In the present study, we hypothesized that postcon (postconditioning) confers cardioprotection in vivo by reducing the production of ONOO (peroxynitrite) and nitro-oxidative stress subsequent to the inhibition of the iNOS (inducible NO synthase). Patients with AMI (acute myocardial infarct) were randomly assigned to undergo percutaneous coronary intervention without (control) or with ischaemic postcon by three episodes of 30-s inflation and 30-s deflation of the angioplasty balloon. Animal models of MI/R (myocardial ischaemia/reperfusion) injury were induced in rats by occluding the left coronary artery for 40 min followed by 4-h reperfusion. Rats were randomized to receive vehicle, postcon (three cycles of 10-s reperfusion and 10-s coronary re-occlusion preceding full reperfusion), the selective iNOS inhibitor 1400W or postcon plus 3-morpholinosydnonimine (an ONOO donor). Postcon in patients reduced iNOS activity in white blood cells, decreased plasma nitrotyrosine, a fingerprint of ONOO and an index of nitro-oxidative stress, and improved cardiac function (P<0.01 compared with control). In rats, postcon reduced post-ischaemic myocardial iNOS activity and nitrotyrosine formation, reduced myocardial infarct size (all P<0.05 compared with control) and improved cardiac function. Administration of 1400W resembled, whereas 3-morpholinosydnonimine abolished, the effects of postcon. In conclusion, reduction in ONOO-induced nitro-oxidative stress subsequent to the inhibition of iNOS represents a major mechanism whereby postcon confers cardioprotection in vivo.

INTRODUCTION

The heart can be protected from reperfusion injury by brief episodes of ischaemia during early reperfusion following a pronounced ischaemic insult, a process referred to as ischaemic postcon (postconditioning) [1,2]. Studies by others [3,4] and ourselves [5] have demonstrated that postcon can reduce infarct size without reported adverse events in humans, indicating that postcon can be a clinically applicable therapeutic approach. However, the mechanisms of postcon, in particular in humans, are uncertain.

NO is a mediator in postcon-mediated cardioprotection [6]. However, NO may react with superoxide, whose production is increased during post-ischaemic reperfusion to form a more toxic nitrating and oxidant agent ONOO (peroxynitrite), an RNS (reactive nitrogen species). ONOO is one of the major triggers of cardiomyocyte apoptosis [7,8]. Hypoxic postcon reduces reoxygenation-induced cardiomyocyte death, which may be attributable to the reduced production of ONOO [9]. It is unknown whether a reduction in ONOO production and the subsequent reduction in nitro-oxidative stress may represent a major mechanism whereby postcon confers cardioprotection in vivo.

PMN (polymorphonuclear leucocyte) accumulation/activation has been implicated as a primary mechanism underlying MI/R (myocardial ischaemia/reperfusion) injury [10,11]. Studies show that PMNs express iNOS (inducible NO synthase) and produce toxic RNS [12,13]. The iNOS, when expressed in isolated cardiomyocyte, is essential for the development of β-adrenergic-receptor-mediated increases in shortening, but becomes essential for the ability of neutrophils to damage myocytes when the neutrophils migrate in proximity to myocytes [14]. Therefore iNOS activation during MI/R may be either beneficial or detrimental depending on the source of origin and/or the magnitude of activation. We hypothesized that postcon confers cardioprotection in vivo primarily by reducing the production of nitrotyrosine, a footprint of ONOO and an index of nitro-oxidative stress, subsequent to the inhibition of PMN-induced iNOS activation.

We designed a small clinical trial in patients with acute myocardial infarction receiving PCI (percutaneous coronary intervention) in parallel with animal experiments of MI/R to address: (i) whether or not postcon can attenuate post-ischaemic myocardial cellular injury and enhance cardiac functional recovery in vivo by reducing nitro-oxidative stress subsequent to the decrease in ONOO formation, and (ii) whether or not postcon is associated with the suppression of iNOS activation and accumulation of PMNs during reperfusion in the myocardium.

MATERIALS AND METHODS

Ethics

The clinical trial was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. The study protocol was approved by the institutional ethics committee. All subjects gave written informed consent after having been given full explanation of the purpose, nature and risk of all procedures used.

Inclusion and exclusion criteria of acute myocardial infarction patients

Patients whose ST segment elevation was >0.1 mV in at least two contiguous electrocardiographic leads from the onset of chest pain were enrolled in the present study. Patients' exclusion criteria were: (i) cardiogenic shock, (ii) left main coronary artery occlusion or severe stenosis, (iii) blood flow in the IRA (infarct-related artery) ≥TIMI (thrombolysis in myocardial infarction) grade 1, (iv) obvious coronary collaterals to the risk region evidenced by Rentrop grade ≥1, (v) previous myocardial infarction, (vi) treatment with glycoprotein IIb/IIIa receptor antagonists before the procedure, and (vii) infection or surgery within 2 weeks [5].

