Supplemental Material can be found at: http://jpet.aspetjournals.org/content/suppl/2013/10/17/jpet.113.207001.DC1.html 1521-0103/348/1/77–85$25.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics

http://dx.doi.org/10.1124/jpet.113.207001 J Pharmacol Exp Ther 348:77–85, January 2014

Dopamine and Lipophilic Derivates Protect Cardiomyocytes against Cold Preservation Injury Christiane Vettel, Maximilia C. Hottenrott, Rahel Spindler, Urs Benck, Peter Schnuelle, Charalambos Tsagogiorgas, Bernhard K. Krämer, Simone Hoeger, Ali El-Armouche, Thomas Wieland, and Benito A. Yard Institute of Experimental and Clinical Pharmacology and Toxicology (C.V., T.W.), Fifth Medical Clinic (M.C.H., R.S., U.B., P.S., B.K.K., S.H., B.A.Y.), and Department of Anesthesiology (C.T.), University Medical Centre Mannheim, Heidelberg University, Mannheim, Germany; and Institute of Pharmacology, University Medical Center Göttingen, Heart Center, Georg-AugustUniversity Medical School Göttingen, Göttingen, Germany (C.V., A.E.-A.) Received June 6, 2013; accepted October 17, 2013

Introduction The insufficient availability of donor heart allografts, a rising waiting list mortality, and increasing demand for donor organs have substantially contributed to extension of traditionally accepted criteria for evaluation of cardiac grafts (Koerner et al., 1997; Tenderich et al., 1998). With this liberalization of donor eligibility criteria, donor hearts are being harvested from remote locations, thereby increasing cold ischemic times. Although static cold storage of solid organs is common practice in organ transplantation, it is generally acknowledged that prolonged hypothermic preservation negatively affects graft quality (Shoskes and Halloran, 1996; Taylor et al., 2009). This work was supported by grants from the German Research Foundation (Deutsche Forschungsgemeinschaft) [Grants SFB 1002 TPA02 and IRTG 1816 (to C.V. and A.E.-A.); Grant GRK880, Project 14 (to B.A.Y.)] and the German Center for Cardiovascular Research [Deutsches Zentrum für Herz-KreislaufForschung (to T.W.)]. C.V. and M.C.H. contributed equally to this study. dx.doi.org/10.1124/jpet.113.207001. s This article has supplemental material available at jpet.aspetjournals.org.

spontaneous contractions after cold storage and rewarming only occurred in treated cardiomyocytes. Hypothermia severely attenuated isoprenaline-induced cAMP formation in control but not in dopamine- or NOD-treated cells. Esterified derivates of NOD with redox potential and lipophilic side chains reduced cell damage during cold storage similarly to NOD. In contrast to dopamine, neither NOD nor its derivates induced a significant b-adrenoceptor–mediated elevation of cellular cAMP levels. The b1adrenoceptor antagonist atenolol and D1/D2 receptor antagonist fluphenazine had no impact on the protective effect of NOD or dopamine. We conclude that dopamine as well as NOD treatment mitigates cold preservation injury to cardiomyocytes. The beneficial effects are independent of b-adrenoceptor or dopaminergic receptor stimulation but correlate with redox potential and lipophilic properties.

Iron-mediated production of reactive oxygen species, impairment of Ca21 homeostasis, and ATP depletion seem to be of instrumental importance in the sequel of hypothermic preservation injury (Rauen et al., 2000; Brinkkoetter et al., 2006; Bartels-Stringer et al., 2007; Radovits et al., 2008). Earlier reports have concluded that prolonged cold ischemic time is not a risk factor for decreased long-term survival in cardiac allograft recipients (Morgan et al., 2003), yet more recent studies have indicated that it is a predictor of a 5-year conditional mortality (Taylor et al., 2008). The effect of cold ischemic time on survival after heart transplantation is dependent on donor age, with greater tolerance among grafts from younger donors (Russo et al., 2007). Cold ischemic time also shows a significant linear relationship with post-transplantation length of stay in the intensive care unit (Rylski et al., 2010). Hence, there is a socioeconomic demand for measures that improve cardiac allograft preservation over longer periods of time without affecting cardiac transplantation outcome (Rao et al., 2001). Clinical studies have shown that dopamine (DA) treatment of the donor reduces the incidence of delayed graft function

ABBREVIATIONS: BB, 2,5-bisacetoxy-benzoic acid; BBNB, 2,5-bisacetoxybenzoyl-N-butylamide; BBNO, 2,5-bisacetoxybenzoyl-N-octanoylamide; CS, cold storage; DA, dopamine; ISO, isoprenaline; LDH, lactate dehydrogenase; NOD, N-octanoyl dopamine; NRCM, neonatal rat cardiomyocyte; SKF-38393, 1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol; UW, University of Wisconsin. 77

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ABSTRACT Donor heart allografts are extremely susceptible to prolonged static cold storage. Because donor treatment with low-dose dopamine improves clinical outcome after heart transplantation, we tested the hypothesis that dopamine and its lipophilic derivate, N-octanoyl dopamine (NOD), protect cardiomyocytes from cold storage injury. Neonatal rat cardiomyocytes were treated with dopamine or NOD or left untreated and subsequently subjected to static cold storage (8–12 hours). Dopamine- and NOD-treated cardiomyocytes displayed a better viability compared with untreated cells after hypothermia. In untreated cardiomyocytes, cell damage was reflected by lactate dehydrogenase (LDH) release and a decrease in intracellular ATP. NOD was approximately 20-fold more potent than dopamine. Similarly to cardiomyocytes in vitro, rat hearts perfused with NOD before explantation showed significantly lower LDH release after static cold storage. ATP regeneration and

