- Email: [email protected]
High pre-ischemic fatty acid levels decrease cardiac recovery in an isolated rat heart model of donation after circulatory death
High Pre-ischemic fatty acid Levels decrease cardiac recovery in an isolated rat heart model of donation after circulatory death Petra Niederberger, Emilie Farine, Maria Arnold, Rahel K. Wyss, Maria Nieves Sanz, Natalia M´endez-Carmona, Brigitta Gahl, Georg M. Fiedler, Thierry P. Carrel, Hendrik T. Tevaearai Stahel, Sarah L. Longnus PII: DOI: Reference:
S0026-0495(17)30087-2 doi: 10.1016/j.metabol.2017.03.007 YMETA 53573
To appear in:
Metabolism
Received date: Accepted date:
1 December 2016 8 March 2017
Please cite this article as: Niederberger Petra, Farine Emilie, Arnold Maria, Wyss Rahel K., Sanz Maria Nieves, M´endez-Carmona Natalia, Gahl Brigitta, Fiedler Georg M., Carrel Thierry P., Tevaearai Stahel Hendrik T., Longnus Sarah L., High Pre-ischemic fatty acid Levels decrease cardiac recovery in an isolated rat heart model of donation after circulatory death, Metabolism (2017), doi: 10.1016/j.metabol.2017.03.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT HIGH PRE-ISCHEMIC FATTY ACID LEVELS DECREASE CARDIAC RECOVERY IN AN ISOLATED RAT HEART MODEL OF DONATION AFTER CIRCULATORY DEATH
RI P
T
Petra Niederberger*a, Emilie Farine*a, Maria Arnolda, Rahel K. Wyssa, Maria Nieves Sanza, Natalia Méndez-Carmonaa, Brigitta Gahla, Georg M. Fiedlerb, Thierry P. Carrela, Hendrik T. Tevaearai Stahela, Sarah L. Longnusa *equally contributed a
SC
Clinic for Cardiovascular Surgery, Inselspital, Bern University Hospital and Department of Clinical Research, University of Bern, Bern, Switzerland b
MA NU
Center of Laboratory Medicine, University Institute of Clinical Chemistry, University Hospital, Inselspital, Bern, Switzerland
CE
PT
ED
E-mail addresses authors: PN: [email protected] EF: [email protected] MA: [email protected] RW: [email protected] MNS: [email protected] NMC: [email protected] BG: [email protected] GF: [email protected] TC: [email protected] HTS: [email protected] SL: [email protected]
AC
Correspondence to: Professor Hendrik T. Tevaearai Stahel Clinic for Cardiovascular Surgery, Inselspital, Bern University Hospital and University of Bern, Murtenstrasse 35, H808 CH-3008, Berne, Switzerland Telephone: +41(0)31 632 12 80; Fax: +41(0)31 632 97 66 Email: [email protected]
1
ACCEPTED MANUSCRIPT Abstract
T
Rationale: Donation after circulatory death (DCD) could improve cardiac graft availability. However, strategies to optimize cardiac graft recovery remain to be established in DCD; these hearts would be expected to be exposed to high levels of circulatory fat immediately prior to the inevitable period of ischemia prior to procurement.
RI P
Objective: We investigated whether acute exposure to high fat prior to warm, global ischemia affects subsequent hemodynamic and metabolic recovery in an isolated rat heart model of DCD.
MA NU
SC
Methods and Results: Hearts of male Wistar rats underwent 20 min baseline perfusion with glucose (11 mM) and either high fat (1.2 mM palmitate; HF) or no fat (NF), 27 min global ischemia (37°C), and 60 min reperfusion with glucose only (n=7-8 per group). Hemodynamic recovery was 50% lower in HF vs NF hearts (34±30% vs 78±8% (60 min reperfusion value of peak pressure*heart rate as percentage of mean baseline); p<0.01). During early reperfusion, glycolysis (0.3±0.3 vs 0.7±0.3 µmol*min-1*g dry-1, p<0.05), glucose oxidation (0.1±0.03 vs 0.4±0.2 µmol*min-1*g dry-1, p<0.01) and pyruvate dehydrogenase activity (1.8±0.6 vs 3.6±0.5 U*min-1*g protein-1, p<0.01) were significantly reduced in HF vs NF groups, respectively, while lactate release was significantly greater (1.8±0.9 vs 0.6±0.2 μmol*g wet-1*min-1; p<0.05).
ED
Conclusions: Acute, pre-ischemic exposure to high fat significantly lowers post-ischemic cardiac recovery vs no fat despite identical reperfusion conditions. These findings support the concept that oxidation of residual fatty acids is rapidly restored upon reperfusion and exacerbates ischemiareperfusion (IR) injury. Strategies to optimize post-ischemic cardiac recovery should take preischemic fat levels into consideration.
AC
CE
PT
Keywords: donation after circulatory death; heart transplantation, ischemia-reperfusion injury; circulating fatty acids, glucose metabolism, pyruvate dehydrogenase
2
ACCEPTED MANUSCRIPT
RI P SC
AC
CE
PT
ED
MA NU
BCA: bicinchoninic acid CF: coronary flow CO: cardiac output cyt c: cytochrome c DCD: donation after circulatory death GIK: glucose-insulin-potassium HF: high fat HR: heart rate IR: ischemia-reperfusion KHB: Krebs-Henseleit bicarbonate LDH: lactate dehydrogenase MI: myocardial infarction mPTP: mitochondrial permeability transition pore NADP: nicotinamide adenine dinucleotide phosphate NF: no fat PCA: perchloric acid PCr: phosphocreatine PDH: pyruvate dehydrogenase PDK: pyruvate dehydrogenase kinase PSP: peak systolic pressure ROS: reactive oxygen species RPP: rate pressure product TnT: troponin T
T
Non-standard Abbreviations and Acronyms
3
ACCEPTED MANUSCRIPT 1 Introduction
SC
RI P
T
Identification of therapeutic strategies to improve ischemic tolerance and/or reduce ischemiareperfusion (IR) injury should enable greater usage of a promising pool of cardiac grafts, those obtained with donation after circulatory death (DCD). DCD heart transplantation has been proposed as a potential solution to reduce the shortage of donor hearts for patients requiring transplantation, the gold standard treatment for patients with severe heart failure. One major concern for the use of cardiac grafts obtained with DCD arises from potential tissue injury caused by the inevitable period of warm ischemia between circulatory arrest and graft procurement1. However, recent reports of successful adult DCD heart transplantations in Australia and the UK demonstrate that cardiac DCD transplantation is feasible and the heart can withstand warm global ischemia for approximately 20-30 min2-5.
PT
ED
MA NU
Investigation into cardiac IR injury and identification of strategies to minimize associated cellular damage has been the subject of intense study for many years; however, the multiple mediators of IR injury and their complex interplay are not fully understood. Thus, despite many promising pre-clinical reports of effective cardioprotective reperfusion strategies, robust approaches are still lacking in clinical practice6-9. During global no-flow ischemia, anaerobic glycolysis of glycogen becomes the primary source of ATP, leading to an acidic environment through accumulation of lactate and protons10,11. Paradoxically, reperfusion is required to salvage ischemic tissue, yet it also leads to further damage, contributing up to 50% of myocardial injury12,13. Upon reperfusion, intracellular pH normalization occurs through removal of protons by Na+/H+ exchanger, which leads to increased intracellular Na+, activation of sarcolemmal Na+/Ca2+ exchangers in reverse mode, and further intracellular Ca2+ overload10,11,14. In addition, reenergization of mitochondria leads to an excessive production of reactive oxygen species (ROS)15. Together, these conditions (pH normalization, Ca2+ overload and ROS production) promote opening of the mitochondrial permeability transition pore (mPTP) in the first minutes of reperfusion, which may induce loss of the mitochondrial membrane potential and cell death13,14.
AC
CE
High levels of circulating fatty acids at reperfusion exacerbate cardiac IR injury. Oxidation of fatty acids is rapidly restored at the onset of reperfusion16, which leads to increased mitochondrial acetylCoA/CoA and NADH/NAD+ ratios, and the activation of pyruvate dehydrogenase kinase (PDK), which phosphorylates and inhibits pyruvate dehydrogenase (PDH)11,17,18. Inhibition of PDH, the rate limiting enzyme of glucose oxidation, results in reduced glucose oxidation rates and a greater uncoupling between glycolysis and glucose oxidation10,19. This uncoupling leads to further production of lactate and protons, thereby contributing to increased Ca2+ overload and IR injury10,17. Furthermore, exposure to high levels of fatty acids has been shown to increase ROS formation at reperfusion in isolated, perfused rat hearts20. In clinical settings, marked increases in circulating fatty acids occur in parallel with cardiac IR. For example, increased levels of circulating free fatty acids were observed in patients with myocardial infarction (MI)21 as well as in patients undergoing heart surgery (1.6-2.2 mmol/L of plasma free fatty acids) 22. This elevation in circulating fatty acid levels results from increased catecholamine levels 21,23 and/or heparin administration24. Correspondingly, increased circulating fat levels would be expected in the clinical setting of DCD. This concept is supported by the observation of White and colleagues who reported markedly increased catecholamine levels prior to cardiac arrest in a pig model of DCD25. Interestingly, preclinical studies suggest that high levels of circulating fatty acids prior to ischemia may also affect post-ischemic cardiac recovery. Indeed, studies in hearts from rats subjected to fasting, which results in elevated circulating fatty acid levels for several hours, have demonstrated both increases26,27 and decreases28 in post-ischemic hemodynamic recovery compared with fed rats. However, to our knowledge, no study has been performed to investigate the effects of a brief preischemic exposure to high circulating fat on post-ischemic cardiac recovery. Therefore, we determined whether acute exposure to high circulating fatty acid levels immediately prior to global ischemia (as would be expected in the clinical setting of DCD) affects hemodynamic and metabolic recovery in our isolated rat heart model of DCD. 4
ACCEPTED MANUSCRIPT 2 Methods 2.1 Materials
RI P
T
Albumin from bovine serum and palmitic acid were purchased from Sigma-Aldrich (Buchs, Switzerland). Unless otherwise stated, all other chemicals were obtained from Merck (Darmstadt, Germany).
2.2 Ethics Statement
MA NU
SC
All animal experimental procedures were performed in compliance with the European Convention for Animal care and approved by the Swiss animal welfare authorities and state veterinary office (Ethics Committee for Animal Experimentation (ECAE), Bern, Switzerland). All surgeries were performed under anesthesia and all efforts were made to minimize suffering.
2.3 Isolated Heart Preparation
PT
ED
An isolated working rat heart system was used to perfuse rat hearts ex vivo, as previously described29. Adult male Wistar rats (mean body weight 397±84 g), fed on a standard laboratory diet ad libitum, were anesthetized using 100 mg/kg of ketamine (Narketan®, Vetoquinol AG, Bern, Switzerland) and 10 mg/kg of xylazine (Xylapan®, Vetoquinol AG, Bern, Switzerland) via an intraperitoneal injection. Hearts were rapidly excised, the aorta cannulated and a retrograde / Langendorff perfusion was initiated with a modified Krebs-Henseleit bicarbonate (KHB) buffer containing: 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4 7H2O, 1.25 mM CaCl2 7H2O, 25 mM NaHCO3 and 11 mM glucose at a constant pressure of 60 mmHg. Time between excision and perfusion was <2 minutes for all hearts. Excess tissue was removed, the left atrium was cannulated, and the perfusion was switched to the loaded (working) mode.
2.4 Experimental Protocol
AC
CE
Hearts underwent a baseline perfusion in the aerobic working-mode with KHB buffer containing 11 mM glucose (as above) and either high fat (1.2 mM palmitate bound to 3% albumin; HF) or no fat (NF) for 20 min. As DCD organs are expected to be exposed to highly elevated circulating fatty acid levels, a concentration of 1.2 mM palmitate was chosen. Hearts were randomly assigned to experimental groups (HF or NF) and care was taken to distribute the experimental groups equally. During the baseline period, heart function was evaluated and hearts with a rate pressure product (RPP=peak systolic pressure * heart rate) below 20,000 mmHg*beats*min-1 were excluded. Hearts were then subjected to a global, no-flow, normothermic ischemia for 27 min by clamping perfusate lines and immersing hearts in a tissue bath containing energy-substrate–free KHB buffer bubbled with 95% N2/5% CO2. Hearts were maintained within a temperature range of 36.5 – 37.2°C during ischemia. 27 min of ischemia was chosen because it leads to an intermediate level of post-ischemic recovery. All hearts were then reperfused under identical conditions; with KHB buffer containing 11 mM glucose in a retrograde, unloaded mode with a constant pressure of 60 mmHg for 10 min, followed by working-mode perfusion for the remaining 50 min of the reperfusion period. Reperfusion with glucose only was chosen for its similarity to the clinical setting of cardiac DCD, in which the heart is often reperfused with a blood- and fat- free cardioplegic solution; however, the optimal composition for such a reperfusion solution remains to be established. Additional series of hearts were perfused identically, but harvested at either end of baseline, end of ischemia, or after 10 min reperfusion (Fig. 1). Hearts were maintained at 37.0°C throughout the experimental protocol and during aerobic perfusions, buffers were gassed with 95% O2/5% CO2. In working-mode, preload and mean afterload pressures were maintained at 11.5 mmHg and 80 mmHg, respectively.
