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Cardiovascular Research Advance Access first published online on October 30, 2007
This version [Corrected Proof] published online on November 22, 2007
Cardiovascular Research, doi:10.1093/cvr/cvm052
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S-nitroso human serum albumin reduces ischaemia/reperfusion injury in the pig heart after unprotected warm ischaemia

Seth Hallström1,*,{dagger}, Maximilian Franz2,{dagger}, Harald Gasser2, Martin Vodrazka2,{ddagger}, Severin Semsroth2, Udo M. Losert3, Markus Haisjackl2, Bruno K. Podesser2,* and Tadeusz Malinski4

1 Institute of Physiological Chemistry, Center for Physiological Medicine, Medical University Graz, Austria
2 Ludwig Boltzmann Cluster for Cardiovascular Research, Medical University of Vienna, Austria
3 Institute for Biomedical Research, Medical University of Vienna, Austria
4 Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio, USA

* Corresponding author. Tel: +43 1 40400 5229; fax: +43 1 40400 5221. E-mail address: b.k.podesser{at}cardiovascular-research.at (Bruno K. Podesser); seth.hallstroem{at}meduni-graz.at (Seth Hallström)

Time for primary review: 19 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Uncoupled endothelial nitric oxide synthase (eNOS) is a major contributor to vascular reactive oxygen species generation in ischaemia/reperfusion (I/R) injury. Supplementation of NO by the novel NO donor S-nitroso human serum albumin (S-NO-HSA) may inhibit uncoupling of eNOS (feedback inhibition).

Methods and results: Pigs (n = 14; 33.1 ± 1.7 kg) were continuously monitored for heart rate (HR), mean arterial pressure (MAP), left ventricular systolic pressure (LVSP), and coronary flow (CF). Infusion of either human serum albumin (n = 8; controls) or S-NO-HSA (n = 6) lasted 60 min (0.1 µmol/kg/h) starting 15 min prior to ischaemia. After clamping the aorta under cardiopulmonary bypass (CPB), the hearts underwent 15 min of warm, unprotected ischaemia (37°C). Reperfusion lasted 150 min (30 min under CPB; 15 min weaning; additional 105 min reperfusion). In biopsies from non-ischaemic hearts and myocardial biopsies taken after 150 min of reperfusion, high-energy phosphates were measured and the calcium ionophore-stimulated release of NO, superoxide, and peroxynitrite (ONOO–) were monitored with nanosensors. Compared with non-ischaemic hearts, the NO level decreased from 930 ± 25 to 600 ± 15 nmol/L (P < 0.001) while the superoxide level increased from 45 ± 5 to 110 ± 10 nmol/L (P < 0.001) after ischaemia. S-NO-HSA restored the NO level to 825 ± 20 nmol/L, shifted favourably the [NO]/[ONOO–] balance (a marker of eNOS uncoupling) from 1.36 ± 0.06 (ischaemia) to 3.59 ± 0.18, significantly improved CF (65 ± 10 vs. control, 43 ± 5 mL/min, P < 0.05), MAP (57 ± 5 vs. 39 ± 3 mm Hg, P < 0.01), LVSP (106 ± 5 vs. 81 ± 4 mm Hg, P < 0.01) and phosphocreatine (PCr) content (41.5 ± 7.3 vs. 18.0 ± 5.6 µmol/g protein; P < 0.01) at 150 min of reperfusion.

Conclusion: Long-lasting release of NO by S-NO-HSA prevented uncoupling of eNOS and thereby improved systolic and diastolic function, myocardial perfusion, and the energetic reserve of the heart after I/R injury.

KEYWORDS Ischaemia; Reperfusion; Nitric oxide; Oxygen radicals; Contractile function

Received June 27, 2007; revised September 4, 2007; accepted October 24, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Changes to the vascular endothelium have been identified as a precursor to reperfusion damage leading to organ failure.1 One important aspect of endothelial dysfunction is the diminished release of nitric oxide (NO) upon reperfusion.2 Due to direct NO measurements with a porphyrinic-based microsensor, the group of Malinski was able to show that NO concentrations dramatically decrease (<1 nmol/L) at the time point of reperfusion in the experimental setting of I/R in skeletal muscle. Calcium-dependent endothelial constitutive nitric oxide synthase (eNOS) was seen as a major source of superoxide (O2–) release.3 In the same experimental setting, supplementation of NO with the novel nitric oxide donor S-nitroso human serum albumin (S-NO-HSA) prior to ischaemia led to a complete protection from vascular constriction with no oedema formation. S-NO-HSA is a high-molecular weight S-nitrosothiol, which has an exact equimolar S-nitrosation, and a high S-nitrosograde (S-NO- in position Cys-34 of HSA: ~0.8 mol/mol protein) due to defined pre-processing. Compared with low-molecular weight S-nitroso thiols S-NO-HSA has a prolonged half-life.4