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after renal transplantation (Schnuelle et al., 2004; Schnuelle et al., 2009). The beneficial effect of DA was more pronounced when cold ischemic time exceeded 17 hours, and this in turn translated into a better graft survival in patients in this particular subgroup (Schnuelle et al., 2009). Based on a substantial number of in vitro studies, current evidence indicates that DA mitigates cell damage related to cold preservation (Yard et al., 2004; Brinkkoetter et al., 2006; Rudic et al., 2010). The cytoprotective properties of DA directly originate from structural entities, which enable the molecule to act as an iron chelator and to scavenge reactive oxygen species (Yard et al., 2004). The analysis of heart transplant recipients who had received a cardiac allograft from a donor enrolled in the randomized dopamine trial revealed that donor DA may also improve the clinical course after heart transplantation, ultimately resulting in superior survival after 3 years (Benck et al., 2011). However, it must be mentioned that, in approximately 12.5% of the donors that enrolled in the randomized dopamine trial, dopamine infusion had to be discontinued because of tachycardia or hypertension. This underscores the need for compounds that have superior cytoprotective properties and yet are devoid of hemodynamic actions. The present study was undertaken to provide a biologic plausibility for the clinical finding that DA improves transplantation outcome in cardiac allograft recipients. In addition, we sought to test the hypothesis that N-octanoyl dopamine (NOD), a lipophilic DA derivative devoid of hemodynamic effects, has superior cytoprotective properties compared with DA in protecting cardiomyocytes against cold preservation injury and that this does not require catecholamine receptor engagement (Lösel et al., 2010).

Materials and Methods Animal Care. Care of the animals was taken in accordance with the Committee on Animal Research of the regional government (Regierunspräsidium Karlsruhe, Germany), which reviewed and approved all experimental protocols according to the Guide for the Care and Use of Laboratory Animals published by the Directive 2010/63/EU of the European Parliament and the corresponding German legislation. Isolation and Culture of Neonatal Rat Cardiomyocytes. One- to 3-day-old female and male Wistar rats were killed by decapitation, and the hearts were removed. Cardiomyocytes were isolated from hearts, as described previously (Will et al., 2010). In brief, hearts were minced and subjected to serial digestion in a mixture of 0.5 mg/ml collagenase type II (Cell Systems, Troisdorf, Germany) and 0.6 mg/ml pancreatin (Sigma-Aldrich, Taufkirchen, Germany) to release single cells. The cell suspension was placed on a Percoll gradient (GE-Healthcare, Freiburg, Germany) to separate cardiomyocytes from other cell types. Thereafter, the myocyte fraction was seeded in 12- or 24-well plates at a density of 300,000 and 150,000 cells/cavity, respectively, and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (PAA Laboratories, Bruschwig, Germany), 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.1 mM 59-bromo-29-deoxyuridine to prevent overgrowth of noncardiomyocyte cell types. In all cultures, cell contractions were observed after 24 hours. For all experiments, the cells were used 3–4 days after isolation. Synthesis of Chemical Compounds. NOD was synthesized, as previously described (Lösel et al., 2010), from commercially available precursors and was purified by 2-fold recrystallization from dichloromethane, as demonstrated by thin-layer chromatography. Octanoic acid was converted to its mixed anhydride derivate by reaction with ethyl chloroformate in the presence of N-ethyl diisopropylamine. The crude mixed anhydride was incubated with dopamine hydrochloride (Sigma-Aldrich) in N,N-dimethylformamide and N-ethyl

diisopropylamine to form NOD. After aqueous preparation and evaporation of the organic solvent, NOD in an overall yield of approximately 60% was obtained. The sample investigated by NMR (Bruker AC250) yielded spectra in accordance with the expected structure. For the synthesis of dihydroxy benzoic acid derivatives, 2 g of 2,5dihydroxybenzoic acid was suspended in 5 ml of acetic anhydride under magnetic stirring. When two drops of sulfuric acid were added, the suspension turned clear and stirring was continued for 1 hour. Diluted hydrochloric acid (5 ml) was added, and 30 minute later, the reaction mixture was poured into 200 ml of ice water. The precipitated product was collected by vacuum filtration and dried under vacuum to yield 2,5bisacetoxybenzoic acid, pure as judged by thin-layer chromatography. Bisacetoxybenzoic acid was reacted with stoichiometric amounts of ethyl chloroformate to obtain the mixed anhydride, which was used without purification. The anhydride was dissolved in dimethyl formamide, and the respective amine was added in equal stoichiometric quantity. After reacting overnight, the mixture was diluted with ethyl acetate, and the organic phase was extracted subsequently with neutral phosphate buffer, brine, diluted sulhuric acid, and again brine. Drying over MgSO4 and removal of the solvent under vacuum yielded the crude product, which was recrystallized from aqueous ethanol. Cold Storage and Rewarming. Cardiomyocytes were stimulated for 1 hour with indicated substances. Hereafter, the cells were extensively washed with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4), and then stored at 4°C in University of Wisconsin (UW) (Southard and Belzer, 1995) solution for 8 to 12 hours. The incubation time was based on initial experiments, showing that preservation less than 6 hours did not significantly damage the cells. Cells were evaluated either directly after cold storage or after a rewarming period of 1 hour at 37°C. Lactate Dehydrogenase Assay. Lactate dehydrogenase (LDH) assays were performed as recommended by the manufacturer (Roche Diagnostics, Mannheim, Germany). A 100-ml aliquot of each supernatant was used to determine LDH release in the preservation solution. In each experiment, 100 ml of preservation solution was used as blank. The results are expressed as OD490 nm, corrected for the blank value. Each concentration was tested in triplicate in all experiments. The EC50 values were estimated from a total of five experiments. Assessment of Intracellular ATP Amount. Intracellular ATP was extracted directly after cold preservation or after 1 hour of rewarming in cell culture medium. ATP was assessed by luciferasedriven bioluminescence using the ATP Bioluminescence Assay Kit CLS II (Roche Diagnostics), according to the manufacturer’s instructions. Absolute ATP concentrations, given in the legends of the figure, were normalized for protein concentrations in the lysates. Determination of cAMP Formation. Cardiomyocytes were subjected to cold preservation conditions for 12 hours. After 30 minutes of rewarming at cell culture conditions, 1 mM 3-isobutyl-1-methylxanthine was applied and cells were incubated for an additional 30 minutes. Thereafter, the cardiomyocytes were stimulated for 10 minutes with the indicated concentrations of isoprenaline and then lysed in 0.1 M HCl. cAMP formation was assessed by using enzyme-linked immunosorbent assay kits (cAMP EIA Kit, Biomol, Hamburg, Germany, for concentration-dependent curves and after cold storage (CS); EIA Kit, Biotrend, Koeln, Germany, for b-adrenergic receptor activation). Determination of Cardiomyocyte Contractility. Cardiomyocyte contractions were determined optically using a phase-contrast microscope. The results are expressed as the percentage of cavities of 24-well plates containing contracting neonatal rat cardiomyocytes (NRCM), irrespective of the number of contracting cells. Cold Preservation of and LDH Measurement in Explanted Rat Hearts. Male Lewis rats, weighing 250–300 g, were used. Animals were kept under standard conditions and fed standard rodent chow and water ad libitum. All procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences and approved by the local authorities.