Coronary angiography and clinical experimental design

Patients were examined at admission and pre-medicated with clopidogrel (300–600 mg) and aspirin before catheterization. They were randomly assigned either to the control group (n=28) or the postcon group (n=22). Clinical PCI was completed as described previously [5]. In the control group, the balloon catheter was withdrawn immediately after the coronary artery dilatation, and no further intervention was applied. A stent was then deployed using a different angioplasty balloon catheter. In the postcon group, the artery was predilated as described previously [5] and, immediately thereafter, three cycles of 30-s deflation and 30-s inflation of the angioplasty balloon were applied at the onset of reperfusion.

Killip class was assessed independently by two cardiologists at admission, 3 and 5 days after PCI. LVEF [LV (left ventricular) ejection fraction] was determined by echocardiogram. Blood samples were taken immediately before PCI and 4 h, 24 h, 3 days and 7 days after PCI under the non-fasting condition for bioanalyses.

Animal experiment protocol

The study was approved by the institutional ethic committee and conforms with U.S. National Institutes of Health guidelines. Male Sprague−Dawley rats (250–300 g) were anaesthetized with sodium pentobarbital. MI was produced by exteriorizing the heart through a left thoracic incision and occluding the LAD (left anterior descending artery) with a silk slipknot. After 40 min of ischaemia, the slipknot was released and the myocardium was reperfused for 4 h. Sham (sham-operated control rats) underwent the same surgical procedures except that the LAD was not occluded.

A total of rats were randomized to receive one of the following treatments (n=16 each): (i) sham, rats receiving vehicle (0.9% NaCl); (ii) MI, rats receiving vehicle during reperfusion; (iii) postcon, three cycles of 10-s reperfusion and 10-s LAD re-occlusion preceding the reperfusion, (iv) 1400W (a selective iNOS inhibitor), MI rats receiving 1400W infusion at 10 μmol·kg−1 of body weight·h−1 during reperfusion, or (v) postcon plus SIN-1 (3-morpholinosydnonimine; an ONOO generator which can simultaneously generate NO and superoxide in aerobic conditions [15]), rats receiving postcon and SIN-1 infusion at 1 mmol·kg−1 of body weight·h−1 during reperfusion. The doses of 1400W and SIN-1 were chosen based on our preliminary dose-finding study, which showed that 1400W at 10 μmol·kg−1 of body weight·h−1 significantly inhibited myocardial iNOS activity and SIN-1 at 1 mmol·kg−1 of body weight·h−1 significantly increased myocardial nitrotyrosine production in the ischaemic-reperfused rat hearts (see the Results section for details), but yet did not cause significant myocardial cellular damage in sham-perfused rat hearts (results not shown).

Determination of myocardial functional recovery in rats

In the rats, LV function was continuously monitored during the entire MI/R period via a Millar Mikro-Tip catheter pressure transducer inserted into the LV via left carotid artery as we have described previously [16]. HR (heart rate), LVDEP (LV end diastolic pressure), LVSP (LV systolic pressure) and LVDP (LV developed pressure) were derived by computer algorithms (Chengdu Instrument).

Determination of myocardial infarct size in rats and cellular injury

The infarct size was measured with a double-staining technique using Evans Blue−TTC (Triphenyltetrazolium Chloride) staining and a digital imaging system, as described previously [16] (Additional n=8 rats per group were used for infarct size determination.). Blood samples were drawn before ligation and at the end of reperfusion.

Plasma cTnI (cardiac-specific troponin I) from rats and patients were measured spectrophotometrically (Beckman DU 640 instrument) with commercially available assay kits (Nanjing Jiancheng). The plasma samples were coded, and the levels of cTnI (cardiac-specific troponin I) were assayed in duplicate by an investigator initially blinded to the research groups.

Determination of rat myocardial apoptosis and caspase 3 activity

Myocardial apoptosis was determined by a combination of histochemical staining [TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling staining)] and caspase 3 activity detection. Immunohistochemical staining was employed to detect apoptotic cardiomyocytes by using an in situ apoptosis detection kit (Roche) according to the manufacturer's instructions.

The activity of caspase 3, a final common pathway in caspase-dependent apoptosis, in ischaemic rat heart tissue, was determined by using a caspase 3 colorimetric assay kit (Chemicon International) according to the manufacturer's instructions as reported previously [17]. Samples were incubated at 37 °C for 2 h, and p-nitroanilide production was measured at 405 nm. The results were presented as the fold increase in caspase 3 activity relative to that in the MI group.

