Effects of inosine on reperfusion injury after heart transplantation§

Gabor Szabo a,*, Nicole Stumpf a, Tamas Radovits a, Karin Sonnenberg a,Domokos Gero a, Siegfried Hagl a, Csaba Szabo b, Susanne Bahrle c

aDepartment of Cardiac Surgery, University of Heidelberg, Im Neuenheimer Feld 326, 69120 Heidelberg, GermanybDepartment of Physiology and Experimental Clinical Research, Semmelweis University, Budapest, Hungary

cDepartment of Cardiology, University of Heidelberg, Heidelberg, Germany

Received 4 October 2005; received in revised form 12 March 2006; accepted 4 April 2006

www.elsevier.com/locate/ejctsEuropean Journal of Cardio-thoracic Surgery 30 (2006) 96—102

Abstract

Objective: Inosine, a break-down product of adenosine, has been recently shown to exert inodilatory and anti-inflammatory properties. Weinvestigated the effects of inosine on ischemia/reperfusion injury in a rat heart transplantation model. Methods: Intraabdominal heterotopictransplantation was performed in Lewis rats. After 1 h of ischemic preservation, reperfusion was started after application of either saline vehicle(control, n = 12) or inosine (100 mg/kg, n = 12). Coronary blood flow, left ventricular function, endothelium-dependent vasodilatation toacetylcholine and endothelium-independent vasodilatation to sodium nitroprusside, and high energy phosphate content were measured after 1and 24 h of reperfusion. In addition, the activation of the poly(ADP-ribose) polymerase was detected by immunhistology. Results: After 1 h,coronary blood flow (4.1 � 0.3 ml/(min g) vs 2.9 � 0.3 ml/(min g), p < 0.05), left ventricular systolic pressure (102 � 9 mmHg vs 83 � 4 mmHg,p < 0.05) and dP/dt (2765 � 609 mmHg/s vs 1740 � 116 mmHg/s, p < 0.05) were significantly higher in the inosine group in comparison tocontrol. Vasodilatatory response to sodium nitroprusside was similar in both groups. Acetylcholine resulted in a significantly higher increase incoronary blood flow in the inosine group (76 � 5% vs 48 � 9%, p < 0.05). Energy charge potential was significantly higher in the inosine group(1.69 � 0.10 mmol/g vs 0.74 � 0.27 mmol/g, p < 0.05). After 24 h, there was no difference between the groups in basal coronary blood flow, leftventricular systolic pressure, dP/dt, and the response to sodium nitroprusside. However, acetylcholine led to a still significantly higher responsein the inosine group (112 � 13% vs 88 � 7%, p < 0.05). Immunhistologic stainings revealed activation of poly(ADP-ribose) polymerase in controlanimals which was abolished by inosine. Conclusions: Thus, inosine improves myocardial and endothelial function during early reperfusion afterheart transplantation with a persisting beneficial effect against reperfusion induced graft coronary endothelial dysfunction. The effects of inosineare mediated at least partly by modulation of the peroxynitrite-poly(ADP-ribose) polymerase pathway.# 2006 Published by Elsevier B.V.

Keywords: Transplantation; Reperfusion injury; Inosine; Endothelial function; Rat

1. Introduction

Ischemia—reperfusion injury is a common condition duringcardiac surgery. Myocardial performance within the first hourafter the surgical procedure determines the patient’s statenot only during the postoperative period but also in the longtime outcome, especially after heart transplantation whenan extended time of ischemia is followed by reperfusion.During ischemia, cellular ATP is degraded into AMP, adenosineinosine, and hypoxanthine. Adenosine and its primarymetabolite inosine are ubiquitous nucleosides that can bereleased from ischemic or reperfused tissue [1]. Adenosine isgenerally considered to act primarily through cell surface

§ Presented at the joint 19th Annual Meeting of the European Association forCardio-thoracic Surgery and the 13th Annual Meeting of the European Societyof Thoracic Surgeons, Barcelona, Spain, September 25—28, 2005.* Corresponding author. Tel.: +49 6221 566246; fax: +49 6221 565585.E-mail address: dzsi@hotmail.com (G. Szabo).

1010-7940/$ — see front matter # 2006 Published by Elsevier B.V.doi:10.1016/j.ejcts.2006.04.003

adenosine receptors [2] modulating numerous physiologicaland pathophysiological events in multiple tissue [3,4].Inosine was generally considered an inactive metabolite.However, some past and recent works suggested that inosinemay exert inotropic, vasodilatoty and anti-inflammatoryeffects [5—7]. However, the exact mechanisms of thebeneficial effects of inosine remain controversial.