Cytoprotection of Cardiomyocytes by Dopamine Analogs Animals were anesthetized with 6 mg/body weight of xylazine (Rompun 2%; Bayer Vital, Leverkusen, Germany) and 100 mg/body weight of ketamine (Ketamin 10%; Intervet, Unterschleißheim, Germany) and heparinized (100 IE, Heparin-Natrium ratiopharm; Ratiopharm, Ulm, Germany). Long midline incision was used to enter the abdominal cavity. The abdominal aorta and inferior vena cava were exposed, and aorta and inferior vena cava were cut to drain the blood. An incision through the thoracic wall was applied to expose the chest organs. The heart was perfused through the superhepatic vena cava with 30 ml of 4°C cold UW solution with and without 50 mM NOD to cool and arrest its beating. The ascending aorta, pulmonary artery, and pulmonary veins were transacted. By cutting distal from the ligature, the heart can be harvested. Subsequently, the explanted hearts were preserved at 4°C in cold 10 ml of UW solution with and without NOD over 4 hours. Thereafter, preservation solution was collected from the heart ventricles to measure LDH release of cardiac cells. LDH was assessed according to the manufacturer’s instructions (Roche Diagnostics). The following groups were investigated: vehicle, hearts in UW solution, n 5 4; NOD, 50 mM in UW solution, n 5 4. Statistical Analysis. Data are presented as mean 6 S.E.M. and were based on three or more separate experiments. Differences between groups were determined by Student’s t test or one-way analysis of variance, followed by Bonferroni’s multiple comparison. A P value of less than 0.05 was considered statistically significant. Mathematical curve fitting and calculation of EC50 or IC50 values were performed with GraphPad Prism5 (GraphPad Software, Inc., San Diego, CA).

Results Dopamine and N-Octanoyl Dopamine Protect Cardiomyocytes from Cold-Inflicted Cell Damage and ATP Depletion. We first assessed the susceptibility of cultured cardiomyocytes for damage to hypothermia. To this end, cardiomyocytes were subjected to cold storage for various time intervals. Thereafter, the supernatants were immediately analyzed for LDH activity. Although cold storage up to 4 hours was not associated with profound cell damage, LDH release in the supernatants significantly increased with increasing preservation time (Fig. 1A). Because LDH release reached a maximum between 8 and 24 hours of cold storage, most experiments were performed using a cold storage time of 8 hours, unless otherwise stated. To study whether treatment of cardiomyocytes with DA or its lipophilic derivative NOD is protective against cold preservation injury, cultured cardiomyocytes were treated for 1 hour with increasing concentrations of DA or NOD and subsequently subjected to 8 hours of cold storage in UW solution. In untreated cardiomyocytes, cold storage resulted in profound cell damage, as demonstrated by a profound release of LDH into the preservation solution. The release of LDH was inhibited in a concentration-dependent manner by prior treatment with either DA or NOD before the start of cold storage (Fig. 1B). In line with the observation that DA or NOD pretreatment was protective, the cellular ATP content was significantly higher in treated than in untreated cells after cold storage (DA or NOD treated vs. untreated, P , 0.01; Fig. 1C). In both settings, NOD was more potent than DA. Half-maximal inhibition of LDH release and ATP depletion occurred at approximately 20-fold lower NOD than DA concentrations (LDH release IC50 DA vs. NOD, 50 6 25 mM vs. 1.3 6 0.3 mM; ATP production EC50 DA vs. NOD, 100 6 25 mM vs. 13 6 3 mM, P , 0.05). No significant difference in the maximally achievable protection was observed between DA and NOD treatment. When the cells were rewarmed

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in culture medium directly after cold storage, ATP regeneration did not occur in untreated cardiomyocytes. In contrast, 1 hour of rewarming was sufficient to regain similar intracellular ATP concentrations in DA- or NOD-pretreated cells as in NRCM not subjected to cold storage (Fig. 1D). NOD was also protective under conditions similar to those occurring during heart explantation and cold storage of donor organs. When rat hearts were perfused with UW solution containing 50 mM NOD prior to explanation and kept at 4°C for 4 hours, the LDH content in the preservation solution taken from the heart ventricles of treated and untreated hearts was significantly lower in the NOD-treated group (approximately 35%; Fig. 1E). A 4-hour time period of cold storage was chosen, as the usually accepted maximal time for organ preservation in human heart transplantation is up to 4 to 5 hours. DA could not be tested in this setting, as the presence of 50 mM DA in the preservation solution gave a strong positive LDH reading, although no heart allografts were stored herein. The false-positive LDH reading was only observed in the heart preservation study for DA but not when cultured cardiomyocytes were pretreated with DA, extensively washed, and subsequently stored at 4°C. NRCM Treated with Dopamine or N-Octanoyl Dopamine Regain Positive Inotropic Capacity after Cold Storage and Rewarming. Even though both DA and NOD are able to protect NRCM against cold storage injury, this does not necessarily imply that protected cells regain full functionality upon rewarming. To address this issue, we studied two aspects of NRCM functionality, i.e., their ability to regain spontaneous contractions after cold storage and to respond to b-adrenoceptor stimulation. Irrespective of the pretreatment, spontaneous cardiomyocyte contractions were not detectable immediately after cold storage. However, when the preservation solution was replaced by culture medium and cells were rewarmed to 37°C for 1 hour, DA and NOD treated cells regained their ability to contract spontaneously (% of wells with spontaneous contractions: 89% for DA and 94% for NOD treated cells vs. 15% for control cells) (Fig. 2A). In untreated NRCM, spontaneous contraction after cold storage and rewarming was only observed in 16% of the cavities. When untreated cardiomyocytes were maintained in culture for another 24 hours after cold preservation, their ability to contract ceased, whereas the beating capacity remained constant in DA- and NODtreated cells. A video of such a comparison is presented in the Supplemental Data (Supplemental Video 1, no treatment; Supplemental Video 2, NOD treatment). To further demonstrate that protected NRCM regain functionality after rewarming, the ability to respond to positive inotropic stimuli was assessed by measuring by b-adrenoceptor–induced cAMP formation after 12 hours of cold storage and 1 hour of rewarming. The b-adrenoceptor agonist isoprenaline (ISO) concentrationdependently increased cAMP production in DA- or NODpretreated cardiomyocytes, whereas the efficacy of ISO to induce cAMP formation was severely impaired in untreated cells that were subjected to cold preservation and rewarming (Fig. 2B). As shown in Fig. 2C, the maximal extent of ISOstimulated cAMP formation in DA- or NOD-pretreated NRCM was not significantly different from cardiomyocytes that were not subjected to cold preservation. Similar to NOD, Lipophilic, Esterified Derivates of Gentisic Acid Protect Cardiomyocytes from Cold Storage Injury. Because NOD contains a redox active catechol structure, we assessed whether the catechol structure is strictly

