Summary
Background
Previous studies have shown protective effects of brain natriuretic peptide (BNP) against the postmyocardial infarction (MI) remodelling process. The transcription factor NF-κB is known to play an important role after MI.
Aims
To investigate if NF-κB is involved in the protective effects of BNP against adverse post-MI remodelling.
Methods
Rats were randomly assigned to five groups: sham-operation; MI by coronary ligation; MI treated with chronic BNP infusion; MI treated with enalapril; MI treated with BNP + enalapril. Rats were closely monitored for survival rate analysis. Rats from each group were sacrificed on days 3, 7 and 28 postoperation.
Results
The results showed that chronic continuous BNP infusion achieved similar effects to enalapril therapy, as evidenced by improved survival rate within the 28-day observation period compared with MI group rats; this effect was closely associated with preserved cardiac geometry and performance. The treatment combination did not offer extra benefits in terms of survival rate. Both BNP and enalapril therapy produced higher heart tissue concentrations of cyclic guanosine monophosphate and lower expression levels of inflammatory cytokines, including tumour necrosis factor-α, interleukin-1 and interleukin-6. These benefits were associated with lower phosphorylation levels of NF-κB subunits IκBα, p50 and p65. While enalapril significantly inhibited extracellular matrix remodelling via regulation of the protein expression ratio of matrix metalloproteinase/tissue inhibitor of metalloproteinase and the activity of matrix metalloproteinase, these variables were not affected by BNP, indicating that the two therapies involve different mechanisms.
Conclusion
Chronic BNP infusion can provide beneficial effects against adverse post-MI remodelling.
Résumé
Justification
Les travaux antérieurs ont montré l’effet protecteur du brain natriuretic peptide (BNP) sur le remodelage post-infarctus du myocarde. Il a été montré que le NF-κB, facteur de transcription, avait un rôle important au décours d’un infarctus.
Objectifs
Dans cette étude, l’objectif était d’évaluer l’implication du NF-κB comme facteur protecteur corrigeant le processus de remodelage, en utilisant un traitement par BNP. Les rats ont été randomisés en premièrement témoins, deuxièmement groupe d’infarctus du myocarde par ligature coronaire, troisièmement infarctus du myocarde recevant des perfusions du BNP en chronique, quatrièmement infarctus du myocarde sous énalapril, cinquièmement infarctus du myocarde traité par BNP et énalapril. Les animaux ont été monitorés, avec évaluation du taux de survie. Tous les rats de cette étude expérimentale ont été sacrifiés à j3, j7 et j28 après l’intervention.
Résultats
Comparativement aux rats ayant un infarctus du myocarde, la perfusion continue de BNP s’accompagnait d’effets similaires à un traitement par énalapril comme cela a été montré par une amélioration du taux de survie à j28, associés avec une préservation de la géométrie ventriculaire gauche et de la fonction myocardique. Cependant, l’association de ces deux traitements, BNP et énalapril n’a pas montré de bénéfice supplémentaire sur le taux de survie. Cette association thérapeutique s’accompagnait d’une concentration tissulaire accrue de cGMP, et une expression moindre des concentrations de cytokine inflammatoire incluant le TNF-α, l’IL-1 et l’IL-6. Ces bénéfices étaient associés avec un taux moindre de phosphorylation des sous-unités NF-κB, IκBα, P50 et P65. L’énalapril a inhibé de façon significative le remodelage de la matrice extracellulaire au travers d’une régulation de l’expression des protéines, exprimée par le rapport métalloprotéinase de la matrice sur métalloprotéinase inhibitrice ainsi que l’activité de la métalloprotéinase matricielle, cependant non affectée par la perfusion de BNP, indiquant qu’il s’agirait de mécanismes différents pour le bénéfice observé de ces deux molécules.
Conclusion
La perfusion chronique du BNP peut provoquer des effets bénéfiques pour corriger le remodelage post-infarctus du myocarde dans un modèle expérimental de rats.
Introduction
BNP, as an important natriuretic peptide secreted from the ventricle, can act via the natriuretic peptide receptor A/guanylyl cyclase/cGMP signalling pathway, maintaining cardiorenal homeostasis through diuresis, natriuresis, vasodilation and inhibition of aldosterone synthesis and renin secretion under physiological and pathological conditions . Release of the stored peptides due to altered chamber loading and myocyte stretch has been observed in MI . Interestingly, exogenous BNP infusion can protect the heart-limiting infarct size in an ischaemia/reperfusion injury rat model, possibly through adenosine triphosphate-sensitive potassium channels and nitric oxide synthase activation . In addition, natriuretic peptide receptor A/cGMP signalling activated by BNP can modulate cardiac responses to hypertrophic stimuli and hence improve cardiac remodelling via mediation of the calcineurin-nuclear factor of activated T cells pathway , indicating that BNP also plays an important role in the post-MI remodelling process.