Recently it has been discovered that purines in generaland inosine in particular (but not adenosine) inhibitpoly(ADP-ribose) polymerase (PARP) activation and modulatecell death [8]. PARP is an abundant nuclear enzyme ofeukarotic cells that has been implicated in response to DNAinjury and oxidant-induced cell death (see overview in Ref.[9]). We and others [10—12] previously showed that PARP-inhibition reduces ischemia—reperfusion injury.

In contrast to adenosine which mainly acts via surfacepurine receptors, inosine might have more direct effect onintracellular signaling. We hypothesized that inosine mayalso exert cytoprotective effects by interfering with the PARP

G. Szabo et al. / European Journal of Cardio-thoracic Surgery 30 (2006) 96—102 97

activation pathway. This assumption is based on thestructural similarity of inosine to part of NAD+, the substrateof PARP. The aim of the present study was to investigate theeffects of inosine on ischemia/reperfusion injury andwhether inosine influences the PARP pathway duringischemia/reperfusion.

Table 1Functional parameters

Recipient Group A Group B Group C Group D

HR (min�1) 295 � 16 280 � 14 300 � 27 313 � 20AoP (mmHg) 91 � 9 86 � 9 98 � 7 88 � 12

GraftHR (min�1) 159 � 23 166 � 15 298 � 21y 266 � 28y

LVSP (mmHg) 83 � 4 102 � 9* 114 � 3y 102 � 11dP/dtmax (mmHg/s) 1740 � 116 2765 � 609 * 3603 � 237y 3708 � 803dP/dtmin (mmHg/s) 1001 � 128 2246 � 394 * 2511 � 208y 2622 � 321t (ms) 22 � 1.5 16 � 1.1 * 12 � 3y 12 � 3LVEDP (mmHg) 5.7 � 1.2 6.1 � 1.5 5.2 � 1.1 4.9 � 1.3CBF (ml/(min g)) 2.9 � 0.3 4.1 � 0.3 4.0 � 0.4 4.2 � 0.3

Group A: control, 60 min of reperfusion; Group B: inosine treatment, 60 min ofreperfusion; Group C: control, 24 h of reperfusion; Group D: inosine treat-ment, 24 h of reperfusion. HR, heart rate; AoP, mean aortic pressure; LVSP, leftventricular systolic pressure; dP/dtmax, maximum rate of pressure develop-ment; dP/dtmin, minimum rate of pressure development; t, time constant ofmonoexponential isovolumetric pressure decay; LVEDP, left ventricular end-diastolic pressure. All values are given as mean � SEM at an intraventricularvolume of 80 ml.

* p < 0.05, inosine versus control transplant.y p < 0.05, 24 h versus 60 min.

2. Materials and methods

2.1. Heterotopic heart transplantation

The experimental model was described elsewhere [10].Briefly, donor hearts were explanted from Lewis rats. After1 h of ischemic preservation at 4 8C, the hearts wereimplanted intraabdominally anastomosing the aorta andthe pulmonary artery of the donor heart with the abdominalaorta or the vena cava of the recipient rat, respectively.

All animals received humane care in compliance with thePrinciples of Laboratory Animal Care formulated by theNational Society of Medical Research and the Guide for theCare and Use of Laboratory Animals prepared by the NationalAcademy of Sciences and published by the National Institutesof Health (NIH Publication No. 86-23, revised 1996).

2.2. Functional measurements in the graft

Left ventricular systolic (LVSP) and end-diastolic pressure(LVEDP), rate of pressure development (dP/dt), and relaxa-tion time constant (t) were measured by a Millar micro-manometer (Millar Instruments Inc., Houston, TX, USA) atdifferent LV volumes using an intraventricular balloon. Totalcoronary blood flow (CBF) was measured by a perivascularultrasonic flow probe on the donor aorta. After baselinemeasurement, the endothelium-dependent vasodilator acet-ylcholine (ACH, 1 nM, 0.2 ml) and bradykinin (BK 0.1 nM,0.2 ml) as well as the endothelium-independent vasodilatorsodium-nitroprusside (SNP, 10 nM, 0.2 ml) were administereddirectly into the coronary arteries of the graft via the donoraorta. Between the infusions, CBF was allowed to return tobaseline levels. Vasodilator response was expressed asmaximum percent change of CBF from baseline.