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Fig. 1. Influence of DA and NOD on cold preservation injury of cultured NRCM and perfused rat hearts. (A) NRCM were subjected to various time intervals of cold storage, and LDH release in the supernatants was assessed hereafter. *P # 0.01 vs. time point 2 hours. (B and C) NRCM were treated for 1 hour with increasing concentrations of DA or NOD, as indicated. Thereafter, the cells were extensively washed with phosphate-buffered saline and stored for 8 hours at 4°C in UW solution. LDH release in the preservation solution was assessed directly after cold preservation, as described in Materials and Methods. The results are expressed as mean OD490 values 6 S.E.M. (B) Intracellular ATP was assessed directly after cold preservation by luciferasedriven bioluminescence. The results are expressed as mean relative light units (RLU) 6 S.E.M. Absolute ATP concentration in nanograms per milligram protein ranged from 13.2 6 2.1 to 2.2 6 0.5 for both DA and NOD (C). A total of five independent experimentswere performed. For each experiment, all conditions were tested in triplicates. *P # 0.01, NOD vs. DA. (D) NRCM were stimulated for 1 hour with DA (100 mM) or NOD (50 mM). Hereafter, the cells were extensively washed with phosphate-buffered saline and stored for 8 hours at 4°C in UW solution. Intracellular ATP was measured either directly before (open bars) or after (filled bars) 1 hour of rewarming in culture medium. NRCM that were not subjected to cold storage (No CS) were included in each experiment. The results are expressed as mean relative light units (RLU) 6 S.E.M. Absolute ATP concentration in nanograms per milligram protein was as follows: 15.4 6 2.2 (No CS), 2.1 6 0.6 [before and after rewarming of untreated cells (C)], 12.9 6 0.8 and 16.8 6 2.1 (DA-treated cells before and after rewarming, respectively), and 13.6 6 2.2 and 17 6 2.1 (NOD-treated cells before and after rewarming, respectively). A total of six independent experiments were performed. For each experiment, all conditions were tested in triplicates. *P # 0.05 vs. no CS; #P # 0.05 vs. DA before rewarming; xP # 0.05 vs. NOD before rewarming. (E) Rat hearts were perfused with UW or UW containing 50 mM NOD prior to explantation. After 4 hours at 4°C, LDH release was determined, as described in Materials and Methods. Results are expressed as mean values 6 S.E.M., n = 4 for each group, *P # 0.05 vs. control.

required for protection. Similar to catechols (ortho-dihydroxyquinones), para-dihydroxyquinones are redox active and hence may have the propensity to act protective in our assays. Meta-Dihydroxyquinones were not tested because these structures have extremely low redox activities. We analyzed the cytoprotective properties of 2,5-acetoxybenzoic acid (BB), 2,5acetoxybenzoyl-N-butylamide (BBNB), and 2,5-acetoxybenzoyl-N-octanoylamide (BBNO). As shown in Fig. 3A, all three substances are derivates of gentisic acid (2,5-hydroxybenzoic acid), a naturally occurring compound with known radical scavenging properties (Chen et al., 2002). The para position and esterification of the hydroxy groups of these compounds make them, however, unlikely candidates for any activation of b-adrenoceptors. Yet, similar to DA and NOD, they have a redox active moiety at the aromatic ring, provided that the acetylated hydroxy groups in the aromatic ring are hydrolyzed by intracellular esterases. BB, BBNB, and BBNO vary in their hydrophobic side chains, which range from eight C-atoms (the same length as NOD) to four and none in the case of BB (Fig. 3A). To compare the cytoprotective effects of these genistic acid derivates, cardiomyocytes were treated with 50 mM BB, BBNB, and BBNO 1 hour prior to cold storage. A comparable protection from cold-induced cell damage by NOD (Fig. 1) was only observed with BBNB and BBNO in which a

hydrophobic side chain was added to the aromatic core (Fig. 3B). Neither b-Adrenoceptor nor D1/D2 Receptor Agonism Is Involved in Protection of NRCM from Cold Storage Injury. To investigate a putative relation between b-adrenergic or dopaminergic agonism and cytoprotection, we first tested whether the different compounds were able to increase intracellular cAMP concentration upon treatment and, if so, to what extent the increased cAMP was inhibited by the b-receptor blocker atenolol. Compared with isoprenaline, which increases intracellular cAMP formation ∼10-fold, only DA led to a significant increase in intracellular cAMP, whereas no significant rise in intracellular cAMP concentration was observed after stimulation with 50 mM NOD, BB, BBNB, or BBNO (Fig. 4A). The DA-induced increase in intracellular cAMP levels could be completely blocked by the addition of the b1-adrenoceptor– specific antagonist atenolol (Fig. 4B). Although we have previously demonstrated that the cytoprotective effect of dopamine could not be overcome by b-adrenoceptor blockade (Yard et al., 2004), this has not been tested to date for NOD. To exclude any receptor engagement in the cytoprotective properties of NOD, cardiomyocytes were treated for 1 hour with NOD alone or with NOD in the presence of 10 mM atenolol before 8 hours of cold storage. As shown in Fig. 4C, pretreated cardiomyocytes were