Determination of MDA (malondialdehyde) in patient plasma and rat heart tissue

MDA is a stable metabolite of the ROS (reactive oxygen species)-mediated lipid peroxidation cascade. Patient blood samples were collected, and the MDA levels were measured with a commercial kit (Baster Biological Tech), expressed as nmol/ml. In rats, the heart tissue was harvested at the end of the experiment and homogenized in ice-cold 150 mM KCl for determination of MDA as described previously [18]. The MDA concentration of the homogenates was determined spectrophotometrically and was expressed as nmol/g of tissue.

Determination of PMN accumulation in vivo

MPO (myeloperoxidase) activity was used as an index of PMN accumulation in the heart, since it correlates closely with the number of neutrophils present in the heart [19]. Cardiac tissue MPO activity was determined using a commercial assay kit (Jiancheng) according to the manufacturers' instructions. Also, the cardiac MPO was also visualized with immunohistochemistrical staining, using specific antibody (Baster Biological Tech).

To assess the effect of postcon on PMN accumulation in patients, WBC (white blood cell) count and neutrophil ratio in patients' peripheral WBCs were measured in the Department of Ecsomatics at the Chaoyang Hospital.

Determination of iNOS activity, NOx (nitrite/nitrate) content in rat ischaemic cardiac tissue and iNOS activity in WBCs in patients

After reperfusion, rat cardiac tissue samples in the ischaemic area were homogenized and centrifuged for 30 min at 12000 g at 4 °C. Protein concentrations in the supernatants were measured using the bicinchoninic acid method. In patients, WBCs were isolated by using a red blood cell lysis solution kit (Genmed Scientifics). The iNOS activities of the rat heart tissue supernatants and of the isolated WBCs were measured with a commercial assay kit (Jiancheng). Specific iNOS enzymatic activity is referred as pmol of L-[3H]citrulline produced·mg−1 of protein·min−1. NOx (stable metabolites of NO) in supernatants was determined by the Griess reaction assayed using an NOx concentration assay kit (R&D Systems).

Determination of nitrotyrosine content in rat ischaemic cardiac tissue and in patient plasma

The nitrotyrosine content in rat cardiac tissue homogenate and in patients plasma was determined with an ELISA kit (Cell Sciences), as described previously [17,20]. The results are presented as nmol of nitrotyrosine/g of protein in tissue homogenate and nmol of nitrotyrosine/litre in the plasma.

Statistical analysis

All values are means±S.E.M. All biochemical assays were performed in duplicate and the results were averaged. Data were subjected to ANOVA, followed by the Bonferroni correction for post-hoc Student's t tests. The correlation between WBC iNOS activity and plasma levels, nitrotyrosine and cTnI in patients was evaluated using the Pearson's test. P<0.05 was considered statistically significant.

RESULTS

Human study data

Patient characteristics

The patients' baseline demographics, cardiovascular risk factors, clinical measures of cardiac function and medications did not differ significantly between the MI control and postcon groups (Table 1). Also, the number of patients who received nitrate and statin administrations did not differ between groups. In addition, treatments after PCI were similar between the MI control and postcon groups in terms of doses of diuretics and vasodilators (results not shown).

Table 1
Baseline characteristics of the study population

Values are means±S.D., and number of patients. ACEI, angiotensin-converting enzyme inhibitors; BMI, body mass index; DBP, diastolic blood pressure; HBP, high blood pressure; SBP, systolic blood pressure. No significant differences were detected between the two groups at baseline.

Characteristic MI (n=28) Postcon (n=22) 
Age (years) 65.9±7.9 65.5±8.1 
Gender (n) (male/female) 28 (17/11) 22 (14/8) 
BMI (kg/m223.1±2.1 23.0±2.5 
SBP (mmHg) 139±19 139±16. 
DBP (mmHg) 71±16 68±14 
Patients with   
 HBP (n17 14 
 Dyslipidaemia (n17 11 
 Diabetes (n11 
Smokers (%) 51 45 
LVEF (%) 53±10 54±7 
Past drug treatment (n  
 Statins 15 11 
 Calcium channel blocker 
 ACEI 16 11 
 β-Adrenoceptor blocker 11 
Drug treatment during the study (n  
 Nitrates 28 22 
 β-Adrenoceptor blocker 27 22 
 ACEI 28 22 
 Statins 23 19 
 Anticoagulants/antithrombotic treatment 28 22 
Characteristic MI (n=28) Postcon (n=22) 
Age (years) 65.9±7.9 65.5±8.1 
Gender (n) (male/female) 28 (17/11) 22 (14/8) 
BMI (kg/m223.1±2.1 23.0±2.5 
SBP (mmHg) 139±19 139±16. 
DBP (mmHg) 71±16 68±14 
Patients with   
 HBP (n17 14 
 Dyslipidaemia (n17 11 
 Diabetes (n11 
Smokers (%) 51 45 
LVEF (%) 53±10 54±7 
Past drug treatment (n  
 Statins 15 11 
 Calcium channel blocker 
 ACEI 16 11 
 β-Adrenoceptor blocker 11 
Drug treatment during the study (n  
 Nitrates 28 22 
 β-Adrenoceptor blocker 27 22 
 ACEI 28 22 
 Statins 23 19 
 Anticoagulants/antithrombotic treatment 28 22 

