FIELD OF THE INVENTION The invention relates to methods of reducing effects of reperfusion injury after ischemic episodes, particularly in the cardiac muscle. More particularly, inhalation of gaseous nitric oxide at the time of reperfusion of the ischemic heart muscle reduces the long-term effects of reperfusion injury, such as reducing the development of long-term postinfarction (left) ventricular remodeling or heart failure.

BACKGROUND TO THE INVENTION

Every year, 500,000 people in the United States suffer from an ST-elevation myocardial infarction (Ml) (Hausenloy et al. 2008). Despite optimal reperfusion therapy, morbidity and mortality remain substantial, with 5 to 6% of patients experiencing a subsequent cardiovascular event by 30 days (Andersen et al; 200). Timely reperfusion of ischemic myocardium following myocardial infarction will halt the wavefront of myocardial tissue injury that extends form the endocardial towards the epicardial layers of the left ventricle and causes inevitable loss of cardiomyocytes. One of the major problems associated with reperfusion therapies, however, is the now recognized paradoxical increase in tissue injury upon reperfusion of an ischemic territory, a phenomenon referred to as reperfusion injury (I/R).

The pathophysiology of reperfusion injury is complex and multi-faceted. Prolonged (>30 min) myocardial ischemia results in increased production of oxygen-derived free radicals (ROS), platelet aggregation, complement activation, neutrophil infiltration, myocardial calcium overload, and myocyte apoptosis causing extensive myocardial infarction, depressed left ventricular contractile function and cardiac arrhythmias (Wang et al. 2001 , Duilio et al. 2001 ). At the molecular and cellular level, activation of nuclear factor-κB (NF- KB) (Iwata et al. 2001 ), increased coronary expression of adhesion molecules (VCAM and ICAM) (Carden et al. 2000, Youker et al. 1994), and activation of platelets and leukocytes were shown to mediate some of the reperfusion- associated inflammatory responses.

Currently, the single established strategy that is able to limit infarct size is established reperfusion using percutaneous coronary intervention (PCI) or thrombolytic therapy (Van de Werf et al. 2003) before the transmural wavefront of tissue injury has set in, an elusive goal for most Ml patients. A large number of pharmacological approaches has been followed in an effort to reduce reperfusion injury but these showed disappointing results in terms of limited experimental efficacy or failure to translate into useful clinical treatments (Ferdinandy et al. 2007, Hausenloy et al. 2008, Baran et al. 2001 , Dirksen et al. 2007, Pache et al. 2004). Cardioprotective effects of ischemic pre- and post- conditioning have been described but the underlying molecular mechanisms are currently very poorly understood and, hence, the development of drugs interacting with such molecular mechanisms is in its infancy (Ferdinandy et al. 2007). A new experimental approach (Thibault et al. 2008) to limit reperfusion injury was mentioned in an editorial by Hausenloy et al. (2008) and consists of temporarily interrupting reperfusion during percutaneous coronary intervention by means of several low-pressure inflations of the angioplasty balloon. This was shown to reduce long-term infarct size and to improve long-term left ventricular function. This procedure, however, requires an invasive intervention.

Overall, cardiologists are still confronted with a substantial patient death rate caused by postinfarcted failing hearts, not only during the standard short-term

1 -mth postinfarction follow-up period (Hausenloy et al. 2008), but, more importantly, also thereafter. In the largest randomized, multicenter international study in more than 14000 patients with acute myocardial infarction complicated by heart failure or left ventricular dysfunction (i.e. ejection fraction less than 35%), the incidence of the combined end-point of death, recurrent infarction or rehospitalization for heart failure at 1 year was -25% (Pfeffer et al. 2003). No drugs are currently available that are capable of further reducing the severity of long-term heart failure that complicates acute ischemic events. The need for such long-term cardioprotective drugs currently clearly remains unmet.

NO is an important signal transduction molecule in a variety of cell types and regulates vasomotor tone, platelet activation, interaction of platelets and leukocytes with the vessel wall, immune and inflammatory responses, and apoptosis. Impaired endogenous NO release by the injured coronary vascular endothelium contributes to several pathological processes in Ml/R injury. Administration of subvasodilator concentrations of NO-donors via an extracorporeal perfusion system in the dog (Lefer et al. 1993), or continuous intracoronary infusion of L-arginine before and at the time of reperfusion (Pernow et al. 1999) preserved coronary vasodilation, inhibited neutrophil accumulation, and reduced myocardial necrosis. The short half life of NO and the need for continuous intravascular infusion and subsequent systemic hypotensive side effects represent major shortcomings of such pharmacological interventions.