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Fig. 2. Influence of DA and NOD treatment on cardiomyocyte function after cold preservation and rewarming. Cardiomyocyte contractions were determined microscopically using a phase-contrast microscope. (A) NRCM were treated with DA (100 mM) or NOD (50 mM) for 1 hour or were left untreated. The percentage of wells containing contracting cardiomyocytes was assessed before cold storage (open bars) or after cold preservation, followed by 1 hour of rewarming (filled bars). The results are expressed as the mean percentage of wells that contain contracting cardiomyocytes 6 S.E.M., irrespective of the number of contracting cells. *P # 0.05 vs. control before CS. (B) Isoprenaline concentration-response curve in DA- or NOD-treated and untreated cells after cold preservation and rewarming. Cardiomyocytes were treated with DA (100 mM) and NOD (50 mM) for 1 hour before cold storage or left untreated. After rewarming, NRCM were stimulated with the indicated concentrations of the b1/b2-adrenoceptor agonist ISO, and intracellular cAMP was quantified. (C) Comparison of cAMP formation in NRCM undergoing cold preservation after DA or NOD treatment or no treatment (C) with cardiomyocytes not being subjected to cold preservation (No CS). Where indicated (filled bars), cells were stimulated with 10 mM ISO. The results are expressed as means 6 S.E.M. *P # 0.05 vs. not treated control. In (B and C), a total of three independent experiments were performed. For each experiment, all conditions were tested in duplicates.

equally protected against cold-induced damage, independent of b1-adrenergic activation. Likewise, the D1/D2-receptor antagonist fluphenazine abrogated neither the protective effect of dopamine nor that of NOD over large range of fluphenazine concentrations (Fig. 4D), the data of which exclude the involvement of dopaminergic receptors on the protection of cardiomyocytes to coldinflicted damage.

Discussion Dopamine and Its Lipophilic Derivates Provide Cytoprotection from Cold Preservation Injury Based on Their Redox Potential and Cellular Uptake. In this study, we sought to explore the biologic plausibility of our clinical observation that treatment of the brain-dead cardiac donor with low-dose DA is associated with an improved clinical outcome after heart transplantation (Benck et al., 2011). We hypothesized that dopamine pretreatment increases the viability of cardiomyocytes during cold preservation and that NOD is superior in this regard. Our data clearly substantiate this hypothesis as pretreatment with DA or NOD concentrationdependently reduces cell damage and enhances tolerance of cardiomyocytes to withstand cold preservation in culture. A

similar loss of damage was seen if NOD was applied to rat hearts before explantation. In cultured cardiomyocytes, ATP depletion was prevented, and, as a consequence, spontaneous contractility as well as responsiveness to adrenergic stimuli is preserved upon rewarming. Our data further indicate that the beneficial effects on NRCM are independent of b1-adrenoceptor or D1/D2 dopaminergic receptor engagement. At the concentrations used, only DA stimulated cAMP formation in NRCM by activation of b1adrenoceptors. It is well documented that dopamine displays b-adrenoceptor agonism at higher concentrations (.3 mM), whereas cardiac effects at lower concentrations are attributed to DA-induced release of noradrenaline in vivo (Habuchi et al., 1997). The DA derivate NOD, in contrast, has no hemodynamic effects when applied in vivo (Lösel et al., 2010) but is more potent with regard to cytoprotection. Similar to the derivates of gentisic acid, NOD did not induce a significant elevation of intracellular cAMP, and its protective effect was unaffected by b1-adrenoceptor antagonists. It has recently been described that various dopamine D1 receptor agonists exert cytoprotective effects on oxidative injury in cultured cardiomyocytes, which raises the question of dopamine receptor involvement (Gerö et al., 2007). Gerö et al. (2007), however, propose that some of the positively tested

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Fig. 3. The cytoprotective effect requires a lipophilic structure with radical scavenging redox potential. (A) Chemical structure of NOD, BB, BBNB, and BBNO. (B) NRCM were treated with the gentisic acid derivates BBNO, BBNB, or BB (50 mM). Thereafter, cells were extensively washed with phosphate-buffered saline and stored for 8 hours at 4°C in UW solution. LDH release in the preservation solution was assessed directly after cold preservation, as described in Materials and Methods. The results are expressed as mean OD490 values 6 S.E.M. A total of six independent experiments were performed. For each experiment, all conditions were tested in triplicates, *P # 0.05 vs. not treated control (C).

D1 receptor agonists confer cytoprotection through indirect inhibition of the poly(ADP-ribose) polymerase without receptor engagement (Gerö et al., 2007). Another study on ischemia/ reperfusion injury in neonate rat cardiomyocytes supports that cardiac D1 receptors are not involved in cytoprotection, because selective activation of D1 receptors with the agonist SKF-38393 (1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol) induces apoptotic cell death (Li et al., 2008). As NOD has been shown not to act on dopamine receptors (Lösel et al., 2010), our data clearly support that DA’s cytoprotective action does not require dopamine receptor engagement (Brinkkoetter et al., 2006, 2008; Benck et al., 2011). Direct evidence to underpin this assumption is provided by the data demonstrating that the protective effects of DA and NOD are not abrogated by the D1/D2 receptor antagonist fluphenazine. The relative hydrophobicity of dopamine derivatives correlates with the efficacy of these compounds to protect against cold-inflicted injury (Lösel et al., 2010) and thus explains the differences in potency between DA and NOD to limit LDH release and the loss of ATP activity following cold storage. Whereas the redox activity of DA and NOD is similar, NOD displays a higher degree of hydrophobicity compared with DA, which allows improved cellular uptake (Lösel et al., 2010). Subsequently, higher intracellular concentrations can be achieved with lower dosages, thus reaching cytoprotective intracellular levels at much lower concentrations (Lösel et al., 2010).

