© Borgis - Postępy Nauk Medycznych 6/2015, s. 426-432
*Monika K. Duda1, Paweł Dobrzyń2, Urszula Mackiewicz1, Agnieszka Dobrzyń3, Michał Mączewski1
Suplementacja wielonienasyconymi kwasami tłuszczowymi ω-3 obniżająca aktywność czynnika transkrypcyjnego κB i zapobiegająca dysfunkcji serca indukowanej przeciążeniem ciśnieniowym
Ω-3 PUFA supplementation decreases nuclear factor κB activity and attenuates pressure overload-induced cardiac dysfunction**
1Department of Clinical Physiology, Medical Centre of Postgraduate Education, Warszawa
Head of Department: Michał Mączewski, MD, PhD
2Laboratory of Molecular and Medical Biochemistry, Nencki Institute of Experimental Biology, Warszawa
Head: Paweł Dobrzyń, MD
3Laboratory of Cell Signaling and Metabolic Disorders, Nencki Institute of Experimental Biology, Warszawa
Head: Agnieszka Dobrzyń, MD, PhD
Streszczenie
Wstęp. Istnieją dowody, że suplementacja diety wielonienasocynymi kwasami ω-3 ma korzystne działanie u pacjentów z niewydolnością serca, jednak mechanizm tego zjawiska nie jest dobrze poznany.
Cel. Celem pracy była ocena wpływu DHA+EPA na aktywność czynnika transkrypcyjnego NF-κB oraz odpowiedzi lewej komory (LV) serca na przeciążenie ciśnieniowe.
Materiał i metody. Badania były przeprowadzone na szczurach rasy Wistar, u których po tygodniu stosowania diety standardowej lub diety z dodatkiem DHA+EPA, wykonano zwężenie aorty brzusznej lub operację pozorną (n = 6-7/grupa). Po 12 tygodniach oceniono echokardiograficznie funkcję LV, oraz sercową aktywność NF-κB i ekspresji genów kontrolowanych przez NF-κB
Wyniki. U szczurów karmionych dietą standardową, przeciążenie ciśnieniowe prowadziło do przerostu, przebudowy i upośledzenia funkcji skurczowej LV, którym towarzyszył wzrost sercowej aktywności NF-κB. Dieta zawierająca DHA+EPA zapobiegała patologiom LV indukowanym przeciążeniem ciśnieniowym i zmniejszała aktywność NF-κB. Stosowanie tej diety było również związane z obniżeniem ekspresji mRNA dla genów kodujących białka regulujące odczyn zapalny (TNFα i IL-1β), apoptozę (Bcl2) i włóknienie (TGF-β1), których ekspresja jest regulowana aktywnością NF-κB.
Wnioski. Suplementacja diety kwasami DHA+EPA zapobiega przerostowi, przebudowie i upośledzeniu czynności LV w odpowiedzi na przeciążenie ciśnieniowe. Efektom tym towarzyszył spadek aktywności NF-κB i zmiany w ekspresji genów kontrolowanych przez NF-κB.
Summary
Introduction. Supplementation with ω-3 polyunsaturated fatty acids (ω-3 PUFA) exerts cardioprotective effects in heart failure patients, however the mechanism is not well know. The present study assessed the effect of DHA+EPA on the activation of nuclear factor (NF)-κB and on the left ventricular (LV) response to pressure overload.
Aim. The present study assessed the effect of DHA+EPA on the activation of nuclear factor (NF)-κB and on the left ventricular (LV) response to pressure overload.
Material and methods. Male Wistar rats were fed a standard chow or DHA+EPA supplemented diet. After 1 week rats underwent abdominal aortic banding or sham surgery (n = 6-7/group). LV function was assessed by echocardiography after 12 weeks. In addition, we studied the effect of ω-3 PUFA on the cardiac NF-κB activity and expression of NF-κB target genes.
Results. Male Wistar rats were fed a standard chow or DHA+EPA supplemented diet. After 1 week rats underwent abdominal aortic banding or sham surgery (n = 6-7/group). On the standard diet, 12 weeks of pressure overload induced significant LV hypertrophy, remodeling and contractile dysfunction, and increased cardiac (NF)-κB activity. Dietary DHA+EPA attenuated pressure overload induced cardiac pathology and inhibited (NF)-κB activity. DHA+EPA decreased mRNA expression of NF-κB-depended genes encoding proteins involved in regulation of inflammation (TNFα and IL-1β), apoptosis (Bcl2), and fibrosis (TGF-β1).
Conclusions. Dietary supplementation with DHA+EPA attenuated pressure overload induced LV hypertrophy, remodeling and contractile dysfunction, which was associated with decreased cardiac (NF)-κB activity and switched expression of NF-κB target genes.