It was previously shown that nitric oxide for inhalation may constitute a novel treatment paradigm in IR injury because of its potent interaction with activated neutrophils and its antiapoptotic and cytoprotective effects on ischemic cardiomyocytes (Liu et al. 2007). These pleiotropic effects resulted in a significant reduction in infarct size 4 h after reperfusion of an occluded coronary artery and improved microvascular blood flow. Long-term effects of such NO inhalation are, however, unknown.

DESCRIPTION OF THE INVENTION

The invention is based on the surprising observation that inhalation of gaseous nitric oxide at the time of myocardial reperfusion after an ischemic episode is translating into improved long-term clinical outcome with lesser degrees of maladaptive ventricular remodeling and reduction of heart failure or reduced risk thereof. Heart failure is a condition caused by a weakened heart muscle implying that the heart needs to work harder to keep blood flowing through the body. Heart failure develops following injury to the heart such as ischemic damage, damage caused by a heart attack, long-term high blood pressure, or an abnormality of one of the heart valves. A functional heart failure stage classification system has been devised by the New York Heart Association (NYHA). According to this system, the stage or class of heart failure symptoms is assessed by the patient's ability to perform everyday activities and by the patient's quality of life. Four classes are discerned in the NYHA classification: - Class I (Mild heart failure): No limitation of physical activity. Ordinary physical activity does not cause undue fatigue, palpitation, or dyspnea (shortness of breath);

- Class Il (Mild heart failure): Slight limitation of physical activity. Comfortable at rest, but ordinary physical activity results in fatigue, palpitation, or dyspnea; - Class III (Moderate heart failure): Marked limitation of physical activity. Comfortable at rest, but less than ordinary activity causes fatigue, palpitation, or dyspnea; and

- Class IV (Severe heart failure): Unable to carry out any physical activity without discomfort. Symptoms of cardiac insufficiency at rest. If any physical activity is undertaken, discomfort is increased.

Heart failure is often not recognized until a more advanced stage of heart failure, commonly referred to as congestive heart failure, in which fluid may leak into the lungs, feet, legs, and in some cases the liver or abdominal cavity.

In the context of the present invention, the development of heart failure is meant to be correlated to an earlier ischemic or hypoxic event. Myocardial ischemia can develop e.g. as a result of a myocardial infarction or as a result of occluded coronary blood supply to the myocardium or by increased oxygen, and thus blood, demand by (a) tissue(s). "Low-flow or no-flow ischemia" or "demand ischemia", respectively, are the terms used for the causes of myocardial ischemia. The ST segment of an electrocardiogram (ECG) is helpful in the diagnosis of ventricular ischemia or hypoxia because under those conditions, ST segment depressions or elevations may occur. When the cause of myocardial ischemia or hypoxia is taken away, i.e. when the myocardial tissue is reperfused, reperfusion injury can occur as described above.

The invention therefore relates in a first aspect to methods of reducing in a mammal the development of heart failure following an acute myocardial ischemic episode, said methods comprising the step of administering to said mammal gaseous nitric oxide via inhalation. More in particular, said heart failure is referring to long-term heart failure such as heart failure complicating an acute ischemic period (such as by myocardial infarction) and occurring or developing at 1 week, 1 month or more after the occurrence of the ischemic or hypoxic episode. A traditional clinical endpoint of heart failure is usually defined as mortality rate 1 year after the occurrence of the acute myocardial ischemic or hypoxic episode. Alternatively, a triple endpoint may be defined including (i) cardiac death (or mortality), (ii) (re)hospitalization in case of deteriorating heart failure (e.g. in case of increase in NYHA classification, e.g. from class Il to III), and (iii) recidive/recurrent myocardial infarction.

In the above-mentioned methods according to the invention said development of heart failure may be (defined as or be expressed as) an increase over time in systolic and diastolic left ventricular volume. This may or may not be further accompanied by a reduction in systolic and diastolic function. Alternatively, but not exclusive, said development of heart failure may be (defined as or be expressed as) an increase in circulating levels of natriuretic peptides.