Gentisic acid is a naturally occurring compound in mold fungi and plants (Chen et al., 2002). It also is a naturally occurring metabolite of acetylsalicylic acid (Cham et al., 1980). Its high redox potential has been clearly demonstrated in vitro; moreover, both in vitro and in vivo findings suggest that this property may convey protection toward geno- and hepatotoxicity of cyclophosphamide and may lower lowdensity lipoprotein oxidation (Chen et al., 2002; Ashidate et al., 2005; Nafees et al., 2012). We have modified gentisic acid in two ways. The free phenolic hydroxy groups have been esterified to protect the compound from oxidation outside of the cell where they cannot be hydrolyzed by esterases and to increase hydrophobicity and thus increase cellular uptake. It should be underscored, however, that cellular uptake of the gentisic acid derivates was not assessed. Nonetheless, it is conceivable that, similarly as shown for cellular uptake of DA and NOD, increased hydrophobicity of the gentisic acid derivates might increase cellular uptake. This study did not identify a putative cellular target by which DA or NOD conveys protection. Yet it suggests that if such a target exists, the redox activity and hydrophobicity of the protective compound seem to have a strong influence on the activity of this target. NOD, BBNB, and BBNO all have the propensity to inhibit nuclear factor kB upon stimulation with tumor necrosis factor a (Hottenrott et al., 2013). Therefore, even though NOD, BBNB, and BBNO are distinct in their chemical structure, their common chemical characteristics, that

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Fig. 4. The cytoprotective effect does not require b-adrenoceptor or D1/D2 receptor engagement. (A) b-Adrenoceptor–induced cAMP formation by ISO (10 mM), DA (100 mM), NOD (50 mM), and the gentisic acid derivates BB, BBNB, and BBNO (50 mM each). (B) cAMP formation in DA-treated NRCM was inhibited by 10 mM atenolol. In A and B, cAMP formation was quantified after 10 minutes of stimulation in the presence of 1 mM 3-isobutyl-1methylxanthine. All samples were tested in duplicate. A total of four experiments was performed, and the results are expressed as mean cAMP concentration (pmol/ml) 6 S.E.M. *P # 0.05 vs. control (C); #P # 0.05. DA vs. DA plus atenolol. (C) NRCM were treated for 1 hour with NOD (50 mM) or left untreated. To each of these conditions, 10 mM atenolol was added. Hereafter, the cells were subjected to 8 hours of cold storage, and supernatants were collected to assess the LDH release. The results are expressed as mean OD490 values 6 S.E.M. A total of three independent experiments were performed. For each experiment, all conditions were tested in triplicates. *P # 0.05 vs. untreated control. (D) NRCM were treated for 1 hour with DA (100 mM), NOD (50 mM) (filled bars), or normal culture medium (open bars) in the presence of different fluphenazine concentrations (F; 10, 100, and 1000 nM). In all experiments, DA, NOD, or culture medium alone (C) without addition of fluphenazine was included. Hereafter, the cells were subjected to 24 hours of cold storage, and supernatants were collected to assess the LDH release. The results are expressed as mean OD490 values 6 S.E.M. A total of three independent experiments were performed. For each experiment, all conditions were tested in triplicates. *P # 0.05 vs. medium controls (open bars).

is, redox activity and hydrophobicity, appear to be sufficient to inhibit the same cellular target. Nakao et al. (2008) have suggested that cold preservation injury occurs as a consequence of destabilization of heme-containing proteins, for example, cytochrome P450. This results in the release of heme and subsequently oxidative cell damage. The requirement for redox activity for cellular protection against cold-inflicted damage fits with the findings of Nakao et al. (2008). All protective compounds that were tested in this study are able to participate in a 2e2 oxidation reaction and therefore have the propensity to reduce reactive oxygen intermediates. Although the less hydrophobic BB compound is also able to participate in such an oxidation reaction, it was not protective in this study. This further supports the notion that the relative hydrophobicity of the protective compound is likely to play part in the protective mechanism. Dopamine and Its Lipophilic Derivates as Potential Drugs to Prevent Graft Dysfunction in Heart Transplantation. There is ample evidence that prolonged hypothermic preservation has detrimental effects on organ quality after transplantation. Unlike in renal transplantation where delayed graft function can be bridged by hemodialysis, graft

dysfunction after heart transplantation has severe consequences, because post-transplant use of cardiac assist devices to aid circulatory demands goes along with high morbidity and mortality. Ischemic tolerance substantially varies between different organs. Because of the enhanced susceptibility of the cardiac allograft, it is of utmost importance to keep cold ischemic time below 4 hours in heart transplantation (Taylor et al., 2009; Jacobs et al., 2010). A considerable number of heart transplants are still performed after prolonged cold ischemia, which promotes early graft dysfunction and increases mortality post-transplantation (Taylor et al., 2009). These sequelae urgently demand a pharmacologic intervention to limit cell damage during cold preservation. DA’s clinical benefit in heart transplantation has recently been shown in a cohort study with 93 patients from 21 centers (Benck et al., 2011). Although these implications have not yet been confirmed by a randomized controlled trial, treatment of the donor with DA is thus a potential option to increase the probability of graft survival after transplantation. Nevertheless, in the clinical setting, intracellular DA accumulation critically depends on the activity of its degrading enzymes monoamine oxidase and catechol-O-methyl transferase. Therefore, a pharmacokinetic