5
ACCEPTED MANUSCRIPT 2.5 Hemodynamic parameters
RI P
T
Heart rate (HR), peak systolic pressure (PSP), RPP, coronary flow (CF), and cardiac output (CO) were assessed. PSP and HR were measured with a pressure transducer attached to the aortic line; perfusate flows in preload and afterload lines were measured using Transonic equipment (Transonic Systems Inc., Ithaca, NY, USA). These parameters were continuously recorded using a PowerLab data acquisition system (ADInstruments, Spechbach, Germany).
2.6 Oxygen consumption, and release of lactate, cytochrome c (cyt c) and necrosis markers
2.7 Glycolysis and glucose oxidation
MA NU
SC
Samples of coronary effluent and circulating buffer were taken at various time points: during the preischemic baseline perfusion at 0, 10 and 20 min; and during reperfusion at 0, 3, 5, 10, 20, 40, and 60 min. These samples were used for quantification of metabolic parameters and cell death markers. Oxygen consumption was determined using the Cobas b 123 blood-gas analyzer (Roche, Basel, Switzerland). Lactate accumulation was measured with a spectrophotometric kit (Sigma-Aldrich, Buchs, Switzerland) and cyt c release was assessed by an enzyme-linked immunosorbent assay (ELISA; Abcam, Cambridge, UK). Lactate dehydrogenase production (LDH) was measured using the Roche MODULAR P800 analyser (Roche Diagnostics Corp, Indianapolis, USA). Troponin T (TnT) production was assessed with the Roche MODULAR E170 analyser (Roche, Basel, Switzerland) and an electro-chemiluminescence immunoassay analyser (Roche, Basel, Switzerland).
AC
CE
PT
ED
Additional series of hearts were perfused to measure myocardial glycolysis and glucose oxidation rates during the first 10 min of reperfusion (Figure 1). To do so, hearts were perfused as described above, but reperfused with 11 mM radiolabelled ([U-14C]- and [5-3H]) glucose (PerkinElmer, Massachusetts, USA) in an air-tight perfusion system with a hyamine hydroxide CO2 trap. Buffer specific activity and hyamine samples were counted directly in Irgasafe Plus Scintillation Cocktail fluid (PerkinElmer, Massachusetts, USA) with a scintillation counter (2200CA TRI-CARB, Toplab, Rickenbach, Switzerland). Dissolved 14C was analyzed by adding the buffer sample into a sealed tube containing 9N H2SO4. The emerging gas was then trapped in a filter paper soaked in hyamine hydroxide. Glycolysis was analyzed by adding the buffer sample to an anion exchange column (Dowex 1x4 chloride form 200-400 mesh; Sigma-Aldrich, Buchs, Switzerland) to separate 3H2O from 3 H-glucose and 14C-glucose30. 2.8 Tissue glycogen
Glycogen content in heart tissue samples was determined as previously described31 using a spectrophotometric kit (Sigma-Aldrich, Buchs, Switzerland).
2.9 PDH activity Heart tissue was homogenized in a Dounce homogenizer and protein content was determined with a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Massachusetts, USA). PDH activity was then measured with an ELISA microplate assay kit (Abcam, Cambridge, UK). 1 U was defined as 1 mM NADH oxidized per min at room temperature.
2.10 ATP and Phosphocreatine (PCr) 100 mg of powdered ventricular tissue was resuspended in 500 µl perchloric acid (PCA) 7%, homogenized in a Dounce homogenizer, and neutralized with 1M KOH-KHCO3. 200 µl of sample was then added to 800 µl assay buffer containing 60 mM Tris (pH 7.5), 40 mM MgSO4, 2 mM glucose, 524 µM nicotinamide adenine dinucleotide phosphate (NADP) and 2 U glucose-6 phosphate dehydrogenase. The ATP assay was initiated by adding 2 U hexokinase and absorbance was measured 6
ACCEPTED MANUSCRIPT at 340 nm over 20 min. The PCr assay was initiated by the addition of 500 µM ADP and 4 U creatine kinase, and the absorbance measured at 340 nm over 1 hour.
2.11 Data analysis
RI P
T
Unless stated otherwise, values are reported as mean±SD. Data analysis was performed with Stata (version 12.0, StataCorp, College Station, Texas, USA).
MA NU
SC
Differences between experimental groups, in body weight, heart weight and baseline measurements were analysed using linear mixed models. Linear mixed model analyses were also used to investigate differences between the HF and NF groups for series of hearts stopped at different time points: at the end of baseline, at the end of ischemia and after 10 min reperfusion. All p values were two sided, adjusted for multiple comparisons (modified, sequential-rejective Bonferroni procedure32). Corrected p values are reported and considered statistically significant if p<0.05.
3 Results
A total of 52 hearts were included in this study. 9 hearts were excluded from the study because RPP was below 20,000 mmHg*beats*min-1 during baseline perfusion.
ED
3.1 Baseline characteristics
PT
The number of hearts per group and baseline characteristics of perfusion series are presented in Table 1. No differences in baseline values between HF and NF hearts were observed.
3.2 Post-ischemic recovery
AC
CE
RPP was significantly lower in HF compared to NF hearts from 20 to 60 minutes of reperfusion (p0.01; Fig. 2a). Indeed, after 60 min of reperfusion, percent recovery of RPP was reduced by approximately half, 34% vs 78% respectively, in HF vs NF hearts (p0.01; Fig. 2a). PSP was significantly lower in HF vs NF hearts (p0.01; Fig. 2b) at 60 min reperfusion, whereas HR was not different (data not shown). CF was similar between groups during the first 10 min of reperfusion when the aortic pressure was maintained at 60 mmHg (Fig. 2c). Once hearts were switched to working mode, CF decreased by approximately 50% in HF vs NF hearts (p0.05). The pattern of oxygen consumption followed that of CF with an approximate 50% reduction in HF vs NF hearts at 60 min reperfusion (p0.01; Fig. 2d).
3.3 Glucose metabolism Several measurements of carbohydrate metabolism were performed during the first 10 min reperfusion when CF was not different between HF and NF groups. Both glycolysis and glucose oxidation rates were significantly lower in HF vs NF hearts at 10 min reperfusion (p<0.05 and p<0.01, respectively; Fig. 3a), while only glucose oxidation rates were significantly lower in HF vs NF at 5 min reperfusion (p<0.05; data not shown). PDH activity was significantly decreased at end-baseline, end-ischemia and 10 min reperfusion in HF compared to NF hearts (p<0.05, p<0.01 and p<0.01, respectively; Fig. 3b). In addition, PDH activity increased from end-ischemia to 10 min reperfusion in NF hearts (p<0.01; Fig. 3b). Lactate release was significantly greater during the first 10 min reperfusion in HF compared to NF hearts (p<0.01; Fig. 3c). Glycogen content was significantly higher in HF vs NF hearts at all time points (p<0.01), however, no difference in glycogen use during ischemia was observed between groups (Fig. 3d).
7
ACCEPTED MANUSCRIPT 3.4 ATP and PCr content
RI P
T
A significant decrease in ATP content after ischemia and an increase from end-ischemia to 10 min reperfusion was observed in both experimental groups (p<0.01; Table 2). ATP content at end-ischemia was significantly higher in HF compared to NF hearts (p<0.01), but ATP consumption during ischemia was not different between groups. A significant decrease in PCr content from end-baseline to end-ischemia and an increase from end-ischemia to 10 min reperfusion were observed in NF hearts (p<0.01; Table 2). PCr content was not different between HF and NF groups at any time point.
3.5 Cyt c and necrosis marker release
MA NU
SC
At 10 min reperfusion, cyt c release in HF hearts was significantly higher compared with both baseline values and release in NF hearts (p<0.01 and p<0.01, respectively; Fig. 4a). Cyt c release in NF hearts was similar before and after ischemia. TnT release was significantly higher in HF vs NF hearts at baseline (p<0.05) and 10 min reperfusion (p<0.01), and was significantly increased at 10 min reperfusion compared to baseline in HF hearts (p<0.05, Fig. 4b). Variability in LDH release was too high to detect differences between experimental groups (Fig. 4c).
4 Discussion
AC
CE
PT
ED
We demonstrate that acute exposure to high levels of circulating fatty acids immediately prior to global warm ischemia leads to a 50% reduction in post-ischemic hemodynamic recovery compared to no fat conditions in an isolated rat heart DCD model. This reduced hemodynamic recovery in HF hearts likely results from the rapid restoration of fatty acid metabolism at reperfusion onset and its subsequent inhibition of glucose metabolism, which is recognized to be detrimental to post-ischemic hemodynamic recovery19,28,33-35. Indeed, during early reperfusion, rates of glycolysis and glucose oxidation were lower, lactate release was higher and PDH activity was reduced in HF compared to NF hearts. Taken together, these results indicate that acute exposure to elevated circulating fatty acids prior to global warm ischemia is sufficient to induce inhibition of glucose oxidation at the level of PDH during early reperfusion, despite identical reperfusion conditions, and thereby potentially exacerbate IR injury. Furthermore, the combination of pre-ischemic high fat exposure and reperfusion injury may stimulate mitochondrial damage, which is suggested in this study by the greater release of cyt c in HF vs NF hearts during early reperfusion. Thus, pre-ischemic energy substrate availability critically affects recovery after a period of warm ischemia and should therefore be considered in preclinical studies and clinical situations involving cardiac IR. Our findings demonstrate that acute pre-ischemic exposure to high levels of fatty acids results in reduced post-ischemic hemodynamic recovery. Indeed, RPP and PSP, but not HR, were reduced by approximately half in HF versus NF hearts after 60 min reperfusion under identical (no fat) reperfusion conditions. To our knowledge, these precise conditions have not previously been investigated. However, in a study of isolated rat hearts with similar conditions (1.2 mM palmitate before global ischemia and no fat during reperfusion) but in the presence of 500 microunits/ml insulin, hearts demonstrated a slightly higher hemodynamic recovery compared to our HF hearts16. In addition, a small number of studies have investigated post-ischemic hemodynamic recovery following fasting, which leads to increased levels of circulating fat for several hours, rather than a brief exposure (20 min) as in this study. Liepinsh and colleagues reported lower hemodynamic recovery after 20 min regional ischemia in hearts from fasted rats perfused with high pre-ischemic fat (1.2 mM palmitate), compared with hearts of fed rats perfused with 0.3 mM palmitate28. Interestingly, Montessuit and colleagues demonstrated that perfusion of hearts from fasted and fed rats with 0.4 mM palmitate prior to 40 min no-flow ischemia, resulted in better hemodynamic recovery in fasted compared to fed rats27. Reasons for the contrasting findings between studies in fasted rats compared with ours may result from differences in experimental conditions, such as the differing exposure times to fat, differing fat concentrations, and the addition of insulin. 8
ACCEPTED MANUSCRIPT
SC
RI P
T
To our knowledge, this is the first report of acute exposure to high levels of fatty acids only prior to ischemia leading to an inhibition of glucose oxidation during early reperfusion. Despite identical postischemic experimental conditions, we observed lower rates of both glycolysis and glucose oxidation. Furthermore, lactate release was increased and PDH activity was decreased in HF hearts during early reperfusion, indicating a lower coupling between glycolysis and glucose oxidation (Fig. 5). Our results support the concept that fatty acid oxidation is rapidly restored at reperfusion, which leads to an inhibition of glucose metabolism, and particularly glucose oxidation via inhibition of PDH, causing an uncoupling of glycolysis and glucose oxidation. Rapid post-ischemic recovery of fatty acid oxidation and inhibition of glucose metabolism in the presence of circulating fat has been previously reported, and has been shown to be associated with reduced post-ischemic hemodynamic recovery19,28,35,36. Inhibition of glucose metabolism by fatty acids is believed to result mainly from inhibition of PDH, the rate-limiting enzyme of glucose oxidation, and to a lesser extent, through inhibition of glucose uptake and glycolysis (6-phosphofructo-1-kinase)18. The uncoupling of glycolysis and glucose oxidation during early reperfusion leads to further production of lactate and protons, which contribute to an increased Ca2+ overload leading to more severe IR injury16,19,36,37.