Previous studies have shown that intravenous infusion of BNP can improve clinical condition in patients with heart failure and MI . The long-term beneficial effect of BNP therapy against ventricular remodelling has also been demonstrated in a rat MI model, which was associated with inhibition of TGFβ1/Smad2 signalling . However, it is still not fully known how BNP infusion attenuates TGFβ expression. NF-κB is a key transcription factor that mediates inflammatory cytokine expression in response to a variety of stimuli , especially after MI . Therefore, using a rat MI model, we aimed to investigate whether modulation of NF-κB activity (and hence pro-inflammatory cytokine expression) is involved in the improved remodelling process associated with chronic BNP infusion. The benefits of BNP infusion were compared with those of the angiotensin-converting enzyme inhibitor enalapril, and additive effects were tested when the two therapies were combined.
Materials and methods
Male, 8-week-old Sprague-Dawly rats (Experimental Animal Center, Fudan University, Shanghai, China) were used in this study. The animal research study protocol complied with The Guide for the Care of Use of Laboratory Animals published by the National Institute of Health (NIH Publication No. 85-23, revised 1996). All rats were housed for a 2-week acclimatization period before the study started.
Animal preparation
The MI model was induced as described in our previous report by LAD coronary ligation. In brief, after anaesthesia with pentobarbital (50 mg/kg, intraperitoneally) all rats (weighing 320–380 g) underwent an open chest operation and the LAD coronary artery was encircled just distal to its first branch. The infarct was confirmed by a pale area below the suture or ST-T elevation on an electrocardiogram. The control group underwent sham operation with passage of a suture around the LAD without tie-down. All rats were then allowed to recover under close observation.
Criteria for rat enrolment
To minimize the variability of infarct size, only rats with moderate infarct size were enrolled. A pilot MI study was performed on 21 rats. Twenty-four hours after LAD ligation, blood serum was collected to measure troponin I concentration (Siemens Medical Solution Diagnostics, Tarrytown, NY, USA). The heart was harvested from each rat and the LV was cut into five parallel slices. Triphenyltetrazolium chloride staining was performed and the extent of necrosis was quantified by computerized planimetry. The total infarct size was expressed as a percentage of the total sum of the infarct area on each slice/LV corrected by weight. The correlation curve was then derived for infarct size and serum troponin I concentration ( Fig. 1 A) with an r value of 0.979 ( P < 0.001). An infarct size of 30–50% was arbitrarily selected as an enrolment criterion, thus a blood serum troponin I concentration of 28–47 g/L 24 hours after LAD ligation was set as the inclusion criterion for MI rats. A total of 286 rats underwent successful coronary ligation; 224 MI rats were enrolled and 62 rats were excluded due to an inappropriate serum troponin I concentration.
Experimental protocols
Protocol I
To evaluate survival rate, 160 MI rats with an appropriate troponin I concentration were randomly assigned to one of the following groups ( n = 40 in each group): MI group; MI + enalapril group (1 day after LAD ligation, MI rats were given enalapril [Berlin-Chemie AG, Berlin, Germany] 10 mg/kg/day by gavage); MI + BNP group (1 day after LAD ligation, MI rats were given continuous intravenous infusion of rat-BNP-32 [Jingmei Biotech Co. Ltd., Shenzhen, China] 0.06 g/kg/minute through an osmotic mini-pump [Alza Corporation, Mountainview, CA, USA] implanted intraperitoneally); MI + combination group (MI rats were given both the enalapril and BNP treatments). All MI rats were rigorously monitored to check the cause of sudden cardiac death. A careful autopsy was performed for each rat with reference to cardiac rupture. Survival rate was analysed to evaluate treatment benefits.
Protocol II
Time-course changes in the post-MI remodelling process were evaluated. The other 64 MI rats were then randomly assigned to the four groups ( n = 16 to each group). Rats were sacrificed on day 3 and day 7 after the MI ( n = 8 for each time point), respectively.
Before the rats were sacrificed, echocardiography (ACUSON SEQUOIA 512 equipped with 14 MHz mini probe) was performed, using the same anaesthesia as described above. Two-dimensional and M-mode echocardiograms were obtained. LVEDD and LVESD were measured in the short-axis view at papillary muscle level. FS was also obtained. All values were averages of three consecutive cardiac cycles and were analysed by two independent blinded researchers. Invasive haemodynamic measurements were also taken by right carotid artery cannulation with a pre-heparinized fine polyethylene tube connected to a fluid-filled pressure transducer (MPA-CFS, Alcott Biotech, Shanghai, China). After euthanization, the heart from each rat was harvested. LV weight was recorded for comparison with body weight, and at papillary muscle level, one LV cross-sectional tissue slice around 5 mm in thickness was obtained, fixed in 4% formalin and embedded in paraffin for the histological examination. Non-infarcted LV tissue was obtained from the rest of the LV, and was snap frozen in liquid nitrogen and stored at −80̊.