2.3. Determination of high energy phosphates

Adenosine triphosphate (ATP), adenosine diphosphate(ADP), and adenosine monophosphate (AMP) contents wereassessed with standard photometry using an enzyme-kineticassay [10]. Energy charge potential was calculated as(ATP + 0.5ADP)/(ATP + ADP + AMP).

2.4. Immunohistochemistry

Formalin-fixed cryostat-fixed sections were stained byprimary mouse monoclonal anti-poly(ADP-ribose) antibody(Alexis, San Diego, CA, USA) to detect the product (poly(ADP-ribose)) of PARP activity [12,13]. Quantitative histomorpho-logic assessment was performed by the COLIM softwarepackage (Pictron Ltd., Budapest, Hungary) based on theintensity and distribution of labeling. The results wereexpressed with a grading system of 0 (no staining) to 4

(extensive staining) based on the measured intensity andarea of positive labelings [10,12].

2.5. Experimental protocol

Four transplant groups were studied (n = 6 for each group).Immediately before releasing the aortic clamp, the slowinjection of either saline (control group) or the inosine(100 mg/kg)was startedandcontinuedduring thefirst 5 minofthe reperfusion period. This dose was chosen based on in vitroand in vivo studies [8], previous efficacy data with thecompound in various models of inflammation and vascularinjury and pilot transplant experiments. In Group A (control)and Group B (inosine) the measurements of systolic anddiastolic function and CBF were performed after 1 h ofreperfusion. In Group C (control) and Group D (inosine) theabdominal cavity was closed and the animals were allowed torecover from the anesthesia. During the following 24 h, theanimals of both groups received the same standard diet andnormal drinking water. After 24 h the animals were reanesthe-tized and the abdominal cavity was reopened. The grafts wereinstrumented and the measurements were performed as inGroups A and B. After the functional measurements the heartswere excised for histologic analysis.

In a separate series of experiments, four groups (n = 6 foreach group) of hearts were transplanted and treated witheither inosine or saline vehicle similarly to the abovementioned protocol. After either 1 or 24 h of reperfusion,the grafts were excised to determine high energy phosphatecontents.

2.6. Statistical analysis

All values were expressed as mean � standard error of themean (SEM). Individual means between the groups werecompared by one-way analysis of variance followed by anunpaired t-test with a Bonferroni correction for multiplecomparisons and the post hoc Scheffe’s test. A value ofp < 0.05 was considered statistically significant.

G. Szabo et al. / European Journal of Cardio-thoracic Surgery 30 (2006) 96—10298

Fig. 1. Left ventricular peak systolic pressure (LVSP)-volume (LVV) (left) and maximum pressure development (dP/dtmax)-LVV (B, right) and left ventricular end-diastolic pressure (LVEDP)-LVV (C) relationships after 1 and 24 h of reperfusion. All values are given as mean � SEM, *p < 0.05 versus other groups.

3. Results

3.1. Early reperfusion (Groups A and B)

The hemodynamic parameters and CBF after 60 min ofreperfusion are shown in Table 1. The recipient’s heart rateand aortic pressure were same in all groups. Systolicfunctional recovery was significantly better in the inosinegroup in comparison to control. LVSP and peak positive dP/dtwere significantly ( p < 0.05) higher in the inosine group.Systolic cardiac function curves showed a significant leftward

Fig. 2. Myocardial relaxation (t, left) and left ventricular end-diastolic pressure (LVEDas mean � SEM, *p < 0.05 inosine versus control at a given time point, yp < 0.05, 24

shift in the inosine group in comparison to the vehicle-treated group (Fig. 1). Peak negative dP/dt was significantlyhigher ( p < 0.05) and t significantly lower ( p < 0.05) in theinosine group indicating a better myocardial relaxation(Table 1, Fig. 2). LVEDP did not differ between the groups.The diastolic compliance curves (end-diastolic pressure—volume relationships) were similar in all groups (Fig. 2). CBFwas significantly higher ( p < 0.05) in the inosine group incomparison to control after 60 min (Table 1). Endothelium-independent vasodilatation after SNP (Fig. 2) was similar inboth groups. In contrast, endothelium-dependent vasodila-

P)-LVV (right) relationships after 1 and 24 h of reperfusion. All values are givenh versus 60 min.

G. Szabo et al. / European Journal of Cardio-thoracic Surgery 30 (2006) 96—102 99

Fig. 3. Vasodilator response after application of the endothelium-dependent vasodilator bradykinin (0.1 nM, left) and acetylcholine (1 nM, mid) and the endotheliumindependent vasodilator sodium nitroprusside (10 nM, right). All values are given asmean � SEM. *p < 0.05 inosine versus control at a given time point, yp < 0.05, 24 hversus 60 min.

tation after ACH and BKwas significantly ( p < 0.05) better inthe inosine group than in the vehicle-treated transplantgroup (Fig. 3).