PT
ED
MA NU
We also demonstrate a significantly higher release of cyt c in HF vs NF hearts during early reperfusion, indicating greater mitochondrial damage. Early reperfusion injury is mediated by the rapid restoration of physiologic pH, intracellular Ca2+ overload and ROS production, which converge at the level of the mitochondria to promote opening of the mitochondrial permeability transition pore (mPTP), which, if sufficiently severe, induces loss of the mitochondrial membrane potential and cell death13. In our HF hearts, greater uncoupling of glycolysis and glucose oxidation would be expected to lead to a more severe Ca2+ overload, thereby promoting mPTP opening and greater mitochondrial damage. In addition, pre- and post- ischemic exposure to 1.5 mM palmitate has been shown to increase ROS production during the first minutes of reperfusion vs hearts perfused with no fat 20. Liepinsh and colleagues demonstrated that long chain acyl-CoA and acylcarnitine, intermediates of fatty acid oxidation, accumulate in the mitochondria during ischemia, and accumulated acylcarnitine results in inhibition of oxidative phosphorylation and stimulation of ROS production upon reperfusion38. Thus in our study, greater mitochondrial damage in HF hearts may occur as a result of greater Ca2+ overload and ROS production during early reperfusion.
AC
CE
In general, tissue content of ATP and PCr were not different between groups. Therefore, the potentially greater mitochondrial damage in HF hearts during early reperfusion does not appear to limit the generation of ATP and PCr in our isolated rat heart model. Although ATP content at the end of ischemia was significantly higher in HF vs. NF hearts, the net reduction in ATP during ischemia was not different between groups. Higher ATP content in HF hearts after ischemia may result from inhibitory effects of long chain fatty acyl CoA and long chain acylcarnitine esters on Na +/K+-ATPase during ischemia39. Indeed, ATPases, such as Na+/K+ ATPase, Ca2+ ATPases of the sarcolemma and sarcoplasmic reticulum consume the majority of ATP generated during ischemia40. Thus, inhibition of ATPases by fatty acid intermediates during ischemia could be a potential cause for higher levels of ATP in HF vs NF hearts after ischemia in our isolated rat heart model. In the context of DCD heart transplantation, cardioprotective strategies applied at the onset of reperfusion hold great potential for improving cardiac recovery. Although the application of cardioprotective interventions prior to circulatory arrest are limited in DCD for ethical reasons, preischemic carbohydrate treatment41,42 or intravenous glucose-insulin-potassium (GIK) administration43, which may promote a shift from fatty acid metabolism towards glucose metabolism, might be permitted as a reasonable strategy. Furthermore, despite much research attention and promising preclinical results, cardioprotective reperfusion therapies have not yet been convincingly demonstrated to improve clinical outcomes, in the setting of MI6,7,9,14; factors that interfere with cardioprotection should be reduced in DCD heart transplantation. Indeed, in the majority of clinical studies investigating reperfusion therapies in MI, patients mainly include the elderly with co-morbidities and/or concomitant therapies and timely administration of reperfusion therapies is not always achievable, which, taken together, could limit cardioprotective effects.7,8,44-46. In contrast, with DCD heart transplantation, donors will be young and healthy and timely administration of reperfusion therapies should generally be achievable. In addition, ex vivo graft perfusion, which is used in the 9
ACCEPTED MANUSCRIPT clinical setting of adult DCD heart transplantation2-5, could allow the control/manipulation of reperfusion conditions, such as the use of strategies to optimize energy substrate availability and use.
SC
RI P
T
Although cellular metabolism has long been recognized as a key mediator of cardiac injury, less is known about its precise role in reperfusion therapies. We recently reported that the efficacy of mechanical postconditioning can be altered by energy substrate availability47. We now demonstrate that pre-ischemic energy substrate availability, even when present for only brief periods, can dramatically influence hemodynamic recovery. Shifting energy substrate metabolism towards glucose oxidation and away from fatty acid oxidation at the time of procurement/ reperfusion is recognized to reduce IR injury19,33,35,37. Furthermore, administration of trimetazidine or ranolazine, two antianginal drugs that are believed to partially inhibit fatty acid oxidation and thereby shift energy metabolism towards glucose oxidation, have provided promising preclinical results48-51. These strategies, among others, that take cardiac energy metabolism into consideration should be of aid in the development of robust cardioprotective reperfusion strategies for DCD heart transplantation.
MA NU
4.1 Strengths and weaknesses
Our findings provide clear evidence that acute cardiac exposure to fatty acids for a brief period prior to ischemia substantially reduces hemodynamic recovery and inhibits glucose metabolism during early reperfusion. To our knowledge, this is the first study specifically designed to investigate effects of acute pre-ischemic high fat exposure on heart recovery and potential associated underlying mechanisms.
PT
ED
Nonetheless, several weaknesses exist. We gave priority to measurements of glycogen, ATP and PCr contents in tissue as well as lactate, cyt c and necrosis release in the coronary effluent, thus it was not possible to measure endogenous triglyceride content and additional mediators of IR such as ROS and Ca2+. In addition, further investigations to extend these studies with blood perfusion, larger animal models and longer reperfusion periods will be necessary to confirm the clinical relevance.
AC
CE
4.2 Conclusions Brief pre-ischemic exposure to high circulating fatty acid levels decreases hemodynamic recovery by approximately 50% compared with hearts not exposed to fat prior to ischemia, despite identical reperfusion conditions. Reduced hemodynamic recovery induced by pre-ischemic high fat conditions likely occurs through the rapid return of residual fatty acid oxidation upon reperfusion, which worsens reperfusion injury through increased Ca2+ overload following inhibition of glucose oxidation. In addition, pre-ischemic high fat conditions may lead to increased mitochondrial damage during early reperfusion. These findings indicate that pre-ischemic levels of circulating fatty acids should be taken into consideration for the development of cardioprotective strategies in pre-clinical models and clinical situations involving cardiac IR, such as DCD heart transplantation. 4.3 Summary of the translational potential of the messages in the paper In DCD heart transplantation, optimizing energy substrate metabolism at the time of procurement may well facilitate use of DCD cardiac grafts. Interventions to lower pre-ischemic circulating fat levels are limited due to ethical reasons, but administration of glucose and/or insulin prior to ischemia are potential strategies that might be permitted. Furthermore, administration of carnitine, trimetazidine, or ranolazine at the onset of reperfusion, could potentially shift energy metabolism away from fatty acid oxidation and towards glucose oxidation, and thereby reduce the detrimental effects of pre-ischemic high fat exposure. Importantly, ex vivo perfusion of DCD hearts used in the clinical setting, should allow/facilitate manipulation of glucose metabolism during early reperfusion.
10
ACCEPTED MANUSCRIPT 5 Acknowledgements
T
The authors would like to thank Mr. Sorin Ciocan and Mr. Adrian Segiser for technical support.
RI P
6 Author Contributions
SC
The authors PN, EF, HTS, and SL contributed to conception and research design, PN, EF, MA, RW, MNS, NMC, BG, GF and SL performed the experiments and data analysis, and all authors drafted, edited, revised and approved the final version of this manuscript.
MA NU
7 Sources of Funding
This work was supported by the Swiss National Science Foundation [310030_149730/1], the Ruth & Arthur Scherbarth Foundation, and the European Society of Cardiology. The funders had no role in study design, data collection and analysis, decision to publish, or in preparation of the manuscript.
8 Conflicts of Interest
AC
CE
PT
ED
None.
11
ACCEPTED MANUSCRIPT 9 References
7 8
9 10 11
12 13 14 15 16
17
18 19
20 21 22
T
RI P
SC
6
MA NU
5
ED
4
PT
3
CE
2
Longnus, S. L. et al. Heart transplantation with donation after circulatory determination of death. Nat. Rev. Cardiol. 11, 354-363 (2014). Dhital, K. K. et al. Adult heart transplantation with distant procurement and ex-vivo preservation of donor hearts after circulatory death: a case series. Lancet 385, 2585-2591 (2015). Messer, S. et al. Functional assessment and transplantation of the donor heart after circulatory death. J. Heart Lung Transplant. 35, 1443-1452 (2016). Leprince, P., Popov, A. F., Simon, A. R., Benk, C. & Siepe, M. Ex vivo perfusion of the heart with the use of the Organ Care System. Eur. J. Cardiothorac. Surg. 49, 1318-1320 (2016). Macdonald, P. S., Chew, H. C., Connellan, M. & Dhital, K. Extracorporeal heart perfusion before heart transplantation: the heart in a box. Curr. Opin. Organ Transplant. 10.1097/MOT.0000000000000309 (2016). Bulluck, H., Yellon, D. M. & Hausenloy, D. J. Reducing myocardial infarct size: challenges and future opportunities. Heart 102, 341-348 (2016). Downey, J. M. & Cohen, M. V. Why do we still not have cardioprotective drugs? Circ. J. 73, 1171-1177 (2009). Heusch, G. & Rassaf, T. Time to Give Up on Cardioprotection? A Critical Appraisal of Clinical Studies on Ischemic Pre-, Post-, and Remote Conditioning. Circ. Res. 119, 676-695 (2016). Kloner, R. A. Current state of clinical translation of cardioprotective agents for acute myocardial infarction. Circ. Res. 113, 451-463 (2013). Lopaschuk, G. D., Ussher, J. R., Folmes, C. D., Jaswal, J. S. & Stanley, W. C. Myocardial fatty acid metabolism in health and disease. Physiol. Rev. 90, 207-258 (2010). Stanley, W. C., Lopaschuk, G. D., Hall, J. L. & McCormack, J. G. Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions. Potential for pharmacological interventions. Cardiovasc. Res. 33, 243-257 (1997). Hausenloy, D. J. & Yellon, D. M. Ischaemic conditioning and reperfusion injury. Nat. Rev. Cardiol. 13, 193-209 (2016). Yellon, D. M. & Hausenloy, D. J. Myocardial reperfusion injury. N. Engl. J. Med. 357, 11211135 (2007). Hausenloy, D. J. & Yellon, D. M. Myocardial ischemia-reperfusion injury: a neglected therapeutic target. J. Clin. Invest. 123, 92-100 (2013). Dhalla, N. S. & Duhamel, T. A. The paradoxes of reperfusion in the ischemic heart. Heart Metab. 37, 31-34 (2007). Saddik, M. & Lopaschuk, G. D. Myocardial triglyceride turnover during reperfusion of isolated rat hearts subjected to a transient period of global ischemia. J. Biol. Chem. 267, 38253831 (1992). Fukushima, A., Milner, K., Gupta, A. & Lopaschuk, G. D. Myocardial Energy Substrate Metabolism in Heart Failure : from Pathways to Therapeutic Targets. Curr. Pharm. Des. 21, 3654-3664 (2015). Hue, L. & Taegtmeyer, H. The Randle cycle revisited: a new head for an old hat. Am. J. Physiol. Endocrinol. Metab. 297, E578-591 (2009). Lopaschuk, G. D., Wambolt, R. B. & Barr, R. L. An imbalance between glycolysis and glucose oxidation is a possible explanation for the detrimental effects of high levels of fatty acids during aerobic reperfusion of ischemic hearts. J. Pharmacol. Exp. Ther. 264, 135-144 (1993). Gambert, S. et al. Adverse effects of free fatty acid associated with increased oxidative stress in postischemic isolated rat hearts. Mol. Cell. Biochem. 283, 147-152 (2006). Oliver, M. F. Fatty acids and the risk of death during acute myocardial ischaemia. Clin. Sci. 128, 349-355 (2015). Lopaschuk, G. D. et al. Plasma fatty acid levels in infants and adults after myocardial ischemia. Am. Heart J. 128, 61-67 (1994).