Eight rats that had completed 28 days of rigorous observation were randomly selected from each group and underwent the same procedure as described above.
Another 24 sham-operated control rats were also sacrificed on day 3, day 7 and day 28; these rats underwent the same procedure as described above.
Quantification of collagen deposition and infarct size
Paraffin-embedded heart tissue was stained by Mallory’s method, which is collagen specific. Three sections per animal and eight fields per section were scanned and computerized with a Leica Q500MC digital image analyser at a magnification of 10. The sections from different groups were stained in one batch. CVF was obtained from the ratio of the connective tissue area divided by the total tissue area within the same microscopic field. The infarct (expressed as fibrotic area) perimeter was traced and the size of the MI was normalized to the LV area using the following equation: percentage infarct perimeter = circumference of infarct scar/[(epicardium perimeter + endocardium perimeter)/2] × 100%.
Immunohistochemistry
After dewaxing, the sections of LV tissue of 5 μm thickness were immersed in 3% hydrogen peroxide for 10 minutes at room temperature. After incubation with 5% bovine serum albumin (Boster, Wuhan, China) for 20 minutes, the tissue sections were probed with rabbit anti-rat type I and type III collagen antibodies (Boster, Wuhan, China) (1:100) at 4̊C overnight. The sections were then incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2000; Boster) for 30 minutes at 37̊C then reacted with streptavidin-biotin-peroxidase complex (Boster) for 20 minutes and coloured by diaminobenzaldehyde (Boster). Quantification of resulting image data from immunohistochemical staining was performed using a Leica Q500MC digital image analyser.
Enzyme-linked immunosorbent assay
A double-antibody sandwich-based enzyme-linked immunosorbent assay kit (USCN Life Science Inc., Wuhan, China) was used to detect angiotensin II (Ang II), cGMP, BNP, TNFα, IL-1 and IL-6 concentrations in non-infarcted cardiac tissue, according to the manufacturer’s instructions. The detection limits of each assay were as follows: Ang II, 1.56 ng/mL; cGMP, 1.56 pmol/mL; BNP, 156 pg/mL; TNFα, 3.9 pg/mL; IL-1, 39 pg/mL; IL-6, 7.8 pg/mL. The intra- and intervariability for each kit were < 8%, respectively.
Western blotting
The detailed method of western blotting has been described previously using the rat MI model . The primary antibodies against different proteins of MMP, including MMP-2 (R&D Systems, Minneapolis, MN, USA), MMP-9, TIMP-1 (Abcam, Cambridge, MA, USA), phosphorylated (p)-NF-κB-p50, NF-κB-p50 (Abcam, USA), p-IκB-α (Ser32), IκB-α, p-NF-κB-p65 and NF-κB-p65 (Cell Signaling Technology, Danvers, MA, USA), were used to probe the target protein. An antibody against β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was also used for protein loading. The bands for different proteins were quantified by densitometry using Image J software (version 1.41, National Institutes of Health, Bethesda, MD, USA) and normalized to the expression of β-actin.
Zymography
Gelatine zymography was used to determine MMP protein activity as described. Approximately 10 mg of myocardial tissue were extracted from the non-infarcted area and equal volumes (10 μL) of samples normalized for protein concentration were subjected to electrophoresis (without boiling or reduction) through a 10% polyacrylamide gel co-polymerized with gelatine (0.5 mg/mL), at 4°C. After electrophoresis, the gel was incubated for 1 hour at 25°C in a 2.5% Triton X-100 solution and then incubated overnight at 37 °C in a 0.05 M Tris-HCl buffer, pH 8.0, containing 5 mM CaCl 2 . The gels were fixed with 40% methanol and 7% acetic acid, stained with 0.25% Coomassie Brilliant Blue R250 and then destained with 10% methanol and 7% acetic acid. The gels were scanned using the EagleEye II imaging system for relative lytic activity. A mixture of human MMP-2 and MMP-9 (Chemicon, Hofheim, Germany) served as a zymography standard.
Statistical analysis
All data are expressed as means ± standard deviations. Kaplan-Meier survival curves were analysed to check the differences between MI rats with and without treatments. Time-course changes in the haemodynamic heart tissue expression of MMP, cGMP, Ang II and BNP, and the extent of fibrosis were compared within groups by using repeated ANOVA measures, and between groups by using two-way ANOVA followed by LSD-corrected multiple comparisons. One-way ANOVA followed by LSD correction was used for comparing the differences in all other variables between groups. Non-parametric data were analysed using Fisher’s exact method. A P -value < 0.05 was considered as statistically significant. Statistical analyses were performed using SPSS 13.0 statistics software (SPSS Inc., Chicago, IL, USA).