The presented results strongly suggest a correlation between high initial NO production during ischaemia followed by increasing release of O2– (decrease in net NO production) and increasing injury to the endothelium. The data also show that I/R injury is initiated by a massive burst of NO production after ischaemia which depletes local L-arginine and/or tetrahydrobiopterin concentrations, followed by high production of O2– after reperfusion and consequently high production of peroxynitrite (ONOO–).3,4 It has become clear from studies with purified eNOS that under substrate or cofactor depletion the enzyme may become ‘uncoupled’. In such an uncoupled state, electrons are rather diverted to molecular oxygen than to L-arginine, resulting in production of superoxide.5,6 One or both subunits of eNOS thereby utilizes only its other substrate O2 to produce O2–. Concomitant with the progressively increasing production of O2– during I/R NO can rapidly react with O2– to form peroxynitrite. This near diffusion limited reaction of O2– with NO to form OONO– (k = 3.8 x 109 L/mol/s) is even faster than the reaction of O2– with superoxide dismutase to form peroxide and oxygen. The reaction of O2– with NO to form peroxynitrite in sufficient concentrations specially occurs under uncoupling conditions of eNOS, when one subunit of eNOS produces NO and the other O2–. When peroxynitrite becomes protonated (pKa = 6.8), the HOONO formed usually undergoes isomerization (t1/2 < 1 s) to form hydrogen cation and nitrate anion. However, as the HOONO concentration increases as maximal O2– accumulations react with freshly synthesized NO during the initial stages of reperfusion local HOONO concentration may become sufficient to ensure its efficient transport to reactive sites as far as several cell diameters away.7 In the vicinity of certain reactive centres, HOONO may undergo homolytic cleavage to a hydroxyl free radical (OH) and nitrogen dioxide free radical (NO2) or heterolytic cleavage to a nitronium cation (NO2+) and hydroxide anion (OH–).7,8 There is also increasing evidence suggesting that physiological levels of ONOO– contribute to regulation of normal cellular function but it is widely accepted that enhanced ONOO– formation is cytotoxic.9,10

We therefore hypothesize that the constitutive eNOS plays a fundamental role in the pathogenesis of I/R injury. The onset of ischaemia leads to elevated intracellular calcium ion concentrations mediated by increased levels of catecholamines. These increased calcium concentrations activate eNOS to generate NO and consequently reactive oxygen species and cytotoxic substances.4 Although the mechanisms underlying endothelial dysfunction are likely multifactorial, it is important to note that increased production of oxygen-derived free radicals by an uncoupled eNOS markedly contributes to this phenomenon.11

Therefore, our strategy for using S-NO-HSA relies on the following deliberations: NO gradually released by S-NO-HSA can actively prevent the dysfunction of the endothelium by preventing eNOS uncoupling due to its negative feedback on the production of NO by eNOS (product inhibition of the enzyme).12 The exogenous NO released by S-NO-HSA can, therefore, (1) decrease production of NO by eNOS, (2) decrease the turnover rate of eNOS substrates and cofactors and prevent/minimize eNOS uncoupling, (3) decrease O2– production by eNOS and generation of cytotoxic ONOO–. Supporting evidence for this concept comes from a recent study in which we have shown the effectiveness of S-NO-HSA after 6 h of hypothermic, cardioplegic arrest in the isolated rabbit heart.13

The rational for conducting the present study is to test the effectiveness of the new substance S-NO-HSA under the most hostile conditions: global, warm, unprotected ischaemia of the heart in a pig model. Based on these results, we intend to test the drug in an orthotopic transplant model. Although rare, there are clinical situations, where warm, unprotected ischaemia exists such as ventricular fibrillation or cardiogenic shock. Under these conditions again the application of a drug like S-NO-HSA may be beneficial. The study therefore addresses the potential role of S-NO-HSA in reducing I/R injury in the detrimental setting of 15 min of unprotected, warm ischaemia in the pig heart based on haemodynamic evaluation and analysis of biochemical parameters.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Animal preparation
This prospective, randomized, blinded study has been approved by the local animal investigation committee. It conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). To determine the optimal dose of the novel NO-donor S-NO-HSA experiments were performed in a monitored pig model. S-NO-HSA applied at 1 µmol/kg/h showed slight systemic vasodilatative effects: the drug was infused over 60 min reaching a stable decreased level of mean arterial pressure (MAP) after 15 min. After stop of infusion, MAP almost returned to pre-infusion levels within 15 min (Figure 1A). In parallel, the kinetic of S-NO-HSA was measured by HPLC (Figure 1B). The dose of 0.1 µmol/kg/h was selected for the warm I/R pig heart experiments as it has absolutely no systemic vasodilatative effects (in contrast to 1 µmol/kg/h) and has been proved to be sufficient to prevent I/R-damage in previous studies.4