AC
1
12
ACCEPTED MANUSCRIPT
29 30 31
32 33 34
35
36
37 38 39 40 41
42 43
44
T
RI P
SC
28
MA NU
27
ED
26
PT
25
CE
24
Spitzer, J. J. & Gold, M. Effect of catecholamines on the individual free fatty acids of plasma. Proc. Soc. Exp. Biol. Med. 110, 645-647 (1962). Grossman, M. I., Palm, L., Becker, G. H. & Moeller, H. C. Effect of lipemia and heparin on free fatty acid content of rat plasma. Proc. Soc. Exp. Biol. Med. 87, 312-315 (1954). White, C. W. et al. Physiologic Changes in the Heart Following Cessation of Mechanical Ventilation in a Porcine Model of Donation After Circulatory Death: Implications for Cardiac Transplantation. Am. J. Transplant. 16, 783-793 (2016). Doenst, T., Guthrie, P. H., Chemnitius, J. M., Zech, R. & Taegtmeyer, H. Fasting, lactate, and insulin improve ischemia tolerance in rat heart: a comparison with ischemic preconditioning. Am. J. Physiol. 270, H1607-1615 (1996). Montessuit, C., Papageorgiou, I., Tardy, I. & Lerch, R. Effect of nutritional state on substrate metabolism and contractile function in postischemic rat myocardium. Am. J. Physiol. 271, H2060-2070 (1996). Liepinsh, E. et al. The heart is better protected against myocardial infarction in the fed state compared to the fasted state. Metab. Clin. Exp. 63, 127-136 (2014). Stadelmann, M. et al. Mild hypothermia during global cardiac ischemia opens a window of opportunity to develop heart donation after cardiac death. Transpl. Int. 26, 339-348 (2013). Barr, R. L. & Lopaschuk, G. D. Direct measurement of energy metabolism in the isolated working rat heart. J. Pharmacol. Toxicol. Methods. 38, 11-17 (1997). Longnus, S. L., Wambolt, R. B., Parsons, H. L., Brownsey, R. W. & Allard, M. F. 5Aminoimidazole-4-carboxamide 1-beta -D-ribofuranoside (AICAR) stimulates myocardial glycogenolysis by allosteric mechanisms. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284, R936-944 (2003). Holland, B. & Copenhaver, M. An improved sequentially rejective Bonferroni test procedure. Biometrics 43, 417-423 (1987). Lopaschuk, G. D. & Saddik, M. The relative contribution of glucose and fatty acids to ATP production in hearts reperfused following ischemia. Mol. Cell. Biochem. 116, 111-116 (1992). Lopaschuk, G. D., Spafford, M. A., Davies, N. J. & Wall, S. R. Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia. Circ. Res. 66, 546-553 (1990). Taniguchi, M. et al. Dichloroacetate improves cardiac efficiency after ischemia independent of changes in mitochondrial proton leak. Am. J. Physiol. Heart and Circ. Physiol. 280, H17621769 (2001). Liu, Q., Docherty, J. C., Rendell, J. C., Clanachan, A. S. & Lopaschuk, G. D. High levels of fatty acids delay the recovery of intracellular pH and cardiac efficiency in post-ischemic hearts by inhibiting glucose oxidation. J. Am. Coll. Cardiol. 39, 718-725 (2002). Ussher, J. R. et al. Stimulation of glucose oxidation protects against acute myocardial infarction and reperfusion injury. Cardiovasc. Res. 94, 359-369 (2012). Liepinsh, E. et al. Long-chain acylcarnitines determine ischemia-reperfusion induced damage in heart mitochondria. Biochem. J. 10.1042/BCJ20160164 (2016). Hendrickson, S. C., St Louis, J. D., Lowe, J. E. & Abdel-aleem, S. Free fatty acid metabolism during myocardial ischemia and reperfusion. Mol. Cell. Biochem. 166, 85-94 (1997). Jennings, R. B. & Reimer, K. A. The cell biology of acute myocardial ischemia. Annu. Rev. Med. 42, 225-246 (1991). Awad, S., Stephens, F., Shannon, C. & Lobo, D. N. Perioperative perturbations in carnitine metabolism are attenuated by preoperative carbohydrate treatment: Another mechanism by which preoperative feeding may attenuate development of postoperative insulin resistance. Clin. Nutr. 31, 717-720 (2012). van Hoorn, D. E. et al. Preoperative feeding preserves heart function and decreases oxidative injury in rats. Nutrition 21, 859-866 (2005). Selker, H. P. et al. Very early administration of glucose-insulin-potassium by emergency medical service for acute coronary syndromes: Biological mechanisms for benefit in the IMMEDIATE Trial. Am. Heart J. 178, 168-175 (2016). Bell, R. M. & Yellon, D. M. Conditioning the whole heart--not just the cardiomyocyte. J. Mol. Cell. Cardiol. 53, 24-32 (2012).
AC
23
13
ACCEPTED MANUSCRIPT
51
T
RI P
SC
50
MA NU
49
ED
48
PT
47
CE
46
Fenton, R. A., Dickson, E. W., Meyer, T. E. & Dobson, J. G., Jr. Aging reduces the cardioprotective effect of ischemic preconditioning in the rat heart. J. Mol. Cell. Cardiol. 32, 1371-1375 (2000). Ferdinandy, P., Hausenloy, D. J., Heusch, G., Baxter, G. F. & Schulz, R. Interaction of risk factors, comorbidities, and comedications with ischemia/reperfusion injury and cardioprotection by preconditioning, postconditioning, and remote conditioning. Pharmacol. Rev. 66, 1142-1174 (2014). Bartkevics, M. et al. Efficacy of mechanical postconditioning following warm, global ischaemia depends on circulating fatty acid levels in an isolated, working rat heart modeldagger. Eur. J. Cardiothorac. Surg. 49, 32-39 (2016). Lionetti, V., Stanley, W. C. & Recchia, F. A. Modulating fatty acid oxidation in heart failure. Cardiovasc. Res. 90, 202-209 (2011). Liu, Z. et al. The protective effect of trimetazidine on myocardial ischemia/reperfusion injury through activating AMPK and ERK signaling pathway. Metab. Clin. Exp. 65,122-130 (2016). Lopaschuk, G. D., Barr, R., Thomas, P. D. & Dyck, J. R. Beneficial effects of trimetazidine in ex vivo working ischemic hearts are due to a stimulation of glucose oxidation secondary to inhibition of long-chain 3-ketoacyl coenzyme a thiolase. Circ. Res. 93, e33-37 (2003). McCormack, J. G., Barr, R. L., Wolff, A. A. & Lopaschuk, G. D. Ranolazine stimulates glucose oxidation in normoxic, ischemic, and reperfused ischemic rat hearts. Circulation 93, 135-142 (1996).
AC
45
14
ACCEPTED MANUSCRIPT
10 min rep
60 min rep
End-baseline
End-Ischemia
10 min rep
60 min rep
5 507±78 2.2±0.2
5 454±132 1.6±0.4
9 409±86 1.7±0.2
8 387±64 1.9±0.2
5 390±92 1.8±0.2
4 369±44 1.5±0.2
9 364±19 1.7±0.2
7 349±74 1.7±0.1
28.0±5.1
30.9±8.9
24.7±3.0
28.3±2.8
23.0±2.1
26.6±3.4
24.5±3.8
25.5±2.5
224±52 126±9 24±6 52±13
273±24 114±7 24±3 56±4
230±37 108±10 ND ND
252±28 113±4 23±3 53±9
200±44 119±16 23±4 41±5
235±34 114±7 24±5 49 ± 8
250±36 99±11 ND ND
241±39 107±9 26±2 50±10
4199±1022
5833±686
ND
4435±643
5005±589
5153±149
ND
5165±505
MA
NU S
CR I
End-Ischemia
D
Number of hearts (n) BW (g) HW (g) RPP (mmHg*beats*min-1*10-3) HR (beats*min-1) PSP (mmHg) CF (mL*min-1) CO (mL*min-1) O2 cons (mmHg*mL*min-1*g wet-1)
End-baseline
TE
Perfusion series
HF
PT
NF
AC
CE P
Table 1: Baseline characteristics in all perfusion series Mean baseline values calculated as mean of 10 and 20 min measurements for the various time points. BW: body weight; CF: coronary flow; CO: cardiac output; HF: high fat; HR: heart rate; HW: heart weight; ND: not determined; NF: no fat; O2 cons: oxygen consumption; PSP: peak systolic pressure; RPP: rate pressure product Data are expressed as mean ± SD.
15
ACCEPTED MANUSCRIPT
NF
HF
AC
CE
PT
ED
MA NU
SC
RI P
T
Perfusion EndEnd10 min rep EndEnd10 min rep series baseline ischemia baseline ischemia 42.6±22.8 9.8±1.5†† 22.7±6.9‡‡ 55.3±27.3 17.8±4.6**†† 27.5±6.9†‡ ATP (µmol *g dry-1) 92.4±24.4 41.3±4.5†† 106.3±38.7‡‡ 111.8±55.7 64.7±45.6 108.9±26.3 PCr (µmol *g dry-1) Table 2: ATP and PCr tissue content Mean tissue content of ATP and PCr measured in ventricular tissue of hearts perfused until different time points: end-baseline, end-ischemia, and 10 min rep. HF: high fat; NF: no fat; PCr: phosphocreatine Data are expressed as mean ± SD. ** p<0.01 vs NF, † p<0.05, †† p<0.01 vs corresponding end-baseline value; ‡ p<0.05, ‡‡ p<0.01 vs corresponding end-ischemia value
16
ACCEPTED MANUSCRIPT
T
Figure 1: Perfusion protocol Hearts underwent 20 min baseline working-mode perfusion with glucose (11 mM) and either high fat (1.2 mM palmitate; HF) or no fat (NF), followed by 27 min global ischemia (37°C) and 60 min glucose-only reperfusion (60 min rep). Additional series of hearts were stopped after baseline (endbaseline), ischemia (end-ischemia), and 10 min reperfusion (10 min rep).
HF
MA NU
NF
SC
RI P
Figure 2: Post-ischemic recovery Hemodynamic and metabolic parameters as a function of perfusion time for: a) Rate pressure product (RPP: peak systolic pressure * heart rate); b) Peak systolic pressure (PSP); c) Coronary flow (CF); d) Oxygen consumption. Percentages indicate post-ischemic recovery as the value at 60 min reperfusion expressed as a percentage of the mean-pre-ischemic value. Data are expressed as mean ± SD. * p<0.05, ** p<0.01 vs NF
HF
PT
NF
ED
Figure 3: Carbohydrate metabolism parameters during early reperfusion a) Glycolysis (GLY) and glucose oxidation (GOX) rates at 10 min reperfusion; b) Pyruvate dehydrogenase (PDH) activity measured in ventricular tissue of hearts stopped at various time points: end-baseline, end-ischemia, and 10 min rep; c) Lactate release measured in buffer samples during baseline perfusion and 5 and 10 min reperfusion; d) Glycogen content measured in ventricular tissue of hearts stopped at various time points: end-baseline, end-ischemia, and 10 min rep, Δ: glycogen use during ischemia. Data are expressed as mean ± SD. * p<0.05, ** p<0.01 vs NF; †† p<0.01 vs corresponding endbaseline value; ‡‡ p<0.01 vs corresponding end-ischemia value
HF
AC
NF
CE
Figure 4: Cyt c and necrosis marker release Release of cyt c, TnT, and LDH at end-baseline and 10 min reperfusion, measured in perfusate samples of hearts perfused until 60 min reperfusion. a) Cytochrome c (Cyt c) release; b) Troponin T (TnT) release; c) Lactate dehydrogenase (LDH) release; BDL: below detection limit (20 U/L) Data are expressed as mean ± SD. * p<0.05, ** p<0.01 vs NF; † p<0.05 vs corresponding end-baseline value
Figure 5: Proposed effect of acute pre-ischemic exposure to high levels of fatty acids. Acute pre-ischemic high fat (1.2 mM palmitate) leads to early reperfusion decreases in activity of pyruvate dehydrogenase (PDH) due to increased acetyl-CoA/CoA and NADH/NAD+ ratios, which cause a strong inhibition of glucose oxidation (GOX). In parallel, increased levels of cytosolic citrate, inhibit glycolysis (GLY). Together these effects lead to uncoupling of GLY/GOX and accumulation of lactate and H+, which worsens Ca2+ overload and exacerbates ischemia-reperfusion (I/R) injury.