Myocardial high energy phosphate content especially ATP-content as well as energy charge potential were betterpreserved by inosine treatment during heart transplantation(Fig. 4).

Histologic findings reveal a slight edema and in some casesa scare inflammatory perivascular infiltrate composedpredominantly of polymorphonuclear neutrophils and lym-phocytes in the transplanted heart which was more prono-unced in the control group. Immunohistochemical stainingshowed increased immunoreactivity for poly(ADP-ribose)(PAR) — indicative of enhanced activation of PARP — in the

Fig. 4. Energy charge potential (left) and total adenylate pool (right). All values are

vehicle-treated transplant group. PAR positive staining wasobserved in the nucleus of the myocytes and in some cases inthe cytosol as an indicator of myocyte cell necrosis.Furthermore, endothelial cell nuclei also showed a strongPAR staining (Fig. 5). The staining for PAR was nearly absent inthe inosine group (Fig. 5).

3.2. Late reperfusion (Groups C and D)

After 24 h of reperfusion, there were no differences inLVSP, peak positive and negative dP/dt, t, and LVEDP betweenthe vehicle- and the inosine-treated transplant groups(Table 1). In the vehicle-treated transplant group, all theseparameters showed a significant improvement in comparison

given as mean � SEM. *p < 0.05 inosine versus control at a given time point.

G. Szabo et al. / European Journal of Cardio-thoracic Surgery 30 (2006) 96—102100

Fig. 5. Immunohistological staining against poly(ADP-ribose), a marker of poly(ADP-ribose) polymerase activation after 1 h of reperfusion. Top panels: lowmagnification (160�) bottom panels: high magnification (400�). The control specimens (A, C) showed positive dark staining in the nuclei of the myocytes andin the capillary endothelium. The inosine group (B, D) showed completely negative staining.

to the values after 60 min of reperfusion ( p < 0.05). In theinosine-treated transplant group, there were no significantdifferences in comparison to the values of 60 min ofreperfusion. Systolic cardiac function curves and diastoliccompliance curves of the control and the inosine group werenearly identical (Figs. 1 and 2). Baseline CBF was also similarin both groups. After 24 h, endothelium-dependent vasodi-latation was significantly increased ( p < 0.05) in both groupsin comparison to the 60 min of reperfusion values. Endothe-lium-dependent vasodilatation after both ACH and BK wasalso significantly higher in the inosine group in comparison tovehicle-treated transplant group (Fig. 3).

After 24 h, total adenylate pool showed no significantdifferences between the groups; however, ATP content wasstill slightly higher in the inosine-treated animals withoutreaching the level of significance (Fig. 4).

Immunohistochemical staining showed almost no activa-tion of PARP in both groups (not shown).

4. Discussion

In this study, the benefits of the application of inosineduring reperfusion were assessed after reversible hypother-mic ischemia in a heterotopic rat heart transplantationmodel. Heterotopic heart transplantation was used tosimulate the clinical conditions in terms of whole bloodreperfusion and to allow an observation time of 24 h, whichis impossible in isolated organ models. Furthermore, theheterotopic situation also allows to assess myocardialfunction independently from the actual loading conditions.We also demonstrated that cardiac preservation (globaldeep hypothermic ischemia) followed by reperfusion leads

to a significant activation of PARP which was blocked byinosine.

The modulation of the ATP degradation pathway atdifferent levels is an important strategy to reduce ische-mia—reperfusion [14]. During the past years, beneficialeffects of adenosine [14,15], adenosine analogues [16],adenosine uptake, and desaminase inhibitors [17] have beendescribed. In contrast, the role of other purine nucleotides ispoorly investigated. An increase of contractile force andcoronary blood flow in response to inosine has beendemonstrated as early as the late 70s [5]. Inosine wassupposed to block the desensitizing action of cyclic AMP onthe reaction between calcium ions and contractile proteins[18]. Furthermore, it was found that inosine infusion resultedin a nonmetabolically coupled coronary vasodilatation indogs [19]. In subsequent studies, inosine exerted beneficialeffects after hypoxia-reoxygenation [20] and ischemia/reperfusion [6] which is in concert with our data.