Figure 1
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  		Figure 1 Changes in mean arterial pressure (A) and plasma concentration profile of S-NO-HSA (B) during and post-infusion of 1 µmol/kg/h in the pig (n = 4). After termination of the infusion at 60 min MAP returns almost to baseline. A concentration of 0.1 µmol/kg/h S-NO-HSA does not influence MAP (data not shown). The initial half-life of S-NO-HSA calculated from the kinetic after stop of infusion is in the range of 15 min.

 
Fourteen pigs (Österreichische Landrasse) weighing 33.1 ± 1.7 kg were sedated with 20 mg/kg ketamin i.m., anaesthetized with 15 mg/kg pentobarbital, intubated and ventilated with a mixture of oxygen in air. Respirator settings were adjusted to achieve normoventilation, normoxia, and keep peak airway pressures below 20 cm H2O. Endtidal pCO2 and pulseoximetry were employed to control respirator variables. Blood samples for blood gas analysis (Blood Gas Analyzer 995, AVL) were drawn from an arterial line. Anaesthesia was maintained with 8 mg/kg/h propofol, 0.7 mg/kg/h piritramide, a synthetic opoid, and 0.1 mg/kg/h pancuronium. Ringer's solution was infused at a rate of 10 mL/kg/h and fluid filled catheters were placed in both internal jugular veins and the right carotid artery. A Swan-Ganz pulmonary artery catheter was introduced via the right external jugular vein into the pulmonary artery to determine cardiac output (CO) by thermodilution (Edwards 9520A thermodilution computer), central venous pressure (CVP), and mean pulmonary artery pressure (mPAP). Blood temperature was kept constant at 37° C with the help of pre-warmed infusions and a heating blanket. After thoracotomy, a tip catheter (Millar) was introduced in the left ventricle via the apex to monitor left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP). Cardiac index (CI), the maximal contraction velocity (dp/dtmax), the maximal relaxation velocity (dp/dtmin), and the total peripheral resistance (TPR) were calculated using standard formulas.

Both venae cavae and the ascending aorta were canulated for cardiopulmonary bypass (CPB). A balanced electrolyte solution with hetastarch was used for CPB priming. Systemic anticoagulation was induced with a bolus injection of 600 U/kg BW heparin and maintained by additional heparin doses (anticoagulation time >400 s). Management of normothermic CPB included the alpha-stat method, the use of membrane oxygenators with arterial line filters, and a pump flow set at 100 mL/kg/h.

After baseline measurements, the pigs were randomly assigned to either the S-NO-HSA (0.1 µmol/kg/h; n = 6) or the control group (human serum albumin 0.1 µmol/kg/h; n = 8). Fifteen minutes after the beginning of infusion, the pigs were subjected to 15 min of warm global cardiac ischaemia by venous inflow obstruction and aortic cross clamping that eventually resulted in asystole. After opening the cross-clamp hearts were sequentially paced at a rate of 120 b.p.m. and defibrillated when necessary. Pump flow was kept at 100 mL/kg/h and the heart, which was still unloaded and not ejecting, was reperfused for 30 min under CPB. MAP was maintained between 50 and 60 mm Hg with the help of intermittent injections of neosynephrine if necessary. Continuing throughout the reperfusion period S-NO-HSA or vehicle was infused until it was stopped at weaning from CPB (see experimental protocol Figure 2). Coronary blood flow (CBF) was measured with an ultrasonic flow probe (3 mm Flowprobe, Transonic Systems) placed around the proximal part of the left anterior descending artery.


Figure 2
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  		Figure 2 Experimental protocol and time points of treatment with S-NO-HSA and human serum albumin (control).

 
After this period, the pigs were weaned from bypass. The blood from the heart lung machine was slowly retransfused to obtain the desired pre-load and 5 min prior to weaning an epinephrine infusion was started at a rate of 0.28 µg/kg/min. Hypotension, i.e. MAP<50 mm Hg, was treated with bolus injections of neosynephrine to obtain a MAP in the range from 50 to 60 mm Hg. Heparin was reversed with 300 U/kg protamine and further volume loading was guided by LVEDP, which was aimed to be at 2 mm Hg above or below baseline level. Respirator settings were adjusted to achieve normoxia and normocapnia. At the end of the experiment, the pigs were sacrificed with a bolus injection of KCl.

