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.

Metabolic Considerations in Direct Procurement and Perfusion Protocols with DCD Heart Transplantation.

Heart transplantation with donation after circulatory death (DCD) provides excellent patient outcomes and increases donor heart availability. However, unlike conventional grafts obtained through donation after brain death, DCD cardiac grafts are not only exposed to warm, unprotected ischemia, but also to a potentially damaging pre-ischemic phase after withdrawal of life-sustaining therapy (WLST). In this review, we aim to bring together knowledge about changes in cardiac energy metabolism and its regulation that occur in DCD donors during WLST, circulatory arrest, and following the onset of warm ischemia. Acute metabolic, hemodynamic, and biochemical changes in the DCD donor expose hearts to high circulating catecholamines, hypoxia, and warm ischemia, all of which can negatively impact the heart. Further metabolic changes and cellular damage occur with reperfusion. The altered energy substrate availability prior to organ procurement likely plays an important role in graft quality and post-ischemic cardiac recovery. These aspects should, therefore, be considered in clinical protocols, as well as in pre-clinical DCD models. Notably, interventions prior to graft procurement are limited for ethical reasons in DCD donors; thus, it is important to understand these mechanisms to optimize conditions during initial reperfusion in concert with graft evaluation and re-evaluation for the purpose of tailoring and adjusting therapies and ensuring optimal graft quality for transplantation.

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.

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.

Cardioprotective reperfusion strategies differentially affect mitochondria: Studies in an isolated rat heart model of donation after circulatory death (DCD).

Donation after circulatory death (DCD) holds great promise for improving cardiac graft availability; however, concerns persist regarding injury following warm ischemia, after donor circulatory arrest, and subsequent reperfusion. Application of preischemic treatments is limited for ethical reasons; thus, cardioprotective strategies applied at graft procurement (reperfusion) are of particular importance in optimizing graft quality. Given the key role of mitochondria in cardiac ischemia-reperfusion injury, we hypothesize that 3 reperfusion strategies-mild hypothermia, mechanical postconditioning, and hypoxia, when briefly applied at reperfusion onset-provoke mitochondrial changes that may underlie their cardioprotective effects. Using an isolated, working rat heart model of DCD, we demonstrate that all 3 strategies improve oxygen-consumption-cardiac-work coupling and increase tissue adenosine triphosphate content, in parallel with increased functional recovery. These reperfusion strategies, however, differentially affect mitochondria; mild hypothermia also increases phosphocreatine content, while mechanical postconditioning stimulates mitochondrial complex I activity and reduces cytochrome c release (marker of mitochondrial damage), whereas hypoxia upregulates the expression of peroxisome proliferator-activated receptor-gamma coactivator (regulator of mitochondrial biogenesis). Characterization of the role of mitochondria in cardioprotective reperfusion strategies should aid in the identification of new, mitochondrial-based therapeutic targets and the development of effective reperfusion strategies that could ultimately facilitate DCD heart transplantation.

Freedom SOLO-Associated Thrombocytopaenia is Valve-Dependent and Not Due to In Vitro Pseudothrombocytopaenia.

BACKGROUND: Use of the Freedom SOLO (FS) stentless aortic bioprosthesis is limited by a unique and as yet unexplained severe decrease in postoperative platelet count in the absence of FS-related excess bleeding or thromboembolism. We investigated whether anticoagulant-associated pseudothrombocytopaenia could explain this complication. METHODS: Thirty consecutive patients (mean age 75.4±7.7 years, 11 [36.7%] female) underwent elective aortic valve replacement (AVR) with either the stented bovine Mitroflow (MF, n=18) or the stentless bovine FS (n=12) aortic valve bioprostheses. Serial platelet counts were performed simultaneously with sampling tubes containing tripotassium (K3-)-EDTA, trisodium (Na3)-citrate, or novel alternative magnesium sulfate (MgSO4, ThromboExact™)-based anticoagulant, respectively. RESULTS: Postoperative platelet counts decreased compared with preoperative values in all patients (p<0.001), but were significantly lower in patients receiving FS compared to MF at all measurement time points until the end of observation (day 9). Lowest platelet counts were seen on the first postoperative day for MF (mean reduction: -41.5%) and on the second postoperative day for FS (mean reduction: -59.9%). Postoperative platelet counts did not correlate with any of the anticoagulants, thereby indicating no pseudothrombocytopaenia in the study population. There was no interaction between anticoagulant and type of valve. Only 1% of variance in platelet counts was caused by the anticoagulant, 46% by the day of measurement relative to baseline, and 20% was caused by the type of valve. CONCLUSIONS: The platelet-lowering effect in patients receiving the FS is valve-dependent and is not caused by systemic preanalytical (laboratory) measurement error such as anticoagulant-dependent pseudothrombocytaemia, particularly with EDTA and citrate.

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.

Circulating factors, in both donor and ex-vivo heart perfusion, correlate with heart recovery in a pig model of DCD.