Natriuretic peptide include atrial natriuretic peptide (ANP) (or atrial natriuretic factor (ANF), or atriopeptin), brain natriuretic peptide (BNP) and the N-terminal portion of pro-BNP (NT-pro-BNP). ANP is a protein (polypeptide) hormone secreted by heart muscle cells. It is involved in the homeostatic control of body water, sodium, potassium and fat (adiposity). ANP is closely related to BNP (brain natriuretic peptide) and CNP (C-type natriuretic peptide), which all share the same amino acid ring. A review of cardiac biomarkers is provided by Carreiro-Lewandowski (2006).

In an alternative aspect, the invention covers methods of reducing or attenuating in a mammal the effects of reperfusion injury following an acute myocardial ischemic episode, said method comprising the step of administering to said mammal gaseous nitric oxide via inhalation. More in particular, said effects of reperfusion injury are long-term effects of reperfusion injury such as referring to effects of reperfusion injury or functionally detrimental effects of reperfusion injury developing or occurring at 1 week, 1 month or longer after the occurrence of the ischemic or hypoxic episode. Alternatively, said long-term effects of reperfusion injury are irreversible effects of reperfusion injury.

In the above-mentioned methods according to the invention said ischemic or hypoxic episode may be caused e.g. by coronary artery occlusion, myocardial infarction or prolonged coronary artery spasm. The coronary artery occlusion may be of any etiology such as of thrombotic or thromboembolic etiology, by spasm, by extrinsic compression or by trauma.

In order to obtain the therapeutic effect, the gaseous nitric oxide (NO) may be administered to the mammal in need thereof in the period coinciding with the reperfusion and ending during reperfusion or together with reperfusion. Alternatively, it may be administered over a period starting shortly prior to start of the reperfusion (pre-reperfusion period) and ending during reperfusion or together with reperfusion. In yet further alternative, the gaseous NO is administered in the period following established reperfusion. The total time of NO inhalation/administration may range from approximately 5 minutes to approximately 6 hours, or from approximately 5 minutes to approximately 5 hours, or may be for approximately 4 hours.

Thus, in any of the above-mentioned methods according to the invention said NO inhalation may be occurring during at least part of the reperfusion period, or may be occurring during a period spanning part of the pre-reperfusion period and at least part of the reperfusion period. The gaseous nitric oxide may be inhaled for a period of between 5 minutes and 5 hours, for instance for a period of 4 hours.

Systemic levels of NO obtainable via inhalation are generally substantially higher than those obtainable via NO-donor compounds without exerting negative side effects of high NO-donor concentrations. It is generally accepted that NO inhalation is safe when inhaled in concentrations ranging between approximately 5 ppm (parts per million) and approximately 100 ppm. Any NO- concentration within this range may be applied as "therapeutically effective dose" (in conjunction with the duration of administration).

Thus, in general, in any of the above-mentioned methods according to the invention, NO-doses ranging between approximately 5 ppm and approximately 100 ppm, or between approximately 5 ppm and approximately 80 ppm are acceptable, in particular this may be a NO-dose of approximately 80 ppm. It will be clear to the skilled person such as the treating physician or cardiologist that the NO-dosing (concentration of the inhaled NO and/or duration of NO- inhalation) may be varied according to the condition of the to-be-treated mammal as judged by said skilled person.

In general myocardial reperfusion may be achieved by standard methods including thrombolytic therapy (with any thrombolytic agent such as tPA, uPA, streptokinase, staphylokinase, plasmin, miniplasmin, microplasmin, or any active variant thereof), percutaneous coronary intervention (PCI), or coronary bypass grafting (which is likely to be emergency coronary bypass grafting).

Thus, any of the above-mentioned methods according to the invention may be combined with a reperfusion method such as thrombolytic therapy, percutaneous coronary intervention, or coronary bypass grafting. Alternatively, in any of the above-mentioned methods according to the invention the reperfusion method is chosen from thrombolytic therapy, percutaneous coronary intervention, or coronary bypass grafting.

Any of the above-mentioned methods according to the invention may be combined with (additional) thrombolytic therapy, anticoagulant therapy or antiplatelet therapy, or with a combination of at least two thereof.