2.2 S-NO-HSA preparation
HSA was processed to yield a maximal free thiol group at position Cys-34 (SH > 0.8 mol/mol protein). Intermolecular disulfides (mixed disulfides) were disassembled prior to nitrosation. The starting material (20% HSA; Baxter) was reduced by mercaptoethanol (10 to 20-fold molar excess; buffer [mmol/L]: sodium phosphate 1, ethylenediaminetetraacetic acid 2, and sodium chloride 150 adjusted to pH = 6.0–6.2 with hydrochloric acid (HCl); 12–48 h at 4°C under nitrogen) and purified by means of gel-permeation chromatography (TSK-HW40F; mobile phase: H2O).

Thiol nitrosation was induced with sodium nitrite at a ratio of 1:1 to 1:1.5 of freely available thiol groups to nitrite in 0.2 mol/L HCl (pH = 1.5–2.5) for 30 min at 25°C. After neutralization with 1 mol/L sodium hydroxide, S-NO-HSA was purified by gel-permeation chromatography (TSK-HW40F; mobile phase: H2O) and lyophilized. S-NO-HSA was dissolved and HSA was diluted with Ringer's solution and continuously infused via a catheter in the jugular vein.

2.3 Biochemical characterization
2.3.1 Determination of S-NO-HSA in plasma
Venous blood samples (3 mL) were taken every 15 min via a catheter in the vena femoralis during and post-continuous infusion. The blood was immediately centrifuged at 3500 r.p.m. in EDTA-tubes. The obtained plasma was shock-frozen in liquid nitrogen and stored at –28°C. Analysis of S-NO-HSA was perfomed by an HPLC technique with post-column derivatization after gel-permeation chromatography utilizing the Saville and Gries reaction.14

2.3.2 Determination of high-energy phosphates
Left ventricular tissue samples (near the apex) were freeze-clamped in situ and stored in liquid nitrogen until further treatment. The weighed tissue (20–100 mg) was homogenized with 250 µL of 0.4 mol/L perchloric acid in a ball mill (Braun) pre-cooled with liquid nitrogen. After thawing (4°C) and centrifugation (12 000 g) 100 µL of the acid extract were neutralized with 10–12.5 µL potassium carbonate (2 mol/L). After centrifugation, the supernatant was stored (–28°C) until analysis. The applied HPLC-method has been reported previously.4 The pellets of the acid extract were dissolved in 1 mL of 0.1 mol/L sodium hydroxide and further diluted 1:10 with physiologic saline for protein determination (BCA Protein Assay, PIERCE).

2.3.3 Preparation of NO, O2–, and ONOO– sensors for in vitro measurements
NO, O2–, and ONOO– release from the heart tissue samples were measured simultaneously with a module of NO, O2–, and ONOO– nanosensors, prepared according to procedures published previously.15–18 The nanosensors operated in a 3-electrode system, consisting of the sensor working electrode, a platinum wire (0.1 mm) counter electrode, and a standard calomel reference electrode. The current proportional to concentration was measured by nanosensors operated in an amperometric mode at constant potentials (0.65 V for NO, –0.23 V for O2–, and –0.40 V for ONOO–) vs. the standard calomel electrode (response time 0.1 ms and detection limit of 1 nmol/L for each sensor). Linear calibration curves were constructed for each sensor from 10 nmol/L to 2 µmol/L before and after measurements with aliquots of NO, O2–, and ONOO– standard solutions.16,17 Thin slices of the biopsies, still in a frozen state, were cut from the epi- to the endocardium with a microtome. Each sample was placed in an organ bath filled with Krebs–Henseleit buffer, which was continuously treated with 95% O2, and 5% CO2. The sample was then allowed to equilibrate for 30 min. Each tissue sample (~0.5 mg) was placed in a Petri dish in oxygenated HBSS adjusted to pH 7.4 at 37°C. A module of NO/O2–/ONOO– nanosensors was positioned close to the surface (6 ± 2 µm) of the tissue sample with the help of a computer-controlled micromanipulator. Release of NO, O2–, and ONOO– was stimulated with 1.0 µmol/L of the Ca2+ ionophore A23187 [GenBank] (a receptor-independent eNOS agonist).3,19,20 The amperometric signals for NO, O2–, and ONOO– were recorded with a computer-based Gamry VF600 voltametric analyser. The amperometric measurements were completed within 10–20 s after stimulation with the Ca2+ ionophore. Under these experimental conditions, a reproducibility of peak concentration is slightly better than the amount calculated from the area of the peak. Therefore, the peak concentration was used in this study.

