphate content correlated positively and cytochrome c, succinate, oxidative damage, and mitochondrial Ca2+ content correlated negatively with cardiac functional recovery (p < 0.05). CONCLUSIONS: Mitochondrial dysfunction occurs with shorter periods of ischemia than cardiac dysfunction. Mitochondrial coupling, ROS emission from reverse electron transfer, and calcium retention are particularly sensitive to early reperfusion injury, reflecting potential targets for cardioprotection. Indicators of mitochondrial integrity may be of aid in evaluating suitability of donation after circulatory death grafts for transplantation.