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steady state needs to be maintained until cross-clamping. In a prospective randomized clinical trial on low-dose donor DA treatment and transplantation outcome in renal transplant recipients, we reported that DA treatment was prematurely discontinued in 12.5% of donors due to circulatory side effects, such as tachycardia and/or hypertension (Schnuelle et al., 2009). Such a discontinuation of DA infusion will, however, result in rapid loss of protection because of monoamine oxidase and catechol-O-methyl transferase activity. Hence, the efficacy of donor preconditioning could be further improved if a DA-like compound would be available, which at cytoprotective concentrations is devoid of dopaminergic and adrenergic action. Although NOD is not yet approved for clinical application, preclinical data on mitigation of ischemia-induced acute kidney injury in rats are promising and suggest superior protection than DA (Tsagogiorgas et al., 2012). Further biological evaluation of NOD, BBNB, and BBNO is ongoing and will clarify whether these compounds can also be considered for clinical testing. In summary, this study provides biologic plausibility for our observation that donor preconditioning with DA may improve the outcome after heart transplantation. Because protection is mediated by antioxidant properties of the tested molecules, this study also provides a rationale for further development and evaluation of DA derivatives devoid of receptor-driven dopaminergic and adrenergic action for clinical use in organ transplantation. Limitations of the Study. We are aware that the data presented in this study do not prove that the beneficial effect of donor dopamine treatment on cardiac transplantation outcome is attributed to the protective properties of dopamine on hypothermic preservation injury. Nonetheless, this study provides a biologic plausibility that urges further research in this context. A prospective trial of donor dopamine administration on outcome of cardiac transplants seems to be justified and should ideally also look at the outcome of all organs transplanted from those donors, to assess the efficacy of donor dopamine pretreatment in general. The in vitro finding that NOD might be superior to dopamine is intriguing but requires further in vivo exploration in relevant transplantation models. Moreover, because the hydrophobicity of drug compounds makes intravenous application complicated, an extensive structure activity study may also further help understanding how structural alterations of NOD affect efficacy of protection and may provide new protective compounds with lower hydrophobicity. Attempts should also be made to provide putative targets by which NOD is mediating its protective effect and how redox activity and hydrophobicity fit in this model.

Acknowledgments

The authors thank Annette Breedijk for help in performing these experiments and the members of the GRK880 for fruitful discussion. Authorship Contributions

Participated in research design: Vettel, Hottenrott, Hoeger, Wieland, Yard. Conducted experiments: Vettel, Hottenrott, Spindler, Hoeger. Contributed new reagents or analytic tools: Tsagogiorgas, Spindler. Performed data analysis: Tsagogiorgas, Hoeger, Benck, Schnuelle. Wrote or contributed to the writing of the manuscript: Vettel, Hottenrott, Benck, Krämer, Schnuelle, El-Armouche, Yard, Wieland.

References Ashidate K, Kawamura M, Mimura D, Tohda H, Miyazaki S, Teramoto T, Yamamoto Y, and Hirata Y (2005) Gentisic acid, an aspirin metabolite, inhibits oxidation of low-density lipoprotein and the formation of cholesterol ester hydroperoxides in human plasma. Eur J Pharmacol 513:173–179. Bartels-Stringer M, Verpalen JT, Wetzels JF, Russel FG, and Kramers C (2007) Iron chelation or anti-oxidants prevent renal cell damage in the rewarming phase after normoxic, but not hypoxic cold incubation. Cryobiology 54:258–264. Benck U, Hoeger S, Brinkkoetter PT, Gottmann U, Doenmez D, Boesebeck D, Lauchart W, Gummert J, Karck M, and Lehmkuhl HB, et al. (2011) Effects of donor pretreatment with dopamine on survival after heart transplantation: a cohort study of heart transplant recipients nested in a randomized controlled multicenter trial. J Am Coll Cardiol 58:1768–1777. Brinkkoetter PT, Beck GC, Gottmann U, Loesel R, Schnetzke U, Rudic B, Hanusch C, Rafat N, Liu Z, and Weiss C, et al. (2006) Hypothermia-induced loss of endothelial barrier function is restored after dopamine pretreatment: role of p42/p44 activation. Transplantation 82:534–542. Brinkkoetter PT, Song H, Lösel R, Schnetzke U, Gottmann U, Feng Y, Hanusch C, Beck GC, Schnuelle P, and Wehling M, et al. (2008) Hypothermic injury: the mitochondrial calcium, ATP and ROS love-hate triangle out of balance. Cell Physiol Biochem 22:195–204. Cham BE, Bochner F, Imhoff DM, Johns D, and Rowland M (1980) Simultaneous liquid-chromatographic quantitation of salicylic acid, salicyluric acid, and gentisic acid in urine. Clin Chem 26:111–114. Chen CH, Shaw CY, Chen CC, and Tsai YC (2002) 2,3,4-Trimethyl-5,7-dihydroxy-2,3dihydrobenzofuran, a novel antioxidant, from Penicillium citrinum F5. J Nat Prod 65:740–741. Gerö D, Módis K, Nagy N, Szoleczky P, Tóth ZD, Dormán G, and Szabó C (2007) Oxidant-induced cardiomyocyte injury: identification of the cytoprotective effect of a dopamine 1 receptor agonist using a cell-based high-throughput assay. Int J Mol Med 20:749–761. Habuchi Y, Tanaka H, Nishio M, Yamamoto T, Komori T, Morikawa J, and Yoshimura M (1997) Dopamine stimulation of cardiac beta-adrenoceptors: the involvement of sympathetic amine transporters and the effect of SKF38393. Br J Pharmacol 122:1669–1678. Hottenrott MC, Wedel J, Gaertner S, Stamellou E, Kraaij T, Mandel L, Loesel R, Sticht C, Hoeger S, and Ait-Hsiko L, et al. (2013) N-Octanoyl dopamine inhibits the expression of a subset of kB regulated genes: potential role of p65 Ser276 phosphorylation. PLoS One 8:e73122. Jacobs S, Rega F, and Meyns B (2010) Current preservation technology and future prospects of thoracic organs. Part 2: heart. Curr Opin Organ Transplant 15: 156–159. Koerner MM, Tenderich G, Minami K, Morshuis M, Mirow N, Arusoglu L, Gromzik H, Wlost S, and Koerfer R (1997) Extended donor criteria: use of cardiac allografts after carbon monoxide poisoning. Transplantation 63:1358–1360. Li HZ, Han LP, Jiang CM, Li H, Zhao YJ, Gao J, Lin Y, Ma SX, Tian Y, and Yang BF, et al. (2008) Effect of dopamine receptor 1 on apoptosis of cultured neonatal rat cardiomyocytes in simulated ischaemia/reperfusion. Basic Clin Pharmacol Toxicol 102:329–336. Lösel RM, Schnetzke U, Brinkkoetter PT, Song H, Beck G, Schnuelle P, Höger S, Wehling M, and Yard BA (2010) N-Octanoyl dopamine, a non-hemodynamic dopamine derivative, for cell protection during hypothermic organ preservation. PLoS One 5:e9713. Morgan JA, John R, Weinberg AD, Kherani AR, Colletti NJ, Vigilance DW, Cheema FH, Bisleri G, Cosola T, and Mancini DM, et al. (2003) Prolonged donor ischemic time does not adversely affect long-term survival in adult patients undergoing cardiac transplantation. J Thorac Cardiovasc Surg 126:1624–1633. Nafees S, Ahmad ST, Arjumand W, Rashid S, Ali N, and Sultana S (2012) Modulatory effects of gentisic acid against genotoxicity and hepatotoxicity induced by cyclophosphamide in Swiss albino mice. J Pharm Pharmacol 64:259–267. Nakao A, Faleo G, Shimizu H, Nakahira K, Kohmoto J, Sugimoto R, Choi AM, McCurry KR, Takahashi T, and Murase N (2008) Ex vivo carbon monoxide prevents cytochrome P450 degradation and ischemia/reperfusion injury of kidney grafts. Kidney Int 74:1009–1016. Radovits T, Lin LN, Zotkina J, Koch A, Rauen U, Köhler G, Karck M, and Szabó G (2008) Endothelial dysfunction after long-term cold storage in HTK organ preservation solutions: effects of iron chelators and N-alpha-acetyl-L-histidine. J Heart Lung Transplant 27:208–216. Rao V, Feindel CM, Cohen G, Borger MA, Boylen P, and Ross HJ (2001) Is profound hypothermia required for storage of cardiac allografts? J Thorac Cardiovasc Surg 122:501–507. Rauen U, Petrat F, Li T, and De Groot H (2000) Hypothermia injury/cold-induced apoptosis—evidence of an increase in chelatable iron causing oxidative injury in spite of low O22/H2O2 formation. FASEB J 14:1953–1964. Rudic B, Song H, Breedijk A, Brinkkoetter P, Beck G, Yard B, and Ponelies N (2010) Hypothermic preservation up-regulates calpain expression and increases ubiquitination in cultured vascular endothelial cells: influence of dopamine pretreatment. J Surg Res 160:325–332. Russo MJ, Chen JM, Sorabella RA, Martens TP, Garrido M, Davies RR, George I, Cheema FH, Mosca RS, and Mital S, et al. (2007) The effect of ischemic time on survival after heart transplantation varies by donor age: an analysis of the United Network for Organ Sharing database. J Thorac Cardiovasc Surg 133:554–559. Rylski B, Berchtold-Herz M, Olschewski M, Zeh W, Schlensak C, Siepe M, and Beyersdorf F (2010) Reducing the ischemic time of donor hearts will decrease morbidity and costs of cardiac transplantations. Interact Cardiovasc Thorac Surg 10:945–947. Schnuelle P, Gottmann U, Hoeger S, Boesebeck D, Lauchart W, Weiss C, Fischereder M, Jauch KW, Heemann U, and Zeier M, et al. (2009) Effects of donor pretreatment with dopamine on graft function after kidney transplantation: a randomized controlled trial. JAMA 302:1067–1075.