It can further be envisaged that a method according to the invention is combined with any other therapy aiming at reducing short-term or long-term reperfusion injury. One example of such other therapy may be the stimulation of angiogenesis by administering growth factors (as protein or via gene therapy) such as vascular endothelial growth factor (VEGF, any isoform) or placental growth factor (PIGF, any isoform), see e.g. US 6,930,089 and US 7,105,168. Another growth factor is granulocyte colony stimulating factor (G-CSF) which is reported to have a beneficial effect by means of mobilizing bone marrow stem cells (e.g. Fukuda et al. 2002).

In order to measure the effect of any of the methods according to the current invention, any suitable method capable of measuring a relevant heart function can be applied. Such methods include assessing ventricular function and/or ventricular architecture, measuring wall thickness in the infarct core and border zone, and/or measuring the size of the infarct zone. Any combination of any of these (i.e. at least two) can be applied. Alternatively, or in combination with the above released cardiac biomarkers can be measured to assess the stage of heart failure or the extent of reperfusion injury. Of particular interest may be the assessment of left ventricular (global and/or regional) function and/or left ventricular architecture. Methods for assessing ventricular function and/or ventricular architecture, measuring wall thickness in the infarct core and border zone, and/or measuring the size of the infarct zone include single photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI) with injection of contrast fluid. Cardiac biomarkers include those circulating in serum (and thus having the advantage of being relatively easily amenable to determination of their concentration). In general, suitable cardiac biomarkers are those reflecting increased left ventricular hemodynamic stress, cardiomyocyte necrosis and inflammation (hsCRP, troponin, adiponectin). Cardiac biomarkers of heart failure include BNP and NT-pro BNP, or ANP. An overview of cardiac biomarkers is provided by Carreiro-Lewandowksi (2006).

The use of gaseous nitric oxide for treatment of any of the indications in any of the above-described methods of the invention is also covered in this invention.

All herein cited references are incorporated by reference.

FIGURE LEGENDS Figure 1. The experimental study protocol is schematically shown. In this prospective, randomized, placebo controlled, chronic experimental study, the effects of inhaled nitric oxide (iNO) are evaluated at baseline, during I/R, and at

2 days and 1 1 weeks after the acute ischemic episode.

Figure 2. Cardiac necrosis markers. Serum CK (Panel A), and Troponin (Panel B) levels were less in iNO pigs than in control after 4h reperfusion (P<0.05) and the CK-MB (Panel C) showed a trend in favour of inhaled NO-treated pigs

(P=O.07 versus control pigs).

Figure 3. Functional analysis of left ventricle (LV) function. LV end-diastolic and end-systolic volumes (LVEDV, panel A; and LVESV, panel B) were calculated. pMI = post myocardial infarction.

Figure 4. Infarct size. In hearts from control pigs (panel A), the infarct zone contains mainly fibrotic scar tissue (white) while more preserved viable myocardial tissue (red) is observed in iNO pigs (panel B). Infarct sizes were calculated (panel C, as % of left ventricle). EXAMPLES

1. MATERIALS AND METHODS

1.1 Animal preparation

The study was approved by the Animal Care and Use Committee of the University of Leuven. Juvenile domestic pigs of both sexes weighing 25-30 kg were used. All animals were studied in accordance with the Belgium National Institute of Health guidelines for care and use of laboratory animals. Pigs were pre-treated for 3 days with amiodarone to reduce life-threatening arrhythmias upon acute vessel occlusion (600 mg/day). Clopidogrel (150 mg/day) and aspirin (300 mg/day) were administered 1 day before and on the day of the procedure. Pigs were sedated using azaperone 3 mg/kg IM (Stresnil, Janssen Pharmaceutics) and anaesthetized using an IV bolus of ketamine (1 mg/kg, Anesketin, Eurovet) followed by a 10 mg/kg/h continuous infusion of 2% propofol (AstraZeneca, SA). Pigs were mechanically ventilated using a 50% oxygen gas mixture. Ventilation was adjusted to maintain physiologic PaCO ₂ and pH. Continuous electrocardiographic monitoring of heart rate, rhythm, and ST-segment changes was performed.

1.2 Experimental design The experimental protocol is schematically shown on Figure 1. In this prospective, randomized, placebo controlled, chronic experimental study, the effects of iNO are evaluated at baseline, during I/R, and at 2 days and 1 1 weeks after the acute ischemic episode.