17
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
Figure 1
18
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
Figure 2
19
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
Figure 3
20
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
Figure 4
21
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
Figure 5
22
ACCEPTED MANUSCRIPT Highlights Effects of pre-ischemic high fat were investigated in a rat heart model of DCD1
Acute pre-ischemic high fat exposure decreases graft hemodynamic recovery by 50%
Pre-ischemic high fat reduces cardiac glucose metabolism during early reperfusion
Pre-ischemic high fat lowers cardiac pyruvate dehydrogenase activity at reperfusion
Pre-ischemic fat may influence cardioprotective reperfusion approaches
AC
CE
PT
ED
MA NU
SC
RI P
T
1
Donation after circulatory death 23
S0026-0495(17)30087-2 doi: 10.1016/j.metabol.2017.03.007 YMETA 53573
To appear in:
Metabolism
Received date: Accepted date:
1 December 2016 8 March 2017
Please cite this article as: Niederberger Petra, Farine Emilie, Arnold Maria, Wyss Rahel K., Sanz Maria Nieves, M´endez-Carmona Natalia, Gahl Brigitta, Fiedler Georg M., Carrel Thierry P., Tevaearai Stahel Hendrik T., Longnus Sarah L., High Pre-ischemic fatty acid Levels decrease cardiac recovery in an isolated rat heart model of donation after circulatory death, Metabolism (2017), doi: 10.1016/j.metabol.2017.03.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT HIGH PRE-ISCHEMIC FATTY ACID LEVELS DECREASE CARDIAC RECOVERY IN AN ISOLATED RAT HEART MODEL OF DONATION AFTER CIRCULATORY DEATH
RI P
T
Petra Niederberger*a, Emilie Farine*a, Maria Arnolda, Rahel K. Wyssa, Maria Nieves Sanza, Natalia Méndez-Carmonaa, Brigitta Gahla, Georg M. Fiedlerb, Thierry P. Carrela, Hendrik T. Tevaearai Stahela, Sarah L. Longnusa *equally contributed a
SC
Clinic for Cardiovascular Surgery, Inselspital, Bern University Hospital and Department of Clinical Research, University of Bern, Bern, Switzerland b
MA NU
Center of Laboratory Medicine, University Institute of Clinical Chemistry, University Hospital, Inselspital, Bern, Switzerland
CE
PT
ED
E-mail addresses authors: PN: [email protected] EF: [email protected] MA: [email protected] RW: [email protected] MNS: [email protected] NMC: [email protected] BG: [email protected] GF: [email protected] TC: [email protected] HTS: [email protected] SL: [email protected]
AC
Correspondence to: Professor Hendrik T. Tevaearai Stahel Clinic for Cardiovascular Surgery, Inselspital, Bern University Hospital and University of Bern, Murtenstrasse 35, H808 CH-3008, Berne, Switzerland Telephone: +41(0)31 632 12 80; Fax: +41(0)31 632 97 66 Email: [email protected]
1
ACCEPTED MANUSCRIPT Abstract
T
Rationale: Donation after circulatory death (DCD) could improve cardiac graft availability. However, strategies to optimize cardiac graft recovery remain to be established in DCD; these hearts would be expected to be exposed to high levels of circulatory fat immediately prior to the inevitable period of ischemia prior to procurement.
RI P
Objective: We investigated whether acute exposure to high fat prior to warm, global ischemia affects subsequent hemodynamic and metabolic recovery in an isolated rat heart model of DCD.
MA NU
SC
Methods and Results: Hearts of male Wistar rats underwent 20 min baseline perfusion with glucose (11 mM) and either high fat (1.2 mM palmitate; HF) or no fat (NF), 27 min global ischemia (37°C), and 60 min reperfusion with glucose only (n=7-8 per group). Hemodynamic recovery was 50% lower in HF vs NF hearts (34±30% vs 78±8% (60 min reperfusion value of peak pressure*heart rate as percentage of mean baseline); p<0.01). During early reperfusion, glycolysis (0.3±0.3 vs 0.7±0.3 µmol*min-1*g dry-1, p<0.05), glucose oxidation (0.1±0.03 vs 0.4±0.2 µmol*min-1*g dry-1, p<0.01) and pyruvate dehydrogenase activity (1.8±0.6 vs 3.6±0.5 U*min-1*g protein-1, p<0.01) were significantly reduced in HF vs NF groups, respectively, while lactate release was significantly greater (1.8±0.9 vs 0.6±0.2 μmol*g wet-1*min-1; p<0.05).
ED
Conclusions: Acute, pre-ischemic exposure to high fat significantly lowers post-ischemic cardiac recovery vs no fat despite identical reperfusion conditions. These findings support the concept that oxidation of residual fatty acids is rapidly restored upon reperfusion and exacerbates ischemiareperfusion (IR) injury. Strategies to optimize post-ischemic cardiac recovery should take preischemic fat levels into consideration.
AC
CE
PT
Keywords: donation after circulatory death; heart transplantation, ischemia-reperfusion injury; circulating fatty acids, glucose metabolism, pyruvate dehydrogenase
2
ACCEPTED MANUSCRIPT
RI P SC
AC
CE
PT
ED
MA NU
BCA: bicinchoninic acid CF: coronary flow CO: cardiac output cyt c: cytochrome c DCD: donation after circulatory death GIK: glucose-insulin-potassium HF: high fat HR: heart rate IR: ischemia-reperfusion KHB: Krebs-Henseleit bicarbonate LDH: lactate dehydrogenase MI: myocardial infarction mPTP: mitochondrial permeability transition pore NADP: nicotinamide adenine dinucleotide phosphate NF: no fat PCA: perchloric acid PCr: phosphocreatine PDH: pyruvate dehydrogenase PDK: pyruvate dehydrogenase kinase PSP: peak systolic pressure ROS: reactive oxygen species RPP: rate pressure product TnT: troponin T
T
Non-standard Abbreviations and Acronyms
3
ACCEPTED MANUSCRIPT 1 Introduction
SC
RI P
T
Identification of therapeutic strategies to improve ischemic tolerance and/or reduce ischemiareperfusion (IR) injury should enable greater usage of a promising pool of cardiac grafts, those obtained with donation after circulatory death (DCD). DCD heart transplantation has been proposed as a potential solution to reduce the shortage of donor hearts for patients requiring transplantation, the gold standard treatment for patients with severe heart failure. One major concern for the use of cardiac grafts obtained with DCD arises from potential tissue injury caused by the inevitable period of warm ischemia between circulatory arrest and graft procurement1. However, recent reports of successful adult DCD heart transplantations in Australia and the UK demonstrate that cardiac DCD transplantation is feasible and the heart can withstand warm global ischemia for approximately 20-30 min2-5.
PT
ED
MA NU
Investigation into cardiac IR injury and identification of strategies to minimize associated cellular damage has been the subject of intense study for many years; however, the multiple mediators of IR injury and their complex interplay are not fully understood. Thus, despite many promising pre-clinical reports of effective cardioprotective reperfusion strategies, robust approaches are still lacking in clinical practice6-9. During global no-flow ischemia, anaerobic glycolysis of glycogen becomes the primary source of ATP, leading to an acidic environment through accumulation of lactate and protons10,11. Paradoxically, reperfusion is required to salvage ischemic tissue, yet it also leads to further damage, contributing up to 50% of myocardial injury12,13. Upon reperfusion, intracellular pH normalization occurs through removal of protons by Na+/H+ exchanger, which leads to increased intracellular Na+, activation of sarcolemmal Na+/Ca2+ exchangers in reverse mode, and further intracellular Ca2+ overload10,11,14. In addition, reenergization of mitochondria leads to an excessive production of reactive oxygen species (ROS)15. Together, these conditions (pH normalization, Ca2+ overload and ROS production) promote opening of the mitochondrial permeability transition pore (mPTP) in the first minutes of reperfusion, which may induce loss of the mitochondrial membrane potential and cell death13,14.
AC
CE
High levels of circulating fatty acids at reperfusion exacerbate cardiac IR injury. Oxidation of fatty acids is rapidly restored at the onset of reperfusion16, which leads to increased mitochondrial acetylCoA/CoA and NADH/NAD+ ratios, and the activation of pyruvate dehydrogenase kinase (PDK), which phosphorylates and inhibits pyruvate dehydrogenase (PDH)11,17,18. Inhibition of PDH, the rate limiting enzyme of glucose oxidation, results in reduced glucose oxidation rates and a greater uncoupling between glycolysis and glucose oxidation10,19. This uncoupling leads to further production of lactate and protons, thereby contributing to increased Ca2+ overload and IR injury10,17. Furthermore, exposure to high levels of fatty acids has been shown to increase ROS formation at reperfusion in isolated, perfused rat hearts20. In clinical settings, marked increases in circulating fatty acids occur in parallel with cardiac IR. For example, increased levels of circulating free fatty acids were observed in patients with myocardial infarction (MI)21 as well as in patients undergoing heart surgery (1.6-2.2 mmol/L of plasma free fatty acids) 22. This elevation in circulating fatty acid levels results from increased catecholamine levels 21,23 and/or heparin administration24. Correspondingly, increased circulating fat levels would be expected in the clinical setting of DCD. This concept is supported by the observation of White and colleagues who reported markedly increased catecholamine levels prior to cardiac arrest in a pig model of DCD25. Interestingly, preclinical studies suggest that high levels of circulating fatty acids prior to ischemia may also affect post-ischemic cardiac recovery. Indeed, studies in hearts from rats subjected to fasting, which results in elevated circulating fatty acid levels for several hours, have demonstrated both increases26,27 and decreases28 in post-ischemic hemodynamic recovery compared with fed rats. However, to our knowledge, no study has been performed to investigate the effects of a brief preischemic exposure to high circulating fat on post-ischemic cardiac recovery. Therefore, we determined whether acute exposure to high circulating fatty acid levels immediately prior to global ischemia (as would be expected in the clinical setting of DCD) affects hemodynamic and metabolic recovery in our isolated rat heart model of DCD. 4
ACCEPTED MANUSCRIPT 2 Methods 2.1 Materials
RI P
T
Albumin from bovine serum and palmitic acid were purchased from Sigma-Aldrich (Buchs, Switzerland). Unless otherwise stated, all other chemicals were obtained from Merck (Darmstadt, Germany).
2.2 Ethics Statement
MA NU
SC
All animal experimental procedures were performed in compliance with the European Convention for Animal care and approved by the Swiss animal welfare authorities and state veterinary office (Ethics Committee for Animal Experimentation (ECAE), Bern, Switzerland). All surgeries were performed under anesthesia and all efforts were made to minimize suffering.
2.3 Isolated Heart Preparation
PT
ED
An isolated working rat heart system was used to perfuse rat hearts ex vivo, as previously described29. Adult male Wistar rats (mean body weight 397±84 g), fed on a standard laboratory diet ad libitum, were anesthetized using 100 mg/kg of ketamine (Narketan®, Vetoquinol AG, Bern, Switzerland) and 10 mg/kg of xylazine (Xylapan®, Vetoquinol AG, Bern, Switzerland) via an intraperitoneal injection. Hearts were rapidly excised, the aorta cannulated and a retrograde / Langendorff perfusion was initiated with a modified Krebs-Henseleit bicarbonate (KHB) buffer containing: 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4 7H2O, 1.25 mM CaCl2 7H2O, 25 mM NaHCO3 and 11 mM glucose at a constant pressure of 60 mmHg. Time between excision and perfusion was <2 minutes for all hearts. Excess tissue was removed, the left atrium was cannulated, and the perfusion was switched to the loaded (working) mode.
2.4 Experimental Protocol
AC
CE
Hearts underwent a baseline perfusion in the aerobic working-mode with KHB buffer containing 11 mM glucose (as above) and either high fat (1.2 mM palmitate bound to 3% albumin; HF) or no fat (NF) for 20 min. As DCD organs are expected to be exposed to highly elevated circulating fatty acid levels, a concentration of 1.2 mM palmitate was chosen. Hearts were randomly assigned to experimental groups (HF or NF) and care was taken to distribute the experimental groups equally. During the baseline period, heart function was evaluated and hearts with a rate pressure product (RPP=peak systolic pressure * heart rate) below 20,000 mmHg*beats*min-1 were excluded. Hearts were then subjected to a global, no-flow, normothermic ischemia for 27 min by clamping perfusate lines and immersing hearts in a tissue bath containing energy-substrate–free KHB buffer bubbled with 95% N2/5% CO2. Hearts were maintained within a temperature range of 36.5 – 37.2°C during ischemia. 27 min of ischemia was chosen because it leads to an intermediate level of post-ischemic recovery. All hearts were then reperfused under identical conditions; with KHB buffer containing 11 mM glucose in a retrograde, unloaded mode with a constant pressure of 60 mmHg for 10 min, followed by working-mode perfusion for the remaining 50 min of the reperfusion period. Reperfusion with glucose only was chosen for its similarity to the clinical setting of cardiac DCD, in which the heart is often reperfused with a blood- and fat- free cardioplegic solution; however, the optimal composition for such a reperfusion solution remains to be established. Additional series of hearts were perfused identically, but harvested at either end of baseline, end of ischemia, or after 10 min reperfusion (Fig. 1). Hearts were maintained at 37.0°C throughout the experimental protocol and during aerobic perfusions, buffers were gassed with 95% O2/5% CO2. In working-mode, preload and mean afterload pressures were maintained at 11.5 mmHg and 80 mmHg, respectively.