Post-ischaemic myocardial function and cellular injury in patients

The Killip class (Figure 1A) and LVEF (Figure 1B) did not differ between the MI control and the postcon group at admission. However, Killip class in the postcon group was significantly lower than that in MI group at days 3 and 5 after PCI. Similarly, at day 7, the LVEF value in the postcon group was higher than that in the control (P=0.034).

Postcon improved cardiac function after PCI in AMI patients
Figure 1
Postcon improved cardiac function after PCI in AMI patients

Postcon improved Killip class (A) and LVEF (B) of AMI patients after PCI. MI indicates patients received ordinary PCI treatment and postcon indicates rats or patients received postcon. The number of patients received postcon is 22 and 28 patients without postcon. *P <0.05 compared with postcon. d, days.

Figure 1
Postcon improved cardiac function after PCI in AMI patients

Postcon improved Killip class (A) and LVEF (B) of AMI patients after PCI. MI indicates patients received ordinary PCI treatment and postcon indicates rats or patients received postcon. The number of patients received postcon is 22 and 28 patients without postcon. *P <0.05 compared with postcon. d, days.

Plasma levels of cTnI, a surrogate reflecting myocardial cellular damage, did not significantly differ between MI control and postcon group at baseline, but it increased 4 h after PCI relative to pre-PCI levels (P<0.001) in both groups and peaked at 24 h after PCI. Postcon attenuated the increase in plasma cTnI after PCI (P<0.05 compared with MI group; Figure 2A).

Effects of postcon on plasma levels of cTnI (A), nitrotyrosine (B), MDA (C), WBC count (D) and WBC iNOS activity (E) in patients receiving PCI
Figure 2
Effects of postcon on plasma levels of cTnI (A), nitrotyrosine (B), MDA (C), WBC count (D) and WBC iNOS activity (E) in patients receiving PCI

MI indicates patients received ordinary PCI treatment (n=28). Postcon indicates patients received postcon (n=22). Blood samples were taken immediately before PCI and 4 h, 24 h, 3 days and 7 days after PCI under the non-fasting condition for bioanalyses. *P <0.05 and **P <0.01 compared with MI; $P<0.001 compared with Pre.

Figure 2
Effects of postcon on plasma levels of cTnI (A), nitrotyrosine (B), MDA (C), WBC count (D) and WBC iNOS activity (E) in patients receiving PCI

MI indicates patients received ordinary PCI treatment (n=28). Postcon indicates patients received postcon (n=22). Blood samples were taken immediately before PCI and 4 h, 24 h, 3 days and 7 days after PCI under the non-fasting condition for bioanalyses. *P <0.05 and **P <0.01 compared with MI; $P<0.001 compared with Pre.

Plasma levels of nitrotyrosine and MDA, WBC count and WBC iNOS activity in patients

Plasma nitrotyrosine in the control (MI) group increased at 4 h after PCI (approx. 3-fold of the value at pre-PCI) and peaked at 24 h after PCI (Figure 2B), which was coincident with the peak level of plasma cTnI (Figure 2A). Postcon significantly attenuated the increases of nitrotyrosine (P<0.05 compared with MI). The profound increase in plasma MDA was seen at 4 h after PCI (approx. 6-fold of the value at pre-PCI; Figure 2C) in the control group, which deceased gradually thereafter and returned to baseline (pre-PCI) values at day 7 after PCI. Postcon attenuated the increase in MDA after PCI (P<0.05 compared with MI). Significant increase in WBC count occurred at 4 and 24 h after PCI in the MI group (Figure 2D), and postcon significantly attenuated the increase. However, PCI did not significantly affect the percentage of neutrophils in WBC (results not shown). The iNOS activity in WBC increased at 4 h after PCI and peaked at 24 h in the MI group, which was reduced by postcon (Figure 2E).