Cytoprotection of Cardiomyocytes by Dopamine Analogs Schnuelle P, Yard BA, Braun C, Dominguez-Fernandez E, Schaub M, Birck R, Sturm J, Post S, and van der Woude FJ (2004) Impact of donor dopamine on immediate graft function after kidney transplantation. Am J Transplant 4:419–426. Shoskes DA and Halloran PF (1996) Delayed graft function in renal transplantation: etiology, management and long-term significance. J Urol 155:1831–1840. Southard JH and Belzer FO (1995) Organ preservation. Annu Rev Med 46:235–247. Taylor DO, Edwards LB, Aurora P, Christie JD, Dobbels F, Kirk R, Rahmel AO, Kucheryavaya AY, and Hertz MI (2008) Registry of the International Society for Heart and Lung Transplantation: twenty-fifth official adult heart transplant report —2008. J Heart Lung Transplant 27:943–956. Taylor DO, Stehlik J, Edwards LB, Aurora P, Christie JD, Dobbels F, Kirk R, Kucheryavaya AY, Rahmel AO, and Hertz MI (2009) Registry of the International Society for Heart and Lung Transplantation: twenty-sixth official adult heart transplant report—2009. J Heart Lung Transplant 28:1007–1022. Tenderich G, Koerner MM, Stuettgen B, Arusoglu L, Bairaktaris A, Hornik L, Wlost S, Mirow N, Minami K, and Koerfer R (1998) Extended donor criteria: hemodynamic follow-up of heart transplant recipients receiving a cardiac allograft from donors . or 5 60 years of age. Transplantation 66:1109–1113.

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Tsagogiorgas C, Wedel J, Hottenrott M, Schneider MO, Binzen U, Greffrath W, Treede RD, Theisinger B, Theisinger S, and Waldherr R, et al. (2012) N-Octanoyldopamine is an agonist at the capsaicin receptor TRPV1 and mitigates ischemiainduced [corrected] acute kidney injury in rat. PLoS One 7:e43525. Will RD, Eden M, Just S, Hansen A, Eder A, Frank D, Kuhn C, Seeger TS, Oehl U, and Wiemann S, et al. (2010) Myomasp/LRRC39, a heart- and muscle-specific protein, is a novel component of the sarcomeric M-band and is involved in stretch sensing. Circ Res 107:1253–1264. Yard B, Beck G, Schnuelle P, Braun C, Schaub M, Bechtler M, Göttmann U, Xiao Y, Breedijk A, and Wandschneider S, et al. (2004) Prevention of cold-preservation injury of cultured endothelial cells by catecholamines and related compounds. Am J Transplant 4:22–30.

Address correspondence to: Dr. Benito A. Yard, University Medical Centre Mannheim, Fifth Medical Clinic, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. E-mail: [email protected]