2.4 Statistical analysis
The means±SEM are given. Comparison between different groups and time points were performed by two-way ANOVA with repeated measures and unpaired and paired t-test as post-hoc analysis. For the biochemical measurements ANOVA with Tukey's HSD post-hoc test was employed (statistical software: SPSS 13.01; 2004). A P-value of <0.05 was considered to be significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Haemodynamic function
3.1.1 Baseline values
Pre-ischaemic baseline values were similar between the S-NO-HSA and control group (all differences not significant) and are listed in Table 1. All post-ischaemic haemodynamic values except coronary flow (see in what follows and Figure 2) were recorded in 15 min intervals beginning at 75 min until termination of the experiment at 180 min.


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  		Table 1 Baseline values

 
3.1.2 Coronary flow, heart rate, mean arterial pressure, and mean pulmonary artery pressure
Coronary blood flow was also evaluated during CPB. In early reperfusion, a dramatic increase of coronary flow (CF) was visible in both groups, representing reactive hyperaemia (86 ± 14 vs. 85 ± 11 mL/min; S-NO-HSA vs. control at 30 min). At 45 min after 15 min of reperfusion on CPB, CF reached its lowest level in both groups (16 ± 1 vs. 19 ± 3 mL/min; P = ns). Then CF constantly increased in both groups reaching a maximum at 75 min (15 min after weaning from CPB; 71 ± 10 vs. 61 ± 7 mL/min; P = ns). In S-NO-HSA-treated pigs, CF remained elevated until the end of the experiment, whereas in controls CF constantly decreased (65 ± 10 vs. 43 ± 5 mL/min; P < 0.05). To illustrate individual changes in CF more accurately, CF data were depicted as recovery of pre-ischaemic baseline values of each individual animal in percent (Figure 3A).


Figure 3
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  		Figure 3 Post-ischaemic recovery of haemodynamic parameters. Time course of coronary flow expressed as percent recovery from baseline values (A), heart rate (B), mean arterial blood pressure (C), and mean pulmonary artery pressure (D). *P < 0.01, {dagger}P < 0.05: S-NO-HSA group (–{circ}–) vs. control group (HSA: – –•– –); §P < 0.01: Control group vs. baseline; IIP < 0.05, #P < 0.01: S-NO-HSA group vs. baseline. Time course of coronary flow is expressed as recovery of pre-ischaemic baseline values of each individual animal in percent.

 
During the 105 min observation period after weaning, heart rate remained stable and did not differ between groups (Figure 3B). The catecholamine support (0.28 µg/kg/min epinephrine infusion starting 5 min prior to weaning) of the heart did not differ between groups and led to the higher heart rate observed after weaning compared with baseline values (75–120 min; both groups vs. baseline; P < 0.01).

After weaning MAP decreased in both groups. However, the decrease in the S-NO-HSA-treated animals was already significantly less pronounced after 15 min post-CPB at 75 min of the experiment (74 ± 11 vs. 52 ± 5 mm Hg; P < 0.05). In the S-NO-HSA-treated pigs, MAP stabilized after 120 min (45 min post-CPB: 59 ± 5 vs. 44 ± 3 mm Hg; P < 0.05) and remained stable until the end of the experiment, whereas in controls MAP further diminished (57 ± 5 vs. 39 ± 3 mm Hg; P < 0.01; Figure 3C). Compared with baseline, MAP in the treatment group was significantly lower between 120 and 180 min (P < 0.05), whereas this significance was persistent at all time points in the control group (75 and 180 min, P < 0.01).

Mean pulmonary artery pressure was increased in both the S-NO-HSA group as well as in the control group compared with baseline after CPB with no significant difference between groups (baseline in both groups: 19 ± 1 mm Hg, endpoints in both groups 26 ± 1 mm Hg; Figure 3D).

3.1.3 Left ventricular systolic pressure, left ventricular end-diastolic pressure, first derivatives of left ventricular systolic pressure, cardiac output, and total peripheral resistance
After weaning LVSP increased in both groups. However, only in S-NO-HSA-treated animals LVSP remained on a significantly elevated level, whereas LVSP returned to baseline values in control animals. At 90 min (30 min after weaning from CPB) the difference between the two groups was significant (123 ± 11 vs. 112 ± 6 mm Hg; P < 0.05). After 135 min, the significance had a value of P < 0.01 and remained at this level until the end of the experiment (106 ± 5 vs. 81 ± 4 mm Hg; P < 0.01; Figure 4A). LVEDP did not differ between groups or to baseline at all measured time points throughout the experiment (Figure 4B). The maximal contraction velocity dP/dtmax became significantly increased in the S-NO-HSA group compared with control at 90 min (P < 0.05). After 135, min the level of significance was P < 0.01 and remained at this level (except the last time point, P < 0.05) until the end of the experiment (Figure 4C). In addition, dP/dtmax in the treatment group was significantly elevated compared with baseline throughout the experiment (P < 0.01; 75–180 min).