5
ACCEPTED MANUSCRIPT 2.5 Hemodynamic parameters
RI P
T
Heart rate (HR), peak systolic pressure (PSP), RPP, coronary flow (CF), and cardiac output (CO) were assessed. PSP and HR were measured with a pressure transducer attached to the aortic line; perfusate flows in preload and afterload lines were measured using Transonic equipment (Transonic Systems Inc., Ithaca, NY, USA). These parameters were continuously recorded using a PowerLab data acquisition system (ADInstruments, Spechbach, Germany).
2.6 Oxygen consumption, and release of lactate, cytochrome c (cyt c) and necrosis markers
2.7 Glycolysis and glucose oxidation
MA NU
SC
Samples of coronary effluent and circulating buffer were taken at various time points: during the preischemic baseline perfusion at 0, 10 and 20 min; and during reperfusion at 0, 3, 5, 10, 20, 40, and 60 min. These samples were used for quantification of metabolic parameters and cell death markers. Oxygen consumption was determined using the Cobas b 123 blood-gas analyzer (Roche, Basel, Switzerland). Lactate accumulation was measured with a spectrophotometric kit (Sigma-Aldrich, Buchs, Switzerland) and cyt c release was assessed by an enzyme-linked immunosorbent assay (ELISA; Abcam, Cambridge, UK). Lactate dehydrogenase production (LDH) was measured using the Roche MODULAR P800 analyser (Roche Diagnostics Corp, Indianapolis, USA). Troponin T (TnT) production was assessed with the Roche MODULAR E170 analyser (Roche, Basel, Switzerland) and an electro-chemiluminescence immunoassay analyser (Roche, Basel, Switzerland).
AC
CE
PT
ED
Additional series of hearts were perfused to measure myocardial glycolysis and glucose oxidation rates during the first 10 min of reperfusion (Figure 1). To do so, hearts were perfused as described above, but reperfused with 11 mM radiolabelled ([U-14C]- and [5-3H]) glucose (PerkinElmer, Massachusetts, USA) in an air-tight perfusion system with a hyamine hydroxide CO2 trap. Buffer specific activity and hyamine samples were counted directly in Irgasafe Plus Scintillation Cocktail fluid (PerkinElmer, Massachusetts, USA) with a scintillation counter (2200CA TRI-CARB, Toplab, Rickenbach, Switzerland). Dissolved 14C was analyzed by adding the buffer sample into a sealed tube containing 9N H2SO4. The emerging gas was then trapped in a filter paper soaked in hyamine hydroxide. Glycolysis was analyzed by adding the buffer sample to an anion exchange column (Dowex 1x4 chloride form 200-400 mesh; Sigma-Aldrich, Buchs, Switzerland) to separate 3H2O from 3 H-glucose and 14C-glucose30. 2.8 Tissue glycogen
Glycogen content in heart tissue samples was determined as previously described31 using a spectrophotometric kit (Sigma-Aldrich, Buchs, Switzerland).
2.9 PDH activity Heart tissue was homogenized in a Dounce homogenizer and protein content was determined with a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific, Massachusetts, USA). PDH activity was then measured with an ELISA microplate assay kit (Abcam, Cambridge, UK). 1 U was defined as 1 mM NADH oxidized per min at room temperature.
2.10 ATP and Phosphocreatine (PCr) 100 mg of powdered ventricular tissue was resuspended in 500 µl perchloric acid (PCA) 7%, homogenized in a Dounce homogenizer, and neutralized with 1M KOH-KHCO3. 200 µl of sample was then added to 800 µl assay buffer containing 60 mM Tris (pH 7.5), 40 mM MgSO4, 2 mM glucose, 524 µM nicotinamide adenine dinucleotide phosphate (NADP) and 2 U glucose-6 phosphate dehydrogenase. The ATP assay was initiated by adding 2 U hexokinase and absorbance was measured 6
ACCEPTED MANUSCRIPT at 340 nm over 20 min. The PCr assay was initiated by the addition of 500 µM ADP and 4 U creatine kinase, and the absorbance measured at 340 nm over 1 hour.
2.11 Data analysis
RI P
T
Unless stated otherwise, values are reported as mean±SD. Data analysis was performed with Stata (version 12.0, StataCorp, College Station, Texas, USA).
MA NU
SC
Differences between experimental groups, in body weight, heart weight and baseline measurements were analysed using linear mixed models. Linear mixed model analyses were also used to investigate differences between the HF and NF groups for series of hearts stopped at different time points: at the end of baseline, at the end of ischemia and after 10 min reperfusion. All p values were two sided, adjusted for multiple comparisons (modified, sequential-rejective Bonferroni procedure32). Corrected p values are reported and considered statistically significant if p<0.05.
3 Results
A total of 52 hearts were included in this study. 9 hearts were excluded from the study because RPP was below 20,000 mmHg*beats*min-1 during baseline perfusion.
ED
3.1 Baseline characteristics
PT
The number of hearts per group and baseline characteristics of perfusion series are presented in Table 1. No differences in baseline values between HF and NF hearts were observed.
3.2 Post-ischemic recovery
AC
CE
RPP was significantly lower in HF compared to NF hearts from 20 to 60 minutes of reperfusion (p0.01; Fig. 2a). Indeed, after 60 min of reperfusion, percent recovery of RPP was reduced by approximately half, 34% vs 78% respectively, in HF vs NF hearts (p0.01; Fig. 2a). PSP was significantly lower in HF vs NF hearts (p0.01; Fig. 2b) at 60 min reperfusion, whereas HR was not different (data not shown). CF was similar between groups during the first 10 min of reperfusion when the aortic pressure was maintained at 60 mmHg (Fig. 2c). Once hearts were switched to working mode, CF decreased by approximately 50% in HF vs NF hearts (p0.05). The pattern of oxygen consumption followed that of CF with an approximate 50% reduction in HF vs NF hearts at 60 min reperfusion (p0.01; Fig. 2d).
3.3 Glucose metabolism Several measurements of carbohydrate metabolism were performed during the first 10 min reperfusion when CF was not different between HF and NF groups. Both glycolysis and glucose oxidation rates were significantly lower in HF vs NF hearts at 10 min reperfusion (p<0.05 and p<0.01, respectively; Fig. 3a), while only glucose oxidation rates were significantly lower in HF vs NF at 5 min reperfusion (p<0.05; data not shown). PDH activity was significantly decreased at end-baseline, end-ischemia and 10 min reperfusion in HF compared to NF hearts (p<0.05, p<0.01 and p<0.01, respectively; Fig. 3b). In addition, PDH activity increased from end-ischemia to 10 min reperfusion in NF hearts (p<0.01; Fig. 3b). Lactate release was significantly greater during the first 10 min reperfusion in HF compared to NF hearts (p<0.01; Fig. 3c). Glycogen content was significantly higher in HF vs NF hearts at all time points (p<0.01), however, no difference in glycogen use during ischemia was observed between groups (Fig. 3d).
7
ACCEPTED MANUSCRIPT 3.4 ATP and PCr content
RI P
T
A significant decrease in ATP content after ischemia and an increase from end-ischemia to 10 min reperfusion was observed in both experimental groups (p<0.01; Table 2). ATP content at end-ischemia was significantly higher in HF compared to NF hearts (p<0.01), but ATP consumption during ischemia was not different between groups. A significant decrease in PCr content from end-baseline to end-ischemia and an increase from end-ischemia to 10 min reperfusion were observed in NF hearts (p<0.01; Table 2). PCr content was not different between HF and NF groups at any time point.
3.5 Cyt c and necrosis marker release
MA NU
SC
At 10 min reperfusion, cyt c release in HF hearts was significantly higher compared with both baseline values and release in NF hearts (p<0.01 and p<0.01, respectively; Fig. 4a). Cyt c release in NF hearts was similar before and after ischemia. TnT release was significantly higher in HF vs NF hearts at baseline (p<0.05) and 10 min reperfusion (p<0.01), and was significantly increased at 10 min reperfusion compared to baseline in HF hearts (p<0.05, Fig. 4b). Variability in LDH release was too high to detect differences between experimental groups (Fig. 4c).
4 Discussion
AC
CE
PT
ED
We demonstrate that acute exposure to high levels of circulating fatty acids immediately prior to global warm ischemia leads to a 50% reduction in post-ischemic hemodynamic recovery compared to no fat conditions in an isolated rat heart DCD model. This reduced hemodynamic recovery in HF hearts likely results from the rapid restoration of fatty acid metabolism at reperfusion onset and its subsequent inhibition of glucose metabolism, which is recognized to be detrimental to post-ischemic hemodynamic recovery19,28,33-35. Indeed, during early reperfusion, rates of glycolysis and glucose oxidation were lower, lactate release was higher and PDH activity was reduced in HF compared to NF hearts. Taken together, these results indicate that acute exposure to elevated circulating fatty acids prior to global warm ischemia is sufficient to induce inhibition of glucose oxidation at the level of PDH during early reperfusion, despite identical reperfusion conditions, and thereby potentially exacerbate IR injury. Furthermore, the combination of pre-ischemic high fat exposure and reperfusion injury may stimulate mitochondrial damage, which is suggested in this study by the greater release of cyt c in HF vs NF hearts during early reperfusion. Thus, pre-ischemic energy substrate availability critically affects recovery after a period of warm ischemia and should therefore be considered in preclinical studies and clinical situations involving cardiac IR. Our findings demonstrate that acute pre-ischemic exposure to high levels of fatty acids results in reduced post-ischemic hemodynamic recovery. Indeed, RPP and PSP, but not HR, were reduced by approximately half in HF versus NF hearts after 60 min reperfusion under identical (no fat) reperfusion conditions. To our knowledge, these precise conditions have not previously been investigated. However, in a study of isolated rat hearts with similar conditions (1.2 mM palmitate before global ischemia and no fat during reperfusion) but in the presence of 500 microunits/ml insulin, hearts demonstrated a slightly higher hemodynamic recovery compared to our HF hearts16. In addition, a small number of studies have investigated post-ischemic hemodynamic recovery following fasting, which leads to increased levels of circulating fat for several hours, rather than a brief exposure (20 min) as in this study. Liepinsh and colleagues reported lower hemodynamic recovery after 20 min regional ischemia in hearts from fasted rats perfused with high pre-ischemic fat (1.2 mM palmitate), compared with hearts of fed rats perfused with 0.3 mM palmitate28. Interestingly, Montessuit and colleagues demonstrated that perfusion of hearts from fasted and fed rats with 0.4 mM palmitate prior to 40 min no-flow ischemia, resulted in better hemodynamic recovery in fasted compared to fed rats27. Reasons for the contrasting findings between studies in fasted rats compared with ours may result from differences in experimental conditions, such as the differing exposure times to fat, differing fat concentrations, and the addition of insulin. 8
ACCEPTED MANUSCRIPT
SC
RI P
T
To our knowledge, this is the first report of acute exposure to high levels of fatty acids only prior to ischemia leading to an inhibition of glucose oxidation during early reperfusion. Despite identical postischemic experimental conditions, we observed lower rates of both glycolysis and glucose oxidation. Furthermore, lactate release was increased and PDH activity was decreased in HF hearts during early reperfusion, indicating a lower coupling between glycolysis and glucose oxidation (Fig. 5). Our results support the concept that fatty acid oxidation is rapidly restored at reperfusion, which leads to an inhibition of glucose metabolism, and particularly glucose oxidation via inhibition of PDH, causing an uncoupling of glycolysis and glucose oxidation. Rapid post-ischemic recovery of fatty acid oxidation and inhibition of glucose metabolism in the presence of circulating fat has been previously reported, and has been shown to be associated with reduced post-ischemic hemodynamic recovery19,28,35,36. Inhibition of glucose metabolism by fatty acids is believed to result mainly from inhibition of PDH, the rate-limiting enzyme of glucose oxidation, and to a lesser extent, through inhibition of glucose uptake and glycolysis (6-phosphofructo-1-kinase)18. The uncoupling of glycolysis and glucose oxidation during early reperfusion leads to further production of lactate and protons, which contribute to an increased Ca2+ overload leading to more severe IR injury16,19,36,37.