On the day of ischemia and reperfusion, an 8 F introduction sheath was introduced into the right carotid artery for blood sampling and an 8 F catheter was introduced to measure blood pressure and engage the coronary arteries for angiography. At selected time points, a 6 F Mikro-Tip® pressure transducer catheter (Millar Instruments, Houston, TX) was inserted via the same catheter into the LV to measure maximum and minimum rates of LV pressure development (indices of systolic and diastolic LV function). All hemodynamic recordings were made for 1 min at a sampling rate of 2000/sec before ischemia, and at 30, 60, and 240 min of reperfusion. Data were processed using PowerLab recording and analysis software (AD Instruments, UK). Transient ischemia over the anterior wall was induced by inflating a properly-sized balloon-mounted stent for 45 min in the proximal part of LAD coronary artery. Coronary artery occlusion was confirmed by contrast injection and by ST- segment elevation on the ECG. After 45 min, the LAD balloon was deflated, and restoration of normal coronary flow for 4h was documented by angiography.

Thirteen pigs underwent a 45 minutes total occlusion of the LAD followed by 4h reperfusion. Pigs were randomized to O ₂ -enriched room air ventilation

(control) or O ₂ -enriched room air containing 80 ppm NO (iNO) using the

INOvent® delivery system starting 40 minutes after balloon occultation. After reperfusion for 240 min, the carotid arteriotomy were repaired and the dermal layers closed using standard techniques and the pigs were allowed to recover. Dual antiplatelet therapy consisting of aspirin (300mg/day) and clopidogrel

(75mg/day) was administered during the entire follow-up.

Cardiac MRI (3.0 T, Siemens, Erlangen, Germany) analysis was performed at 2 days and 1 1 weeks after the acute event.

After 1 1 weeks, an 8 F introduction sheath was introduced into the left carotid artery for blood sampling, hemodynamic measurements and coronary angiography, as described above. At the end of the protocol, the pigs were euthanized using overdose of propofol and the hearts were excised for postmortem analysis.

1.3 Hemodynamic measurements

Heart rate, mean blood pressure, LVEDP, LV pressures and maximum and minimum rates of pressure development (dP/dt _ma χ and dP/dt _m in) were determined using a microtip catheter (Millar® Instruments Inc., USA) at a sampling rate of 2000/sec. Data were processed using PowerLab recording and analysis software (AD Instruments, United Kingdom). 1.4 Cardiac necrosis markers

Arterial blood samples were collected at baseline, after 40 min of ischemia, and 30 min, 2h and 4h during reperfusion and 1 1 weeks post Ml for cardiac CK-MB measurements, and all samples were analyzed at the central core clinical chemistry laboratory (Gasthuisberg University Hospital, Leuven).

To quantitate the overall release of cardiac necrosis over the 4 hour reperfusion period, CK, CPK-MB and Troponin versus time curves were plot-fitted, and area under the curve (AUC) was derived, using the method reported by Vollmer et al.

(1993). Liver function tests, ANP, BNP and pro-BNP/NT-BNP were measured at baseline during I/R and at 1 1 weeks follow-up.

1.5 Magnetic resonance imaging: cine-angiography, delayed enhancement infarct and perfusion studies

Cardiac MRI (3.0 T, Sonata, Siemens, Erlangen, Germany) was performed at 2 days and 1 1 weeks after AMI. All studies were done with Siemens Numaris 4 cardiac MRI software, electrocardiographic triggering, and cardiac-dedicated surface coils. Global and regional function was assessed with breath-hold cineMRI in the cardiac short axis, vertical axis, and horizontal long axis. In the cardiac short axis, the LV was completely encompassed by contiguous 6-mm thick slices. Infarct area was defined as the zone of bright signal on late-enhanced images ie, 10-20 min after injection of 0.15 mmol/kg of gadopentetate dimeglumine (Gd-DTPA), using an inversion-recovery gradient- echo technique. All MRI studies were analysed on an off-line workstation (Cardioviewer) by investigators unaware of the treatment allocation. For assessment of global LV function and calculation of infarct volume, endocardial and epicardial borders were traced in end-diastolic and end-systolic short-axis slices. We calculated LV end-diastolic and end-systolic volumes (LVEDV and LVESV), ejection fraction and infarct size. Infarct volume, LVEDV and LVESV were indexed to body-surface area. We used the AHA 16 segments model and the classification of the coronary supplied region (Cerqueira et al. 2002, Kim et al. 2000). Transmural extent of late hyperenhancement was graded according to the following classification: <50% and >50 hyperenhancement. We calculated end-diastolic wall thickness and wall motion in the infarct zone. Changes in infarct size were expressed quantitatively (ml/m ² ). First-pass perfusion imaging was performed continuously for 1 minute obtaining 80 measurements (0.8 s temporal resolution) using a dual bolus injection of Gd-DTPA (0.0015/kg, and 0.05 at 3.5 cc/sec), using an ECG-gated steady state free precession (SSFP) imaging pulse sequence in cardiac short-axis direction. Regional perfusion was calculated for segments 1 -2-7-8-13 (perfusion area of the LAD artery) and used as most reliable parameter for perfusion (Barkhausen et al. 2004, Nagel et al; 2003).