Figure 4
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  		Figure 4 Post-ischaemic recovery of haemodynamic parameters. Time course of left ventricular systolic pressure (A), left ventricular end-diastolic pressure (B), maximal contraction velocity –dP/dtmax (C), maximal relaxation velocity –dP/dtmin (D), cardiac output (E), and total peripheral resistance (F). {dagger}P < 0.05, *P < 0.01: S-NO-HSA (–{circ}–) vs. control group (– –•– –). {ddagger}P < 0.05, §P < 0.01: Control group vs. baseline; IIP < 0.05, #P < 0.01: S-NO-HSA group vs. baseline.

 
The maximal relaxation velocity dP/dtmin was significantly lower in the S-NO- HSA group compared with control at the last two time points at 165 and 180 min (Figure 4D). In the S-NO-HSA group dP/dtmin did not differ throughout the experiment compared with baseline. In the control group dP/dtmin increased over time and was significantly elevated compared with baseline after 105 min until the end of the experiment (P < 0.01).

Similarly after weaning from CPB, cardiac index increased in both groups but returned to baseline values until the end of the experiment. There was no statistically significant difference at any time points between groups (endpoints in both groups: 135 ± 27 vs. 96 ± 9 mL/kg/min; P = ns; Figure 4E).

There was no significant difference in total peripheral resistance between treatment and control group. Compared with baseline both groups showed in mean lower values but only the control group significantly differed from the baseline value from 75–180 min at all time points (P < 0.01; Figure 4F).

3.2 Biochemical parameters
3.2.1 High-energy phosphates
PCr, the buffering energy source for ATP in situations of energy demand, was significantly higher in S-NO-HSA-treated animals (41.5 ± 7.31 vs. control, 18.03 ± 5.63 µmol/g protein, P < 0.01; Figure 5A). Also the energy charge [EC=(ATP+0.5ADP)/(AMP+ADP+ATP)], was significantly higher in S-NO-HSA-treated pigs. (EC = 0.900 ± 0.001 vs. control, EC = 0.870 ± 0.009, P < 0.05; Figure 5B).


Figure 5
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  		Figure 5 High-energy phosphates in biopsies of the left ventricle in the treatment group receiving S-NO-HSA compared with the control group. Changes in PCr and adenine nucleotide levels (A) and energy charge (B) in S-NO-HSA vs. control group *P < 0.01 vs. control.

 
3.2.2 Nitric oxide, superoxide, and peroxynitrite release
Typical amperometric curves showing changes in calciumionophore stimulated NO, O2–, and ONOO– release from a non-ischaemic heart (recipient heart; see legend Figure 6) and an ischaemic heart (control group) are represented in Figure 6A and B. The maximal NO concentration was 930 ± 25 nmol/L in non-ischaemic hearts (recipient hearts–baseline values) and decreased dramatically to 600 ± 15 nmol/L in ischaemic hearts (Figure 6C). The decrease in NO in ischaemic heart was accompanied by the increase in O2– (from 45 ± 5 to 110 ± 10 nmol/L) and the increase in ONOO– (from 180 ± 10 to 440 ± 15 nmol/L). S-NO-HSA treatment significantly increased NO concentration to 825 ± 20 nmol/L and concomitantly decreased O2– and ONOO– concentrations to levels about 20% lower than in non-ischaemic hearts (recipient hearts). The NO concentration reflects NO bioavailability, whereas the O2– and ONOO– concentrations reflect the extent of oxidative/nitroxidative stress in the myocardium. Therefore, the ratio of NO to ONOO– concentrations ([NO]/[ONOO–]) was used here as a marker of eNOS coupling/uncoupling.


Figure 6
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  		Figure 6 Typical amperograms showing changes of NO, O2–, and ONOO– concentrations recorded in vitro after calciumionophore (CaI) stimulation with nanosensors in the myocardium of a non-ischaemic heart (A) and in an ischaemic heart (B, control group). Non-ischaemic heart biopsies (‘baseline values’; n = 6) were from recipient hearts obtained from an ongoing study of orthotropic heart transplantation. Peak NO, O2–, and ONOO– concentrations in non-ischaemic hearts, ischaemic hearts (control group), and S-NO-HSA treated hearts; (C) control group vs. non-ischaemic hearts *P < 0.001 and S-NO-HSA vs. control group **P < 0.001. The concentration ratio: [NO]/[ONOO–] in non-ischaemic hearts, ischaemic (control), and S-NO-HSA treated hearts; (D) *P < 0.001 control group vs. non-ischaemic hearts and S-NO-HSA vs. control group **P < 0.001.