Correlation between WBC iNOS activity and plasma levels of nitrotyrosine and cTnI in patients

As plasma levels of cTnI and nitrotyrosine and WBC iNOS activity all coincidently peaked at 24 h after PCI in patients (Figure 2), we analysed their correlation. Plasma levels of nitrotyrosine were strongly correlated with WBC iNOS activity [r=0.73 (95% confidence interval, 0.66−0.79); P<0.0001] and plasma levels of cTnI [r=0.72 (95% confidence interval, 0.66−0.78); P<0.0001]. In addition, a strong correlation existed between WBC iNOS activity and plasma cTnI [r=0.72 (95% confidence interval, 0.65−0.78); P<0.0001].

Animal study data

Effects of postcon, 1400W and SIN-1 on post-ischaemic functional recovery in rats

Our dose-finding study shows that 1400W at 10 μmol·kg−1 of body weight·h−1 significantly inhibited myocardial iNOS activity and SIN-1 at 1 mmol·kg−1 of body weight ·h−1 significantly increased myocardial nitrotyrosine production in the ischaemic reperfused rat hearts when administrated during reperfusion (Figure 3). Therefore the above doses of 1400W and SIN-1 were used in the subsequent experiments.

Effects of different doses of 1400W (A) and SIN-1 (B) on rat myocardial iNOS activity (A) and nitrotyrosine content (B)
Figure 3
Effects of different doses of 1400W (A) and SIN-1 (B) on rat myocardial iNOS activity (A) and nitrotyrosine content (B)

Various doses of 1400W or SIN-1 were infused intravenously during reperfusion in rats subjected to 40 min of coronary ligation and 4 h of reperfusion. *P <0.05 compared with saline (n=8 in each group).

Figure 3
Effects of different doses of 1400W (A) and SIN-1 (B) on rat myocardial iNOS activity (A) and nitrotyrosine content (B)

Various doses of 1400W or SIN-1 were infused intravenously during reperfusion in rats subjected to 40 min of coronary ligation and 4 h of reperfusion. *P <0.05 compared with saline (n=8 in each group).

In the ischaemic-reperfused rat hearts, myocardial ischaemia significantly reduced HR, decreased LVSP and increased LVEDP compared with the baseline (Figure 4). Postcon did not significantly affect LVSP and HR after ischaemia (Figures 4A and 4E), but improved myocardial functional recovery by reducing LVEDP in the postcon group compared with the control group (Figure 4B; P<0.01). Consequently, the LVDP in the postcon group was significantly higher than that in the control group after 60 min of reperfusion and thereafter during reperfusion (P<0.01). Postcon significantly improved cardiac contractility (Figure 4C) and relaxation (Figure 4D), respectively, after 120 and 60 min of reperfusion compared with control (P<0.05). The effects of postcon on haemodynamic improvements were counteracted by SIN-1 infusion, whereas 1400W exerted a postcon-like effect on the recovery of myocardial function.

Postcon improved cardiac function during reperfusion in rats
Figure 4
Postcon improved cardiac function during reperfusion in rats

Effect of postcon on LVSP (A), (B) LVEDP, rate of increase in LV pressure [+dp/dt (C) and −dp/dt (D)] and heart rate (HR) (E). MI indicates only MI/R group in animal experiment; postcon indicates postcon group; 1400W, MI plus 1400W and SIN-1, postcon plus SIN-1. Results are means±S.E.M., n=16/group. *P <0.05, **P <0.01 and ***P <0.001 for postcon compared with MI. #P <0.05, ##P <0.01 and ###P <0.001 for 1400W compared with MI. P <0.05, ●●P <0.01 and ●●●P <0.001 for SIN-1 compared with postcon. BMP, beats/min I40, 40 min of ischaemia; R60 etc., 60 min of reperfusion etc.

Figure 4
Postcon improved cardiac function during reperfusion in rats

Effect of postcon on LVSP (A), (B) LVEDP, rate of increase in LV pressure [+dp/dt (C) and −dp/dt (D)] and heart rate (HR) (E). MI indicates only MI/R group in animal experiment; postcon indicates postcon group; 1400W, MI plus 1400W and SIN-1, postcon plus SIN-1. Results are means±S.E.M., n=16/group. *P <0.05, **P <0.01 and ***P <0.001 for postcon compared with MI. #P <0.05, ##P <0.01 and ###P <0.001 for 1400W compared with MI. P <0.05, ●●P <0.01 and ●●●P <0.001 for SIN-1 compared with postcon. BMP, beats/min I40, 40 min of ischaemia; R60 etc., 60 min of reperfusion etc.