1.6 Determination of infarct size

After the pigs were euthanized using an overdose of propofol, and the hearts were excised. The LV was sectioned into 5 slices perpendicular to the heart base-apex axis. Three transversely sectioned slice (basal, mid and apex) of the explanted heart were then incubated in 2,3,5 triphenyltetrazolium chloride (1.4 %, TTC) at 37 ^° C to evaluate viability. The extent of the infarct size (% of LV) was determined by planimetry on a Zeiss KS300 microscope using NIH image software independently by 3 experienced investigators blinded to the treatment group. 1.7 Blood and Tissue NOx measurements

Myocardial transmural biopsies taken from the infarct, border, and remote zones were weighted and homogenized in 400 μl 0.5 M NaOH using a Ribolyzer (Hybaid, Ashford, UK). Samples kept on ice for 15 minutes were deproteinized with an equal volume of 10% zinc sulfate, precipitates were centrifuged at 14,000 g, and total oxidized NO species (NO _x ) were determined in the supernatant using ozone-based chemiluminescence following injection in vanadium (lll)-chloride reductants in-line with the Sievers Model 280 NO analyzer (Boulder, CO). This assay method detects predominantly nitrite and nitrate in tissue (Yang et al. 2003, Lopez-Ramos et al. 2005).

1.8 Histological markers of myocardial fibrosis At 1 1 weeks, biopsies were taken from the infarct core, infarct border zone and remote area, and stored in 10% formalin tissue fixative for histological analysis. Replacement fibrosis will be assessed on 50 randomly-selected high power fields of myocardial 5-μm sections from paraffin-embedded biopsy specimens of the infarct, border, and remote zones from the anterior wall using a Sirius red stain (Serotec, UK). In addition, H&E, lectin and CD45, stains will be performed to evaluate the cellularity in the scar tissue and expression of matrix metalloproteinases will be investigated.

Apoptosis will be evaluated on different sections of the peri-infarct areas following terminal dUTP nick end-labeling (TUNEL) using the in situ cell death detection kit (Roche, Belgium). TUNEL-positive nuclei from these regions will be counted in 20-25 microscopic fields, and the percentage of apoptotic nuclei will be calculated from a total number of more than 2.500 nuclei for each animal.

1.9 Statistical analysis

Data are expressed as mean±SEM. ANOVA followed by a Bonferroni correction was used to analyze differences between groups with normally- distributed data. Repeated measurement analysis of variance was used to test serial hemodynamic and MRI values obtained at different time points during the experimental protocol. When data did not follow a normal distribution, Kruskal- Wallis non-parametric statistics were reported, and differences between groups were identified using a Mann-Whitney or Wilcoxon test. P<0.05 was considered statistically significant.

2. RESULTS

2.1 Acute complications and adverse events during follow-up

Of the 13 pigs subjected to coronary artery occlusion and randomized, 1 animal in the control group died because of ventilation problem during the MRI scanning 2 days after the acute event. One pig in the iNO group was euthanized 3 weeks after AMI /treatment because of a severe chronic knee infection in the right hindlimb. Both adverse events were not related to the experimental treatment. Of the 1 1 pigs, which thus far completed the endpoint of the study, blood gas analysis performed at baseline and during reperfusion confirmed normal oxygenation and ventilatory parameters, and no increase in methemoglobin levels was observed with NO inhalation, suggesting effective methemoglobin reductase activity (data not shown).