 
The [NO]/[ONOO–] ratio in ischaemic hearts (controls) was low (1.36 ± 0.06) when compared with non-ischaemic recipient hearts (5.17 ± 0.27). S-NO-HSA treatment partially, but significantly, restored the [NO]/[ONOO–] ratio to a level of 3.59 ± 0.18 (Figure 6D).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
Data presented here clearly show that in the detrimental setting of warm, unprotected ischaemia in the pig heart, the infusion of 0.1 µmol/kg/h S-NO-HSA from 15 min prior to ischaemia to 30 min of reperfusion preserves the function of eNOS, stabilizes the basal production of NO, decreases production of O2– and cytotoxic ONOO–, and therefore has beneficial effects on haemodynamics due to reduction of I/R injury.

In the S-NO-HSA treatment group, the maximal concentration of NO-release in heart biopsies after stimulation with the calcium ionophore A23187 [GenBank] reveals that endothelial NO production is near the range observed in biopsies from healthy animals, whereas the biopsies of untreated animals show a significantly decreased NO production. The maximal concentration of both O2– and ONOO– in the S-NO-HSA treatment group is slightly higher than in normal myocardium but significantly lower as in the untreated group.

NO deficiency due to its consumption by O2–, produced in high concentrations during ischaemia and reperfusion, plays an important role in the pathophysiology of I/R injury.2 The mechanism by which S-NO-HSA as an exogenous NO-donor can protect the dysfunction of the endothelium and prevent excessive O2– formation is based on prevention of eNOS uncoupling. The uncoupled eNOS can intermittently produce both NO and superoxide.3,21

In the present study, exogenous NO generated by S-NO-HSA prevents eNOS uncoupling in 15 min warm ischaemia of the heart. This further prevents the formation of excessive local peroxynitrite concentrations and consequent cleavage products. Three of these cleavage products (hydroxyl free radical, nitrogen dioxide free radical, and nitronium cation) are among the most reactive and damaging species and may be major contributors to the severe I/R damage.2,19 It is also important to emphasize once more that continuous infusion of 0.1 µmol/kg/h S-NO-HSA does not influence blood pressure.

From the results obtained with the calcium ionophore stimulation of the control heart biopsies it is evident that there is a deficiency in bioavailable NO in ischaemic hearts and enhanced O2– and ONOO– production. The production of NO and O2– by uncoupled eNOS increases the probability of a collision of these two molecules and rapid generation of ONOO–. There are several other potential sources of O2– in the myocardium in addition to eNOS, including NAD(P)H oxidase. However, uncoupled eNOS seems to be the most efficient generator of O2– and ONOO– in the endothelium as indicated by calcium ionophore stimulation of eNOS.17 We have demonstrated previously that, inhibition of NAD(P)H oxidase with apocynin, an NAD(P)H oxidase inhibitor, decreased the release of O2– by 25%, while inhibition of eNOS with N-nitro-L-arginine methyl ester (L-NAME) reduced O2– production by 70%.18 This provides direct evidence that uncoupled eNOS in the endothelium is probably the most significant source of cytotoxic ONOO–.

The present results are in accordance with the data obtained in the isolated rabbit heart: after 6 h of cold, cardioplegic arrest, where NO release was significantly higher and the corresponding O2– release significantly lower in S-NO-HSA-treated hearts compared with controls.13 Diminished reperfusion damage could also be demonstrated by the high-energy phosphates. There was a significant preservation of PCr and energy charge, a crucial parameter reflecting the energetic situation of the cell. Pre-treatment with S-NO-HSA prior to I/R is beneficial as the released NO can counteract the initial excessive NO production by eNOS, prevent uncoupling of eNOS at an earlier stage, and actively scavenge superoxide from other sources.12

Similarly, in failing rat hearts after 1 h of cold, cardioplegic arrest, the application of the ACE-inhibitor quinaprilat, an indirect NO-donor, during ischaemia and reperfusion resulted in a significantly higher preservation of myocardial high-energy phosphates compared with controls.22

In the present study there is an improvement in post-ischaemic haemodynamic performance. However, the results have to be separated in those during early reperfusion and those after weaning from CPB. During the initial reperfusion the pig is still on CPB at constant heart rate and constant flow. Coronary flow during this early period depicts the initial reactive hyperaemia that is seen in both groups irrespective of treatment followed by a decrease in flow, again in both groups. The massive burst of NO at onset of ischaemia, leads as a consequence on the one hand to the initially observed reactive hyperaemia upon reperfusion, on the other hand to depletion of L-arginine (and tetrahydrobiopterin) stores with the already described sequel.3