SIN-1 counteracted, whereas 1400W mimicked, postcon-mediated reductions in myocardial infarct size and apoptosis following MI/R in rats

To determine whether attenuation of ONOO formation and the subsequent reduction in nitro-oxidative stress play a major role in postcon-mediated cardioprotection, we tested whether or not the ONOO donor SIN-1 could reverse postcon cardioprotection. Postcon reduced the increase in myocardial infarct size compared with the MI group which was accompanied with reduction in plasma levels of cTnI (Figures 5A–5C; P<0.05). These protective effects of postcon were abolished by treatment with SIN-1, but mimicked by 1400W. MI/R was associated with significant increases of myocardial apoptotic cell death and caspase 3 activity (P<0.001, MI compared with sham; Figures 5D–5F), which was attenuated by postcon. SIN-1 abolished, whereas 1400W mimicked, the effects of postcon in attenuating myocardial apoptosis.

Effects of rat myocardial necrosis and apoptosis after MI/R
Figure 5
Effects of rat myocardial necrosis and apoptosis after MI/R

(A) Representative photomicrographs of Evans Blue−TTC staining in heart tissue slices from rats. (B) Myocardial infarct size (IS) expressed as percentage of area at risk (AAR) of the ischaemic−reperfused myocardium. (C) Plasma cTnI concentration in different groups. (D) Representative photomicrographs of TUNEL staining in all five groups. Total nuclei were labelled with PI (propidium iodide) (red), and apoptotic nuclei were detected by TUNEL staining (green). (E and F) Summary of TUNEL-positive myocytes (E) and caspase 3 activity (F). Sham represents sham operation; MI, myocardial ischaemia reperfusion alone; post, postcon group; 1400W, MI plus 1400W group; SIN-1, postcan plus SIN-1 group. Results are means±S.E.M., n=16/group. *P <0.05 or **P <0.01 or ***P <0.001 compared with MI. ##P <0.01 or ###P <0.001 compared with postcon (Post); $P<0.001 compared with Sham.

Figure 5
Effects of rat myocardial necrosis and apoptosis after MI/R

(A) Representative photomicrographs of Evans Blue−TTC staining in heart tissue slices from rats. (B) Myocardial infarct size (IS) expressed as percentage of area at risk (AAR) of the ischaemic−reperfused myocardium. (C) Plasma cTnI concentration in different groups. (D) Representative photomicrographs of TUNEL staining in all five groups. Total nuclei were labelled with PI (propidium iodide) (red), and apoptotic nuclei were detected by TUNEL staining (green). (E and F) Summary of TUNEL-positive myocytes (E) and caspase 3 activity (F). Sham represents sham operation; MI, myocardial ischaemia reperfusion alone; post, postcon group; 1400W, MI plus 1400W group; SIN-1, postcan plus SIN-1 group. Results are means±S.E.M., n=16/group. *P <0.05 or **P <0.01 or ***P <0.001 compared with MI. ##P <0.01 or ###P <0.001 compared with postcon (Post); $P<0.001 compared with Sham.

Effects of postcon on myocardial MPO activity, iNOS activity and NOx content following MI/R in rats

MI/R caused a marked increase in myocardial MPO activity (Figures 6A and 6B) and a concomitant increase in myocardial iNOS activity (Figure 5C; P<0.01 compared with sham), which was inhibited by postcon (P<0.01 postcon compared with MI). The effect of postcon on MPO activity and iNOS activity was not significantly affected by SIN-1. 1400W did not significantly affect cardiac MPO activity (Figure 6B), but decreased myocardial iNOS activity to a level comparable with the postcon group. MI/R resulted in significant increase in myocardial NOx content in the MI group (approx. 7-fold of the sham group, Figure 6D). Both postcon and 1400W attenuated (P<0.05 compared with MI), but did not completely block, the increase in myocardial NOx (P<0.05 compared with sham). In contrast, SIN-1 abolished the effect of postcon in attenuating the increase in NOx in the infarcted myocardium (P<0.05, SIN-1 compared with postcon; Figure 6D).

Effects of postcon on rat myocardial MPO activity (A and B), myocardial iNOS activity (C), NOx content (D), MDA concentration (E) and nitrotyrosine content (F) after 40 min of ischaemia and 4 h reperfusion
Figure 6
Effects of postcon on rat myocardial MPO activity (A and B), myocardial iNOS activity (C), NOx content (D), MDA concentration (E) and nitrotyrosine content (F) after 40 min of ischaemia and 4 h reperfusion

In (A), representative photomicrographs of immunochemical staining of MPO activity in cardiac tissue are shown. Values are means±S.E.M., n=16/group. *P <0.05 and **P <0.01 compared with MI; #P <0.05 and ##P <0.01 compared with postcon (Post); $P<0.001 compared with sham.