PT
ED
MA NU
We also demonstrate a significantly higher release of cyt c in HF vs NF hearts during early reperfusion, indicating greater mitochondrial damage. Early reperfusion injury is mediated by the rapid restoration of physiologic pH, intracellular Ca2+ overload and ROS production, which converge at the level of the mitochondria to promote opening of the mitochondrial permeability transition pore (mPTP), which, if sufficiently severe, induces loss of the mitochondrial membrane potential and cell death13. In our HF hearts, greater uncoupling of glycolysis and glucose oxidation would be expected to lead to a more severe Ca2+ overload, thereby promoting mPTP opening and greater mitochondrial damage. In addition, pre- and post- ischemic exposure to 1.5 mM palmitate has been shown to increase ROS production during the first minutes of reperfusion vs hearts perfused with no fat 20. Liepinsh and colleagues demonstrated that long chain acyl-CoA and acylcarnitine, intermediates of fatty acid oxidation, accumulate in the mitochondria during ischemia, and accumulated acylcarnitine results in inhibition of oxidative phosphorylation and stimulation of ROS production upon reperfusion38. Thus in our study, greater mitochondrial damage in HF hearts may occur as a result of greater Ca2+ overload and ROS production during early reperfusion.
AC
CE
In general, tissue content of ATP and PCr were not different between groups. Therefore, the potentially greater mitochondrial damage in HF hearts during early reperfusion does not appear to limit the generation of ATP and PCr in our isolated rat heart model. Although ATP content at the end of ischemia was significantly higher in HF vs. NF hearts, the net reduction in ATP during ischemia was not different between groups. Higher ATP content in HF hearts after ischemia may result from inhibitory effects of long chain fatty acyl CoA and long chain acylcarnitine esters on Na +/K+-ATPase during ischemia39. Indeed, ATPases, such as Na+/K+ ATPase, Ca2+ ATPases of the sarcolemma and sarcoplasmic reticulum consume the majority of ATP generated during ischemia40. Thus, inhibition of ATPases by fatty acid intermediates during ischemia could be a potential cause for higher levels of ATP in HF vs NF hearts after ischemia in our isolated rat heart model. In the context of DCD heart transplantation, cardioprotective strategies applied at the onset of reperfusion hold great potential for improving cardiac recovery. Although the application of cardioprotective interventions prior to circulatory arrest are limited in DCD for ethical reasons, preischemic carbohydrate treatment41,42 or intravenous glucose-insulin-potassium (GIK) administration43, which may promote a shift from fatty acid metabolism towards glucose metabolism, might be permitted as a reasonable strategy. Furthermore, despite much research attention and promising preclinical results, cardioprotective reperfusion therapies have not yet been convincingly demonstrated to improve clinical outcomes, in the setting of MI6,7,9,14; factors that interfere with cardioprotection should be reduced in DCD heart transplantation. Indeed, in the majority of clinical studies investigating reperfusion therapies in MI, patients mainly include the elderly with co-morbidities and/or concomitant therapies and timely administration of reperfusion therapies is not always achievable, which, taken together, could limit cardioprotective effects.7,8,44-46. In contrast, with DCD heart transplantation, donors will be young and healthy and timely administration of reperfusion therapies should generally be achievable. In addition, ex vivo graft perfusion, which is used in the 9
ACCEPTED MANUSCRIPT clinical setting of adult DCD heart transplantation2-5, could allow the control/manipulation of reperfusion conditions, such as the use of strategies to optimize energy substrate availability and use.
SC
RI P
T
Although cellular metabolism has long been recognized as a key mediator of cardiac injury, less is known about its precise role in reperfusion therapies. We recently reported that the efficacy of mechanical postconditioning can be altered by energy substrate availability47. We now demonstrate that pre-ischemic energy substrate availability, even when present for only brief periods, can dramatically influence hemodynamic recovery. Shifting energy substrate metabolism towards glucose oxidation and away from fatty acid oxidation at the time of procurement/ reperfusion is recognized to reduce IR injury19,33,35,37. Furthermore, administration of trimetazidine or ranolazine, two antianginal drugs that are believed to partially inhibit fatty acid oxidation and thereby shift energy metabolism towards glucose oxidation, have provided promising preclinical results48-51. These strategies, among others, that take cardiac energy metabolism into consideration should be of aid in the development of robust cardioprotective reperfusion strategies for DCD heart transplantation.
MA NU
4.1 Strengths and weaknesses
Our findings provide clear evidence that acute cardiac exposure to fatty acids for a brief period prior to ischemia substantially reduces hemodynamic recovery and inhibits glucose metabolism during early reperfusion. To our knowledge, this is the first study specifically designed to investigate effects of acute pre-ischemic high fat exposure on heart recovery and potential associated underlying mechanisms.
PT
ED
Nonetheless, several weaknesses exist. We gave priority to measurements of glycogen, ATP and PCr contents in tissue as well as lactate, cyt c and necrosis release in the coronary effluent, thus it was not possible to measure endogenous triglyceride content and additional mediators of IR such as ROS and Ca2+. In addition, further investigations to extend these studies with blood perfusion, larger animal models and longer reperfusion periods will be necessary to confirm the clinical relevance.
AC
CE
4.2 Conclusions Brief pre-ischemic exposure to high circulating fatty acid levels decreases hemodynamic recovery by approximately 50% compared with hearts not exposed to fat prior to ischemia, despite identical reperfusion conditions. Reduced hemodynamic recovery induced by pre-ischemic high fat conditions likely occurs through the rapid return of residual fatty acid oxidation upon reperfusion, which worsens reperfusion injury through increased Ca2+ overload following inhibition of glucose oxidation. In addition, pre-ischemic high fat conditions may lead to increased mitochondrial damage during early reperfusion. These findings indicate that pre-ischemic levels of circulating fatty acids should be taken into consideration for the development of cardioprotective strategies in pre-clinical models and clinical situations involving cardiac IR, such as DCD heart transplantation. 4.3 Summary of the translational potential of the messages in the paper In DCD heart transplantation, optimizing energy substrate metabolism at the time of procurement may well facilitate use of DCD cardiac grafts. Interventions to lower pre-ischemic circulating fat levels are limited due to ethical reasons, but administration of glucose and/or insulin prior to ischemia are potential strategies that might be permitted. Furthermore, administration of carnitine, trimetazidine, or ranolazine at the onset of reperfusion, could potentially shift energy metabolism away from fatty acid oxidation and towards glucose oxidation, and thereby reduce the detrimental effects of pre-ischemic high fat exposure. Importantly, ex vivo perfusion of DCD hearts used in the clinical setting, should allow/facilitate manipulation of glucose metabolism during early reperfusion.
10
ACCEPTED MANUSCRIPT 5 Acknowledgements
T
The authors would like to thank Mr. Sorin Ciocan and Mr. Adrian Segiser for technical support.
RI P
6 Author Contributions
SC
The authors PN, EF, HTS, and SL contributed to conception and research design, PN, EF, MA, RW, MNS, NMC, BG, GF and SL performed the experiments and data analysis, and all authors drafted, edited, revised and approved the final version of this manuscript.
MA NU
7 Sources of Funding
This work was supported by the Swiss National Science Foundation [310030_149730/1], the Ruth & Arthur Scherbarth Foundation, and the European Society of Cardiology. The funders had no role in study design, data collection and analysis, decision to publish, or in preparation of the manuscript.
8 Conflicts of Interest
AC
CE
PT
ED
None.
11
ACCEPTED MANUSCRIPT 9 References
7 8
9 10 11
12 13 14 15 16
17
18 19
20 21 22
T
RI P
SC
6
MA NU
5
ED
4
PT
3
CE
2
Longnus, S. L. et al. Heart transplantation with donation after circulatory determination of death. Nat. Rev. Cardiol. 11, 354-363 (2014). Dhital, K. K. et al. Adult heart transplantation with distant procurement and ex-vivo preservation of donor hearts after circulatory death: a case series. Lancet 385, 2585-2591 (2015). Messer, S. et al. Functional assessment and transplantation of the donor heart after circulatory death. J. Heart Lung Transplant. 35, 1443-1452 (2016). Leprince, P., Popov, A. F., Simon, A. R., Benk, C. & Siepe, M. Ex vivo perfusion of the heart with the use of the Organ Care System. Eur. J. Cardiothorac. Surg. 49, 1318-1320 (2016). Macdonald, P. S., Chew, H. C., Connellan, M. & Dhital, K. Extracorporeal heart perfusion before heart transplantation: the heart in a box. Curr. Opin. Organ Transplant. 10.1097/MOT.0000000000000309 (2016). Bulluck, H., Yellon, D. M. & Hausenloy, D. J. Reducing myocardial infarct size: challenges and future opportunities. Heart 102, 341-348 (2016). Downey, J. M. & Cohen, M. V. Why do we still not have cardioprotective drugs? Circ. J. 73, 1171-1177 (2009). Heusch, G. & Rassaf, T. Time to Give Up on Cardioprotection? A Critical Appraisal of Clinical Studies on Ischemic Pre-, Post-, and Remote Conditioning. Circ. Res. 119, 676-695 (2016). Kloner, R. A. Current state of clinical translation of cardioprotective agents for acute myocardial infarction. Circ. Res. 113, 451-463 (2013). Lopaschuk, G. D., Ussher, J. R., Folmes, C. D., Jaswal, J. S. & Stanley, W. C. Myocardial fatty acid metabolism in health and disease. Physiol. Rev. 90, 207-258 (2010). Stanley, W. C., Lopaschuk, G. D., Hall, J. L. & McCormack, J. G. Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions. Potential for pharmacological interventions. Cardiovasc. Res. 33, 243-257 (1997). Hausenloy, D. J. & Yellon, D. M. Ischaemic conditioning and reperfusion injury. Nat. Rev. Cardiol. 13, 193-209 (2016). Yellon, D. M. & Hausenloy, D. J. Myocardial reperfusion injury. N. Engl. J. Med. 357, 11211135 (2007). Hausenloy, D. J. & Yellon, D. M. Myocardial ischemia-reperfusion injury: a neglected therapeutic target. J. Clin. Invest. 123, 92-100 (2013). Dhalla, N. S. & Duhamel, T. A. The paradoxes of reperfusion in the ischemic heart. Heart Metab. 37, 31-34 (2007). Saddik, M. & Lopaschuk, G. D. Myocardial triglyceride turnover during reperfusion of isolated rat hearts subjected to a transient period of global ischemia. J. Biol. Chem. 267, 38253831 (1992). Fukushima, A., Milner, K., Gupta, A. & Lopaschuk, G. D. Myocardial Energy Substrate Metabolism in Heart Failure : from Pathways to Therapeutic Targets. Curr. Pharm. Des. 21, 3654-3664 (2015). Hue, L. & Taegtmeyer, H. The Randle cycle revisited: a new head for an old hat. Am. J. Physiol. Endocrinol. Metab. 297, E578-591 (2009). Lopaschuk, G. D., Wambolt, R. B. & Barr, R. L. An imbalance between glycolysis and glucose oxidation is a possible explanation for the detrimental effects of high levels of fatty acids during aerobic reperfusion of ischemic hearts. J. Pharmacol. Exp. Ther. 264, 135-144 (1993). Gambert, S. et al. Adverse effects of free fatty acid associated with increased oxidative stress in postischemic isolated rat hearts. Mol. Cell. Biochem. 283, 147-152 (2006). Oliver, M. F. Fatty acids and the risk of death during acute myocardial ischaemia. Clin. Sci. 128, 349-355 (2015). Lopaschuk, G. D. et al. Plasma fatty acid levels in infants and adults after myocardial ischemia. Am. Heart J. 128, 61-67 (1994).