The mechanism of action of inosine is multiple and notcompletely understood. It has been shown previously thatintermediate periods of ischemia induce a severe loss ofcellular NAD+ and ATP levels. The loss of cellular energeticpools, in turn, importantly affects myocardial function [10].We demonstrated that a major action of inosine is thepreservation of myocardial of ATP contents during reperfu-sion. This is in accordance with a 31P magnetic resonancespectroscopic study in which significantly higher levels of ATPwas detected after ischemia/reperfusion in crystalloidperfused isolated hearts after application of inosine[21].The increase of coronary blood flow during reperfusioncontribute also to the improvement of cardiac function.Previous studies [6,21] demonstrated that inosine increasecoronary flow dose dependently and, as a consequence, the

G. Szabo et al. / European Journal of Cardio-thoracic Surgery 30 (2006) 96—102 101

function of the reperfused heart. These effects are alsocomparable with that of adenosine: Galinanes and Hearse[15] showed in a similar model of heterotopic transplantationthat after 8 h of cardiac preservation and 1 h of reperfusion,that adenosine significantly improved coronary blood flowand myocardial function. Interestingly they did not found anydifference in high energy phosphate contents which impli-cate that increased coronary blood flow is an independentfactor of enhanced functional recovery.

Similar to our previous studies, endothelial function wasseverely impaired after 1 h of reperfusion and was sustainedafter 24 h. The present study is the first which demonstratesthat inosine improves not onlymyocardial but also endothelialfunction.This effect is comparable to thosewith applicationofnitric oxide donors [22], endothelin receptor antagonists [23],or PARP-inhibitors [9,10,12]. How inosine protects theendothelium remains not completely understood. Previousdata suggest that energy depletion severely impairs endothe-lial function [10]. As inosine restores ATP levels, this maycontribute to improved endothelial function. If inosine has adirect effect on nitric oxide synthesis remains to be clarified.

Neutrophil—endothelial interaction is a central stepduring the development of reperfusion injury which leadsto endothelial and subsequent myocardial injury. Novelstudies implicate that purin nucleotides in general andinosine in particular may reduce neutrophil-mediated injury.In isolated cell cultures, inosine dose-dependently inhibitedLPS-induced monocyte activation [24]. In a rodent model ofendotoxin shock, inosine improved vascular function andreduced neutrophil accumulation in selected organs [13].Recently, it has been shown that inosine-monophosphate, anintermediate metabolite of the AMP-inosine degredationpathway and inosine itself inhibits neutrophil rolling andtrafficking into the reperfused tissue [25].

A very important new finding of the present study thatinosine acts as an endogen PARP inhibitor. The immunhisto-logic examinations indicated extensive PARP activation in thevehicle-treated group, which is in accordance with ourprevious studies [9,10]. Inosine treatment almost completelyabolished PARP activation. Recently we were able todemonstrate in vitro that inosine (and other purine nucleo-tides such as hypoxanthine) reduce cellular PARP activity inperoxynitrite-treated macrophages and improves mitochon-drial respiration.

The activation of PARP is currently described to be a finalcommon effector in various types of tissue injury includingsystemic inflammation, circulatory shock, and ischemia/reperfusion (see Section 1). Both genetic disruption andpharmacologic inhibition of the PARP pathway effectivelyprotect against oxygen radical and nitric oxide toxicity indifferent cell cultures, attenuate regional myocardialischemia/reperfusion and global hypoxia-reoxygenationinjury [5,9]. The pathomechanisms of PARP inhibition arediscussed in detail elsewhere [9]. Briefly, PARP-inhibitorsexert their beneficial effects at multiple levels by preserva-tion of cellular ATP, downregulation of adhesion molecules,improvement of mitochondrial respiration, and regulation ofnecrosis and apoptosis. The observed changes after inosinetreatment are in concert with previous studies using PARPinhibitors in similar models of ischemia/reperfusion [10]. Thecurrent results are the first to demonstrate that an

endogenous molecule (other than nicotinamide), namelyinosine, inhibits PARP activation in vivo and thereforemodulates oxidant-induced cell death [8]. We propose thehypothesis that inosine appears to work, at least in thecurrently used model systems, as nonprofessional, butreasonably potent inhibitor of PARP activation; it modulatescell death in a fashion that is entirely consistent with its PARPinhibitory effect. Nevertheless, further studies are necessaryto elucidate the exact mode of action of inosine treatment.

Acknowledgements

This work was supported by ForschungsschwerpunktTransplantation of the University of Heidelberg to G.S.and, in part, by grants from the National Institutes of Health(HL59266 and GM 60915) to C.S.

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