Results
Survival rate
Troponin I values were similar in the four groups of rats with MI (MI group, 35.8 ± 6.0 g/L; MI + enalapril group, 33.6 ± 4.7 g/L; MI + BNP group, 36.8 ± 5.8 g/L; MI + combination group, 37.0 ± 5.4 g/L), with no significant differences ( P > 0.05, respectively). However, during the 28-day observation period, survival rates improved in rats treated with BNP or enalapril, with no significant difference between these two groups ( P = 1.000; Fig. 1 B). The combination of BNP and enalapril did not provide additional benefits in terms of survival rate during this period. Rigorous monitoring combined with autopsy showed that cardiac ruptures occurred within 7 days after MI (two in the MI + enalapril group, two in the MI + BNP group, two in the MI + combination group and three in the MI group) with no differences between the four groups ( P = 1.000, respectively).
Tissue weight and percentage infarct perimeter
There were no significant differences in infarct perimeter between the four MI groups at any time point post MI. Neither enalapril nor BNP therapy exerted any protective effects on infarct size ( Table 1 ) and their combination did not produce additive effects in terms of limiting infarct size.
| Observation period | Groups | IP (%) | MAP (mmHg) | LVEDP (mmHg) | LVW/BW (mg/g) | LVEDD (mm) | FS (%) |
|---|---|---|---|---|---|---|---|
| 3 days | Sham | – | 114 ± 12 | 3.4 ± 1.0 | 1.76 ± 0.11 | 5.33 ± 0.33 | 61.7 ± 4.9 |
| MI | 38.35 ± 4.93 | 103 ± 10 | 8.3 ± 1.3 a | 1.89 ± 0.09 | 6.93 ± 0.92 a | 34.7 ± 9.3 a | |
| MI + enalapril | 37.00 ± 3.45 | 95 ± 6 a | 8.9 ± 1.5 a | 1.91 ± 0.08 | 6.97 ± 0.37 a | 33.7 ± 12.2 a | |
| MI + BNP | 39.13 ± 4.56 | 92 ± 5 a | 7.2 ± 0.9 a | 1.87 ± 0.06 | 7.20 ± 0.54 a | 37.2 ± 8.3 a | |
| Combination | 38.27 ± 6.21 | 88 ± 5 a,b | 6.9 ± 1.0 a | 1.91 ± 0.07 | 7.10 ± 0.82 a | 38.6 ± 6.9 a | |
| 7 days | Sham | – | 114 ± 6 | 2.9 ± 0.8 | 1.76 ± 0.08 | 5.32 ± 0.338 | 60.3 ± 4.5 |
| MI | 42.10 ± 5.78 | 104 ± 7 | 10.2 ± 1.1 a | 1.93 ± 0.09 | 7.85 ± 0.58 a | 30.9 ± 6.6 a | |
| MI + enalapril | 41.11 ± 7.33 | 93 ± 5 a | 8.4 ± 1.3 a | 1.86 ± 0.14 | 7.00 ± 0.53 a | 33.3 ± 6.1 a | |
| MI + BNP | 42.13 ± 6.23 | 103 ± 8 | 7.2 ± 1.5 a,b | 1.89 ± 0.08 | 7.13 ± 0.63 a | 31.9 ± 7.0 a | |
| Combination | 41.35 ± 3.46 | 92 ± 7 a | 6.5 ± 1.2 a,b | 1.85 ± 0.08 | 6.77 ± 0.54 a,b | 36.3 ± 4.4 a | |
| 28 days | Sham | – | 116 ± 12 | 2.8 ± 1.0 | 1.71 ± 0.13 | 5.68 ± 0.62 | 62.5 ± 4.2 |
| MI | 41.18 ± 5.92 | 106 ± 8 | 14.2 ± 1.7 a,d,e | 2.19 ± 0.12 a | 8.83 ± 0.61 a,d | 19.2 ± 2.6 a,d,e | |
| MI + enalapril | 41.14 ± 6.25 | 96 ± 5 a | 9.5 ± 1.0 a,b | 1.90 ± 0.25 b | 7.47 ± 0.73 a,b | 27.7 ± 5.6 a,b | |
| MI + BNP | 42.05 ± 6.08 | 104 ± 9 | 7.7 ± 1.2 a,b | 1.82 ± 0.11 b | 7.52 ± 0.97 a,b | 27.5 ± 3.9 a,b | |
| Combination | 42.63 ± 7.31 | 97 ± 7 a | 6.8 ± 1.5 a,b,c | 1.75 ± 0.07 b | 6.92 ± 0.65 a,b | 31.4 ± 5.9 a,b | |
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