Figure 6
Effects of postcon on rat myocardial MPO activity (A and B), myocardial iNOS activity (C), NOx content (D), MDA concentration (E) and nitrotyrosine content (F) after 40 min of ischaemia and 4 h reperfusion

In (A), representative photomicrographs of immunochemical staining of MPO activity in cardiac tissue are shown. Values are means±S.E.M., n=16/group. *P <0.05 and **P <0.01 compared with MI; #P <0.05 and ##P <0.01 compared with postcon (Post); $P<0.001 compared with sham.

Effects of postcon on myocardial MDA and nitrotyrosine content following MI/R in rats

MI/R resulted in a 2.5-fold increase in MDA level in the cardiac tissue (P<0.05 compared with sham; Figure 6E). Postcon minimally but significantly reduced MI/R-induced MDA increase (P<0.05 postcon compared with MI). 1400W did not exert significant effect on cardiac MDA content, whereas SIN-1 counteracted the effect of postcon in reducing cardiac MDA. In contrast, MI/R resulted in an approx. 7-fold increase in nitrotyrosine level in the cardiac tissue (P<0.01 compared with sham; Figure 5F), which was profoundly decreased by either postcon or 1400W. SIN-1 abolished the effect of postcon in decreasing cardiac nitrotyrosine levels.

DISCUSSION

Studies show the beneficial effects of postcon in patients with AMI undergoing reperfusion therapy [3,5]. Postcon may not only exert short-term cardioprotection but may also improve cardiac function of patients after AMI at 1 year after PCI [21]. However, many fundamental questions regarding what is the predominant protective mechanism of postcon remain unanswered. Experimental studies show that postischaemic myocardial apoptosis is one of the major pathways of cell death after reperfusion and that ONOO is a major trigger of cardiomyocyte apoptosis in vitro and in vivo [7]. However, ONOO may act as a ‘double-edged sword’ in postischaemic myocardial injury: although being directly toxic to the cardiac tissue at a relatively high concentration, it may indirectly protect myocardial cells from neutrophil-induced injury at a much lower concentration [22]. As such, it is important to answer the question of whether reduction in ONOO production [23] and the subsequent decrease in ONOO-induced nitro-oxidative stress play a major role in postcon-mediated cardioprotection in vivo in humans. Further, a better understanding of the signalling pathway by which postcon reduces ONOO toxicity may lead to the development of comprehensive, therapeutic regimens.

The present study has ended with several novel observations. To our knowledge, we are the first to demonstrate that postcon cannot only enhance short-term postischaemic myocardial function in patients receiving PCI evidenced as improvement of LVEF at day 7, but most meaningfully, it can significantly reduce the Killip class, which is recently identified as a strong predictor of long-term mortality in patients with AMI [24]. Patients with Killip class >2 and LVEF <50% have approximately double the 10-year mortality than patients with lower Killip class and higher LVEF [24]. Secondly, we identified that the increase in plasma nitrotyrosine peaked coincidently with cTnI at 24 h after PCI, which is highly correlated, suggestive of causal relationship between ONOO-induced nitro-oxidative toxicity and myocardial cellular damage. By contrast, plasma MDA peaked early at 4 h after PCI and was reduced by postcon. It is plausible that postcon may have conferred its early cardioprotection primarily by reducing ROS toxicity[25,26] and conferred a delayed protection primarily by reducing nitro-oxidative damage. Further, we have provided evidence, in an in vivo animal model of MI/R, that postcon-mediated reduction in myocardial infarct may be primarily attributable to its ability to decrease myocardial apoptotic cell death and that inhibition of PMN accumulation in cardiac tissue during reperfusion and, in particular, the inhibition of iNOS activation and the subsequent decrease in ONOO production may represent an important upstream effector in the protective signalling. It should be noted that administration of nitrates or commencement of statin may have, respectively, potentially influenced the production of ONOO and plasma MDA, given that nitrates can increase the production of nitric oxide, and statin has antioxidant properties [27]. However, the number of patients who received nitrate and statin administrations did not differ between groups. Consequently, postcon should be the major factor that led to the decrease in plasma ONOO and MDA seen in the postcon group. In addition, we measured MDA using the conventional spectrophotometric procedure based on absorbance of the thiobarbituric acid−malonaldehyde complex at 532 nm. This methodology is not highly specific to MDA and is prone to the interference by other non-lipid-related compounds. An HPLC-based assay of MDA [28] would be superior and should be applied in future related studies to confirm the finding of the present study.