2.2. Hemodynamic measurements and left ventricular function

There were no differences in heart rate (HR), mean arterial blood pressure (MAP), or maximal and minimal rates of pressure development (dP/dtmax and dP/dt _mi n, respectively) between the groups at baseline. Heart rate increased modestly in all groups during ischemia and reperfusion (Table 1 ). LV dP/dtmax decreased and LVEDP increased 30 min after reperfusion in the control pigs, suggesting a reduction in both systolic and diastolic function (Table 1 ). In contrast, this progressive decline in LV function was not observed in iNO pigs, suggesting NO inhalation is safe and may have a favorable effect on LV contractile function (Table 1 ).

To investigate whether or not inhaled NO affects blood pressure in the setting of myocardial ischemia, MAP was measured for 10 min starting 5 min before NO was given. No blood pressure decline was observed in iNO pigs, suggesting that the treatment did not cause systemic vasodilation or cardiodepressant, hypotensive side effects (data not shown).

Table 1. Hemodynamic measurements before, during, and 11 weeks after myocardial ischemia-reperfusion injury in pigs.

^* p<0.05 vs. baseline

2.3 Evaluation of myocardial injury during I/R

To investigate whether or not inhaled NO reduces cardiac injury upon reperfusion. Release of cardiac necrosis markers (CK, CK-MB and Tropoin) in serum was followed over time. Serum CK, and Tropoin levels were less in iNO pigs than in control after 4h reperfusion (P<0.05; Figure 2) and the CK-MB showed a trend in favour of inhaled NO-treated pigs (P=O.07 versus control pigs). 2.4 Evaluation of global myocardial function (MRI)

For assessment of global LV function and calculation of infarct volume, endocardial and epicardial borders were traced in end-diastolic and end-systolic short-axis slices at 2 days and 1 1 weeks post Ml. We calculated LV end- diastolic, end-systolic volumes (LVEDV and LVESV), ejection fraction (EF) and infarct volume (Table 2 and Figure 3). Infarct volume, LVEDV and LVESV were indexed to body-surface area. MRI measurements at 2d after I/R showed LV ejection fraction (LVEF) decreased similarly in control and iNO pigs (28±2 vs 32±3%, respectively). Infarct size tended to be less in iNO pigs in control pigs (15±2 vs 22±3%, P=0.06). After 1 1 weeks, LVEDV increased in control from 74±5 to 210±33 ml (P<0.05), whereas in iNO pigs, LVEDV increased from 83±6 to 150±14 ml (P<0.05). LVEDV increased to a greater extent in control than in iNO pigs (P <0.05). After 1 1 weeks, LVESV increased from 53±3 to 147±23 ml in control and from 57±6 to 97±14 ml in iNO (P<0.05). LVESV increased to a greater extent in control than in iNO pigs (P<0.05). LVEF at 1 1 weeks was similar in both groups (36±3 vs 30±2% in iNO and control, respectively), while infarct size tended to decrease more in iNO than control (20±3 vs 13±2%, P=0.06) (Table 2 and Figure 3). Table 2. MRI-based Analysis of Global LV Function at 2 days (baseline) and 11 weeks after m ocardial IR in ur in i s

^* p<0.05 vs. BL; # p<0.05 vs. control; ^** p<0.06

2.5 Evaluation of regional myocardial function (MRI)

To assess regional LV function, we used the AHA 16 segments model and the classification of the coronary supplied region. Transmural extent of late hyperenhancement was graded according to the following classification: <50% and >50 hyperenhancement. We calculated end-diastolic wall thickness, systolic wall thickening (dWTr) and wall motion in the infarct zone. Of the segments with >50% transmural extent of infarction, end-diastolic wall thickness 1 1 weeks after I/R was greater in iNO than in control pigs (9.6±0.3 vs 6.9±0.2, P<0.001 ) (Table 3).

Table 3. Regional function analysis by MRI

"P^.Oδj ^O.OI , ANCOVA

2.6 Histological markers of myocardial fibrosis

To investigate whether or not inhaled NO reduce infarct size and prevent maladaptive remodeling, transverse sections of the heart were obtained and stained with 1 % 2,3,5-triphenyltetrazolium chloride (TTC). In hearts from control pigs, the infarct zone contains mainly fibrotic scar tissue (white) while more preserved viable myocardial tissue (red) is observed in iNO pigs (Figure 4). We also observed a trend towards a smaller Infarct size as a fraction of LV in the iNO pigs (p=0.07) (Figure 4).

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