BACKGROUND: Heart transplantation with donation after circulatory death and ex-situ heart perfusion offers excellent outcomes and increased transplantation rates. However, improved graft evaluation techniques are required to ensure effective utilization of grafts. Therefore, we investigated circulating factors, both in-situ and ex-situ, as potential biomarkers for cardiac graft quality. METHODS: Circulatory death was simulated in anesthetized male pigs with warm ischemic durations of 0, 10, 20, or 30 min. Hearts were explanted and underwent ex-situ perfusion for 3h in an unloaded mode, followed by left ventricular loading for 1h, to evaluate cardiac recovery (outcomes). Multiple donor blood and ex-situ perfusate samples were used for biomarker evaluation with either standard biochemical techniques or nuclear magnetic resonance spectroscopy. RESULTS: Circulating adrenaline, both in the donor and at 10 min ex-situ heart perfusion, negatively correlated with cardiac recovery (p <0.05 for all). We identified several new potential biomarkers for cardiac graft quality that can be measured rapidly and simultaneously with nuclear magnetic resonance spectroscopy. At multiple timepoints during unloaded ex-situ heart perfusion, perfusate levels of acetone, betaine, creatine, creatinine, fumarate, hypoxanthine, lactate, pyruvate and succinate (p <0.05 for all) significantly correlated with outcomes; the optimal timepoint being 60 min. CONCLUSIONS: In heart donation after circulatory death, circulating adrenaline levels are valuable for cardiac graft evaluation. Nuclear magnetic resonance spectroscopy is of particular interest, as it measures multiple metabolites in a short timeframe. Improved biomarkers may allow more precision and therefore better support clinical decisions about transplantation suitability.

Open- vs. closed-chest pig models of donation after circulatory death.

Background: During donation after circulatory death (DCD), cardiac grafts are exposed to potentially damaging conditions that can impact their quality and post-transplantation outcomes. In a clinical DCD setting, patients have closed chests in most cases, while many experimental models have used open-chest conditions. We therefore aimed to investigate and characterize differences in open- vs. closed-chest porcine models. Methods: Withdrawal of life-sustaining therapy (WLST) was simulated in anesthetized juvenile male pigs by stopping mechanical ventilation following the administration of a neuromuscular block. Functional warm ischemic time (fWIT) was defined to start when systolic arterial pressure was <50 mmHg. Hemodynamic changes and blood chemistry were analyzed. Two experimental groups were compared: (i) an open-chest group with sternotomy prior to WLST and (ii) a closed-chest group with sternotomy after fWIT. Results: Hemodynamic changes during the progression from WLST to fWIT were initiated by a rapid decline in blood oxygen saturation and a subsequent cardiovascular hyperdynamic (HD) period characterized by temporary elevations in heart rates and arterial pressures in both groups. Subsequently, heart rate and systolic arterial pressure decreased until fWIT was reached. Pigs in the open-chest group displayed a more rapid transition to the HD phase after WLST, with peak heart rate and peak rate-pressure product occurring significantly earlier. Furthermore, the HD phase duration tended to be shorter and less intense (lower peak rate-pressure product) in the open-chest group than in the closed-chest group. Discussion: Progression from WLST to fWIT was more rapid, and the hemodynamic changes tended to be less pronounced in the open-chest group than in the closed-chest group. Our findings support clear differences between open- and closed-chest models of DCD. Therefore, recommendations for clinical DCD protocols based on findings in open-chest models must be interpreted with care.

Mild hypothermia during global cardiac ischemia opens a window of opportunity to develop heart donation after cardiac death.

Although heart donation after cardiac death (DCD) could greatly improve graft availability, concerns regarding warm ischemic damage typically preclude transplantation. Improving tolerance to warm ischemia may thus open a window of opportunity for DCD hearts. We investigated the hypothesis that, compared with normothermia, mild hypothermia (32° C) initiated after ischemic onset improves cardiac functional recovery upon reperfusion. Isolated, working hearts from adult, male Wistar rats underwent global, no-flow ischemia, and reperfusion (n = 28). After ischemic onset, temperature was maintained at either 37° C for 20 or 30 min or reduced to 32° C for 40, 50, or 60 min. Recovery was measured after 60-min reperfusion. Following normothermic ischemia, recovery of rate-pressure product (RPP; per cent of preischemic value) was almost complete after 20-min ischemia (97 ± 9%), whereas no recovery was detectable after 30-min ischemia. After mildly hypothermic ischemia (32° C), RPP also recovered well after 40 min (86 ± 4%). Markers of metabolism and necrosis were similar in 37° C/20 min and 32° C/40 min groups. Simple reduction in cardiac temperature by a few degrees after the onset of global ischemia dramatically prolongs the interval during which the heart remains resistant to functional deterioration. Preservation of hemodynamic function is associated with improved metabolic recovery and reduced necrosis. The application of mild hypothermia may be a simple first step towards development of clinical protocols for DCD heart recovery.

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.].

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.

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.

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.

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.

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.

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.