Elevated WBC and neutrophils in patient [29,30] peripheral circulation have been shown to be associated with a greater risk of cardiovascular events in many epidemiological and clinical studies [31,32]. In our study, peripheral blood WBCs are elevated 4 to 24 h after PCI in patients. This result is in line with that reported by Mariani et al. [33], who found that neutrophils and monocyte counts were positively related to markers of myocardial reperfusion injury on the first days after acute MI treated with primary PCI. Postcon-mediated inhibition of peripheral WBC elevation and the potential reduction in neutrophil migration into the ischaemic heart tissue should have contributed to its cardioprotection seen in the postcon group in patients. WBC is a critical mediator of inflammation during reperfusion and a major resource of ROS and iNOS [13]. However, the strong correlation between WBC iNOS activity and plasma nitrotyrosine and cTnI suggests that iNOS activation may have played a critical or essential role in myocardial reperfusion injury. To investigate the effect of postcon on PMN accumulation and the relative role of iNOS inhibition in postcon-mediated cardioprotection, we further measured the MPO activity in rat ischaemic cardiac tissue and found that postcon indeed decreased postischaemic heart tissue PMN accumulation that is associated with reduced myocardial iNOS activity, reduced NOx content and reduced ONOO production. The fact that the iNOS inhibitor 1400W reduced myocardial infarct and apoptosis to a similar extent as postcon in the rats without reducing myocardial MPO activity compared with MI group suggests that postcon-mediated inhibition of iNOS activity (subsequent to the reduction in PMN accumulation) in the myocardium and the resultant decrease in ONOO production play a key role in cardioprotection. Indeed, Poon et al. [14] have found that wild-type neutrophils bound to wild-type myocytes caused severe cardiomyocyte dysfunction, while iNOS-deficient neutrophils did not depress myocyte function. This highlights a central role of iNOS activation in PMN-accumulation-mediated cardiac injury. Further, SIN-1, an ONOO donor that simultaneously produces superoxide and NO was applied to elucidate whether the decrease in ONOO production and the subsequent reduction in nitro-oxidative stress may present a major mechanism or one of the end effectors in postcon-mediated cardiac protection. Of note, SIN-1 abolished postcon-mediated cardiac protection in reducing myocardial infarct without significantly affecting postcon effect in reducing myocardial MPO and iNOS activity.

Taken together, results from the present study indicate that inhibition of ONOO-induced nitro-oxidative stress may pay a central role in postcon-mediated cardioprotection. Our findings extend a most recent study by Iliodromitis et al. [34], who reported that postcon-mediated cardioprotection was associated with reduced nitro-oxidative stress in vivo. Acknowledging that iNOS activation in some cell types (such as cardiac myocytes [14]) could be beneficial and that nitriate or NO could be both beneficial and detrimental, the dose of the iNOS inhibitor 1400W was chosen to significantly inhibit, but not prevent, the increase in myocardial iNOS activity after myocardial infarction. The new insights gained from the present study should promote large-scale and/or longer-term clinical trials to further confirm and enhance the effectiveness of postcon based on a better understanding of the mechanisms involved.

FUNDING

This study was supported, in part, by the National Natural Sciences Foundation of China (NSFC) [grant numbers 30800448 (to Q.F.), 30770875 (to X.-C.Y.)]; and the Research Grants Council of Hong Kong General Research Fund [grant number 782910 M (to Z.X.)].

Abbreviations

     
  • AMI

    acute myocardial infarct

  •  
  • cTnI

    cardiac-specific troponin I

  •  
  • HR

    heart rate

  •  
  • iNOS

    inducible NO synthase

  •  
  • LAD

    left anterior descending artery

  •  
  • LV

    left ventricular

  •  
  • LVDP

    LV developed pressure

  •  
  • LVEDP

    LV end-diastolic pressure

  •  
  • LVEF

    LV ejection fraction

  •  
  • LVSP

    LV pressure

  •  
  • MDA

    malondialdehyde

  •  
  • MI/R

    myocardial ischaemia/reperfusion

  •  
  • MPO

    myeloperoxidase

  •  
  • postcon

    postconditioning

  •  
  • NOx

    nitrite/nitrate

  •  
  • ONOO

    peroxynitrite

  •  
  • PCI

    percutaneous coronary intervention

  •  
  • PMN

    polymorphonuclear leucocyte

  •  
  • RNS

    reactive nitrogen species

  •  
  • ROS

    reactive oxygen species

  •  
  • SIN-1

    3-morpholinosydnonimine

  •  
  • TTC

    Triphenyltetrazolium Chloride

  •  
  • WBC

    white blood cell

AUTHOR CONTRIBUTION

Xin-Chun Yang and Zhengyuan Xia designed the research; Qian Fang, Yu Liu, Le-Feng Wang, Sheng-Hui Liu, Yong-Gui Ge, Mu-Lie Chen and Wen Wang performed the research; Qian Fan, Xin-Chun Yang, Li-Ke Zhang and Michael Irwin analysed the data; and Qian Fan, Xin-Chun Yang and Zhengyuan Xia wrote the paper.

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