After weaning from CPB at constant heart rate and similar TPR, coronary flow starts to deteriorate in controls but remains elevated in the treatment group. Under the constant conditions mentioned earlier, there is a significant increase in LVSP and MAP, dP/dtmax and CO (in part) until the end of the experiment, indicating improved systolic function. Diastolic parameters such as LVEDP do only partly reflect this improvement although at the end of the experiment there is a clear trend in the treatment group. In contrast, dP/dtmin clearly shows the positive effects of S-NO-HSA treatment on diastolic function. Taken together, these results can only be explained by an indirect/direct, positive inotropic and lusitropic effect of S-NO-HSA on the myocardium, as a reduction of TPR was not seen under the dose of S-NO-HSA used. The lusitropic effects of NO are well known.23 Similarly, the positive-mediated inotropic potential of NO and the way how NO modulates myocardial contractility are well documented.24–26

CPB itself induces inflammatory reactions and can be involved in the generation of free radicals, mainly by leucocytes.27 Exogenous NO released from S-NO-HSA can also be beneficial as it may prevent the adherence of leucocytes to the endothelium as demonstrated in an intravital microscopy study of the liver in haemorrhagic shock.28 Enhanced NO concentrations may actually inhibit other sources of O2–, such as NADPH oxidase.29 Additionally, platelet aggregation is both impaired by a functioning eNOS and exogenous NO.

In the isolated rabbit heart, after 6 h of cold ischaemia, there was a similar picture: S-NO-HSA increased CO, a surrogate for systolic function. More importantly, left atrial pressure, a surrogate for diastolic function, remained constant during reperfusion in the treatment group, whereas it increased constantly in controls.13 These findings were underlined by a reduction of myocardial oxygen consumption (MVO2) during early reperfusion. A similar picture was observed in a recent study from our group focusing on MVO2 and delivery in the pig model of warm ischaemia: again S-NO-HSA led to a higher oxygen extraction during initial reperfusion.30 The reduction of MVO2 and the higher oxygen extraction is desirable during early reperfusion as thereby the ischaemic burden to the tissue is reduced. Loke et al. have shown that the MVO2-sparing effects of bradykinin, the ACE-inhibitor ramiprilate, and amlodipine are mediated via the B2-kinin receptor and NO.31

One limitation of the present study lies in the model itself: there is hardly any clinical situation comparable with global, warm, unprotected ischaemia, except ventricular fibrillation or cardiogenic shock. However, we aimed to test the effectiveness of the new substance S-NO-HSA under these hostile conditions. In preliminary studies we first tested, how long a pig heart can stand unprotected, warm ischaemia, and we had to realize that the maximum tolerated time span is between 15 and 20 min. A second limitation is the measurement of coronary flow by selective measurements on the LAD using ultrasonic technology. One can argue that the measurements only reflect the flow in the epicardial conductance vessels and not in the capillaries. However, if flow is reduced by oedema in the capillary vessels, also the flow in the main coronary artery branches will be reduced. Therefore we decided to take this clinically relevant measurement of coronary flow as a surrogate for myocardial perfusion.

In conclusion, the present study demonstrates a protective effect of S-NO-HSA in the heart in the detrimental setting of warm I/R, if applied already prior and during ischaemia, as well as during early reperfusion. The concentration of NO delivered to endothelial cells by this novel NO donor is in the range of physiological concentrations and does not affect systemic blood pressure. S-NO-HSA prevented uncoupling of eNOS by supplementing NO, decreasing oxidative/nitroxidative stress induced by O2–, and ONOO–, thereby improved (i) systolic and diastolic function, (ii) myocardial perfusion, and (iii) the energetic reserve of the heart. These beneficial effects can only be explained by a dual mechanism: on the one hand S-NO-HSA by supplementing NO stabilizes the endothelial cell leading to sufficient NO production after offset of NO supplementation, on the other hand the improved NO production has a indirect/direct positive inotropic and lusitropic effect on the myocardial cell. Therefore, S-NO-HSA treatment has the potential to prevent or mollify I/R injury.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
The study was supported by the Austrian Science Fund Grant (FWF #P 15194), the United States Public Health Service (HL-55397), Marvin & Ann Diley White Endowment, and the Biomimetic Nanoscience and Nanoscale Technology Program (Ohio University).


   Acknowledgements
 
This article is dedicated to Martin Vodrazka, our friend and colleague, who passed away too early for all of us.

Conflict of interest: none declared.


   Notes
 
{dagger} These authors contributed equally to this work. Back

{ddagger} Deceased. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

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