AC
1
12
ACCEPTED MANUSCRIPT
29 30 31
32 33 34
35
36
37 38 39 40 41
42 43
44
T
RI P
SC
28
MA NU
27
ED
26
PT
25
CE
24
Spitzer, J. J. & Gold, M. Effect of catecholamines on the individual free fatty acids of plasma. Proc. Soc. Exp. Biol. Med. 110, 645-647 (1962). Grossman, M. I., Palm, L., Becker, G. H. & Moeller, H. C. Effect of lipemia and heparin on free fatty acid content of rat plasma. Proc. Soc. Exp. Biol. Med. 87, 312-315 (1954). White, C. W. et al. Physiologic Changes in the Heart Following Cessation of Mechanical Ventilation in a Porcine Model of Donation After Circulatory Death: Implications for Cardiac Transplantation. Am. J. Transplant. 16, 783-793 (2016). Doenst, T., Guthrie, P. H., Chemnitius, J. M., Zech, R. & Taegtmeyer, H. Fasting, lactate, and insulin improve ischemia tolerance in rat heart: a comparison with ischemic preconditioning. Am. J. Physiol. 270, H1607-1615 (1996). Montessuit, C., Papageorgiou, I., Tardy, I. & Lerch, R. Effect of nutritional state on substrate metabolism and contractile function in postischemic rat myocardium. Am. J. Physiol. 271, H2060-2070 (1996). Liepinsh, E. et al. The heart is better protected against myocardial infarction in the fed state compared to the fasted state. Metab. Clin. Exp. 63, 127-136 (2014). Stadelmann, M. et al. Mild hypothermia during global cardiac ischemia opens a window of opportunity to develop heart donation after cardiac death. Transpl. Int. 26, 339-348 (2013). Barr, R. L. & Lopaschuk, G. D. Direct measurement of energy metabolism in the isolated working rat heart. J. Pharmacol. Toxicol. Methods. 38, 11-17 (1997). Longnus, S. L., Wambolt, R. B., Parsons, H. L., Brownsey, R. W. & Allard, M. F. 5Aminoimidazole-4-carboxamide 1-beta -D-ribofuranoside (AICAR) stimulates myocardial glycogenolysis by allosteric mechanisms. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284, R936-944 (2003). Holland, B. & Copenhaver, M. An improved sequentially rejective Bonferroni test procedure. Biometrics 43, 417-423 (1987). Lopaschuk, G. D. & Saddik, M. The relative contribution of glucose and fatty acids to ATP production in hearts reperfused following ischemia. Mol. Cell. Biochem. 116, 111-116 (1992). Lopaschuk, G. D., Spafford, M. A., Davies, N. J. & Wall, S. R. Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia. Circ. Res. 66, 546-553 (1990). Taniguchi, M. et al. Dichloroacetate improves cardiac efficiency after ischemia independent of changes in mitochondrial proton leak. Am. J. Physiol. Heart and Circ. Physiol. 280, H17621769 (2001). Liu, Q., Docherty, J. C., Rendell, J. C., Clanachan, A. S. & Lopaschuk, G. D. High levels of fatty acids delay the recovery of intracellular pH and cardiac efficiency in post-ischemic hearts by inhibiting glucose oxidation. J. Am. Coll. Cardiol. 39, 718-725 (2002). Ussher, J. R. et al. Stimulation of glucose oxidation protects against acute myocardial infarction and reperfusion injury. Cardiovasc. Res. 94, 359-369 (2012). Liepinsh, E. et al. Long-chain acylcarnitines determine ischemia-reperfusion induced damage in heart mitochondria. Biochem. J. 10.1042/BCJ20160164 (2016). Hendrickson, S. C., St Louis, J. D., Lowe, J. E. & Abdel-aleem, S. Free fatty acid metabolism during myocardial ischemia and reperfusion. Mol. Cell. Biochem. 166, 85-94 (1997). Jennings, R. B. & Reimer, K. A. The cell biology of acute myocardial ischemia. Annu. Rev. Med. 42, 225-246 (1991). Awad, S., Stephens, F., Shannon, C. & Lobo, D. N. Perioperative perturbations in carnitine metabolism are attenuated by preoperative carbohydrate treatment: Another mechanism by which preoperative feeding may attenuate development of postoperative insulin resistance. Clin. Nutr. 31, 717-720 (2012). van Hoorn, D. E. et al. Preoperative feeding preserves heart function and decreases oxidative injury in rats. Nutrition 21, 859-866 (2005). Selker, H. P. et al. Very early administration of glucose-insulin-potassium by emergency medical service for acute coronary syndromes: Biological mechanisms for benefit in the IMMEDIATE Trial. Am. Heart J. 178, 168-175 (2016). Bell, R. M. & Yellon, D. M. Conditioning the whole heart--not just the cardiomyocyte. J. Mol. Cell. Cardiol. 53, 24-32 (2012).
AC
23
13
ACCEPTED MANUSCRIPT
51
T
RI P
SC
50
MA NU
49
ED
48
PT
47
CE
46
Fenton, R. A., Dickson, E. W., Meyer, T. E. & Dobson, J. G., Jr. Aging reduces the cardioprotective effect of ischemic preconditioning in the rat heart. J. Mol. Cell. Cardiol. 32, 1371-1375 (2000). Ferdinandy, P., Hausenloy, D. J., Heusch, G., Baxter, G. F. & Schulz, R. Interaction of risk factors, comorbidities, and comedications with ischemia/reperfusion injury and cardioprotection by preconditioning, postconditioning, and remote conditioning. Pharmacol. Rev. 66, 1142-1174 (2014). Bartkevics, M. et al. Efficacy of mechanical postconditioning following warm, global ischaemia depends on circulating fatty acid levels in an isolated, working rat heart modeldagger. Eur. J. Cardiothorac. Surg. 49, 32-39 (2016). Lionetti, V., Stanley, W. C. & Recchia, F. A. Modulating fatty acid oxidation in heart failure. Cardiovasc. Res. 90, 202-209 (2011). Liu, Z. et al. The protective effect of trimetazidine on myocardial ischemia/reperfusion injury through activating AMPK and ERK signaling pathway. Metab. Clin. Exp. 65,122-130 (2016). Lopaschuk, G. D., Barr, R., Thomas, P. D. & Dyck, J. R. Beneficial effects of trimetazidine in ex vivo working ischemic hearts are due to a stimulation of glucose oxidation secondary to inhibition of long-chain 3-ketoacyl coenzyme a thiolase. Circ. Res. 93, e33-37 (2003). McCormack, J. G., Barr, R. L., Wolff, A. A. & Lopaschuk, G. D. Ranolazine stimulates glucose oxidation in normoxic, ischemic, and reperfused ischemic rat hearts. Circulation 93, 135-142 (1996).
AC
45
14
ACCEPTED MANUSCRIPT
10 min rep
60 min rep
End-baseline
End-Ischemia
10 min rep
60 min rep
5 507±78 2.2±0.2
5 454±132 1.6±0.4
9 409±86 1.7±0.2
8 387±64 1.9±0.2
5 390±92 1.8±0.2
4 369±44 1.5±0.2
9 364±19 1.7±0.2
7 349±74 1.7±0.1
28.0±5.1
30.9±8.9
24.7±3.0
28.3±2.8
23.0±2.1
26.6±3.4
24.5±3.8
25.5±2.5
224±52 126±9 24±6 52±13
273±24 114±7 24±3 56±4
230±37 108±10 ND ND
252±28 113±4 23±3 53±9
200±44 119±16 23±4 41±5
235±34 114±7 24±5 49 ± 8
250±36 99±11 ND ND
241±39 107±9 26±2 50±10
4199±1022
5833±686
ND
4435±643
5005±589
5153±149
ND
5165±505
MA
NU S
CR I
End-Ischemia
D
Number of hearts (n) BW (g) HW (g) RPP (mmHg*beats*min-1*10-3) HR (beats*min-1) PSP (mmHg) CF (mL*min-1) CO (mL*min-1) O2 cons (mmHg*mL*min-1*g wet-1)
End-baseline
TE
Perfusion series
HF
PT
NF
AC
CE P
Table 1: Baseline characteristics in all perfusion series Mean baseline values calculated as mean of 10 and 20 min measurements for the various time points. BW: body weight; CF: coronary flow; CO: cardiac output; HF: high fat; HR: heart rate; HW: heart weight; ND: not determined; NF: no fat; O2 cons: oxygen consumption; PSP: peak systolic pressure; RPP: rate pressure product Data are expressed as mean ± SD.
15
ACCEPTED MANUSCRIPT
NF
HF
AC
CE
PT
ED
MA NU
SC
RI P
T
Perfusion EndEnd10 min rep EndEnd10 min rep series baseline ischemia baseline ischemia 42.6±22.8 9.8±1.5†† 22.7±6.9‡‡ 55.3±27.3 17.8±4.6**†† 27.5±6.9†‡ ATP (µmol *g dry-1) 92.4±24.4 41.3±4.5†† 106.3±38.7‡‡ 111.8±55.7 64.7±45.6 108.9±26.3 PCr (µmol *g dry-1) Table 2: ATP and PCr tissue content Mean tissue content of ATP and PCr measured in ventricular tissue of hearts perfused until different time points: end-baseline, end-ischemia, and 10 min rep. HF: high fat; NF: no fat; PCr: phosphocreatine Data are expressed as mean ± SD. ** p<0.01 vs NF, † p<0.05, †† p<0.01 vs corresponding end-baseline value; ‡ p<0.05, ‡‡ p<0.01 vs corresponding end-ischemia value
16
ACCEPTED MANUSCRIPT
T
Figure 1: Perfusion protocol Hearts underwent 20 min baseline working-mode perfusion with glucose (11 mM) and either high fat (1.2 mM palmitate; HF) or no fat (NF), followed by 27 min global ischemia (37°C) and 60 min glucose-only reperfusion (60 min rep). Additional series of hearts were stopped after baseline (endbaseline), ischemia (end-ischemia), and 10 min reperfusion (10 min rep).
HF
MA NU
NF
SC
RI P
Figure 2: Post-ischemic recovery Hemodynamic and metabolic parameters as a function of perfusion time for: a) Rate pressure product (RPP: peak systolic pressure * heart rate); b) Peak systolic pressure (PSP); c) Coronary flow (CF); d) Oxygen consumption. Percentages indicate post-ischemic recovery as the value at 60 min reperfusion expressed as a percentage of the mean-pre-ischemic value. Data are expressed as mean ± SD. * p<0.05, ** p<0.01 vs NF
HF
PT
NF
ED
Figure 3: Carbohydrate metabolism parameters during early reperfusion a) Glycolysis (GLY) and glucose oxidation (GOX) rates at 10 min reperfusion; b) Pyruvate dehydrogenase (PDH) activity measured in ventricular tissue of hearts stopped at various time points: end-baseline, end-ischemia, and 10 min rep; c) Lactate release measured in buffer samples during baseline perfusion and 5 and 10 min reperfusion; d) Glycogen content measured in ventricular tissue of hearts stopped at various time points: end-baseline, end-ischemia, and 10 min rep, Δ: glycogen use during ischemia. Data are expressed as mean ± SD. * p<0.05, ** p<0.01 vs NF; †† p<0.01 vs corresponding endbaseline value; ‡‡ p<0.01 vs corresponding end-ischemia value
HF
AC
NF
CE
Figure 4: Cyt c and necrosis marker release Release of cyt c, TnT, and LDH at end-baseline and 10 min reperfusion, measured in perfusate samples of hearts perfused until 60 min reperfusion. a) Cytochrome c (Cyt c) release; b) Troponin T (TnT) release; c) Lactate dehydrogenase (LDH) release; BDL: below detection limit (20 U/L) Data are expressed as mean ± SD. * p<0.05, ** p<0.01 vs NF; † p<0.05 vs corresponding end-baseline value
Figure 5: Proposed effect of acute pre-ischemic exposure to high levels of fatty acids. Acute pre-ischemic high fat (1.2 mM palmitate) leads to early reperfusion decreases in activity of pyruvate dehydrogenase (PDH) due to increased acetyl-CoA/CoA and NADH/NAD+ ratios, which cause a strong inhibition of glucose oxidation (GOX). In parallel, increased levels of cytosolic citrate, inhibit glycolysis (GLY). Together these effects lead to uncoupling of GLY/GOX and accumulation of lactate and H+, which worsens Ca2+ overload and exacerbates ischemia-reperfusion (I/R) injury.
17
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
Figure 1
18
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
Figure 2
19
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
Figure 3
20
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
Figure 4
21
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
MA NU
SC
RI P
T
Figure 5
22
ACCEPTED MANUSCRIPT Highlights Effects of pre-ischemic high fat were investigated in a rat heart model of DCD1
Acute pre-ischemic high fat exposure decreases graft hemodynamic recovery by 50%
Pre-ischemic high fat reduces cardiac glucose metabolism during early reperfusion
Pre-ischemic high fat lowers cardiac pyruvate dehydrogenase activity at reperfusion
Pre-ischemic fat may influence cardioprotective reperfusion approaches
AC
CE
PT
ED
MA NU
SC
RI P
T
1
Donation after circulatory death 23