Introduction
Heart failure is the most common cause for hospitalization and death within Medicare population despite optimal treatment, therefore new therapeutic approaches are needed. Nutritional therapies aimed at stopping or slowing the development of heart failure are particularly attractive because they could work additively with current drugs. Some epidemiological and animal researches suggest that dietary supplementation with the ω-3 polyunsaturated fatty acids (ω-3 PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are highly concentrated in fish oil, may be effective in preventing the development and progression of heart failure (1, 2). Result from clinical studies show that treatment with DHA+EPA can improve left ventricular function and modestly reduce clinical events in heart failure patients (3, 4). However, there is modest evidence supporting the effectiveness of ω-3 PUFA in heart failure (5).
The mechanisms for a potential favorable effect of ω-3 PUFA in prevention the development and progression of heart failure are likely multifactorial and not well established. We previously reported that ω-3 PUFA supplementation increased adiponectin concentration in the plasma in a dose-dependent manner in rats, indicating a potential role for adiponectin in mediating the beneficial cardioprotective effect of ω-3 PUFA (6). Adiponectin is adipocyte-derived hormone that can limit the LV hypertrophy, remodeling and contractile dysfunction in response to pressure overload (7) or after myocardial infarction (8). The cardioprotective action of adiponectin has been linked to inhibition of the nuclear transcription factor kappa B (NF)-κB (9), which activity increased in the failing myocardium (10, 11). NF-κB exists in the cytoplasm in an inactive form, bound to the inhibitory protein, IκB. On stimulation, IκB is phosphorylated leading to the release of IκB, which then translocates to the nucleus and regulates expression an array of genes involved in inflammation, apoptosis and collagen metabolism (12). Activation of these mechanisms in myocardium, under increased hemodynamic load condition, initiates hypertrophy, inflammation, apoptosis, and fibrosis, culminating in contractile dysfunction and overt HF.
Aim
The goal of the present investigation was to assess the ability of clinical relevant dose of ω-3 PUFA (DHA+EPA) to prevent LV pathology and NF-κB activation in response to pressure overload. We hypothesize that DHA+EPA supplementation decreases cardiac activity of NF-κB and modulates the expression of NF-κB target genes, which attenuates LV hypertrophy, remodeling and contractile dysfunction. This effect corresponds to the increased in plasma adiponectin concentration. Studies were performed in an established rat model of chronic pressure overload induced by abdominal aortic banding. This model results in the LV hypertrophy and the development of heart failure as evidenced by an increase in LV end-diastolic and end-systolic, and expansion of fetal genes.
Material and methods
Experimental design
The animal protocol was conducted according to the local and institutional regulation. The study conformed to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23). Animals were maintained on a reverse 12-hour light-dark cycle.
Five-week old male Wistar rats were fed either a standard chow or a modified standard chow containing ω-3 PUFA from fish oil. After one week on the assigned diet, rats were randomly assigned to either sham surgery or abdominal aortic banding (AAB) (n = 6-7/group), and dietary treatment was continued for 12 wks. Echocardiographic assessment of LV function was performed 1-2 days before and 12 weeks post-surgery. Twelve weeks after surgery, rats were weighed and anesthetized with 1.5-2.0% isoflurane, and 3 mL blood was drawn from the inferior vena cava for biochemical measurements. The LV tissue was quickly removed, weighed, freeze clamped and stored at -80°C for biochemical analysis.
Diets
All chows were custom manufactured (Research Diets Inc., USA). The standard chow was similar to typical commercial rodent chows, with 70% of total energy from carbohydrate (75% from cornstarch, 15% maltodextrin and 10% from sucrose by energy), 10% of energy from fat (78% from cocoa butter and 22% from soybean oil) and 20% protein (casein supplemented with L-cystine). ω-3 PUFA diet also derived 10% of the total energy from fat, with 3% the total energy as DHA+EPA (1:1) from fish oil (gift from KD Pharma, Germany), 4.8% from cocoa butter, and 2.2% from soybean oil. The protein and carbohydrate composition of the ω-3 PUFA diet matched the standard chow.
Abdominal aortic banding
The rats (170-220 g) were anesthetized with 2.0-2.5% isoflurane by mask. A midline abdominal incision was used to expose the suprarenal abdominal aorta. The aorta was tied with a 3-0 silk suture against a blunt needle (21G). The needle was immediately removed, leaving the aortic lumen constricted to the diameter of the needle. Sham surgery animals were subjected to the same procedure without the aortic banding.
Echocardiography
Echocardiography was performed using MYLab25 (Esaote, Italy) with a 13-MHz linear array transducer. Briefly, rats were anesthetized with 1.5-2.0% isoflurane by mask, the chest was shaved and the animal was situated in the supine position. Two-dimensional cine loops and guided M-mode frames were recorded from parasternal short and long axis. All data were analyzed offline with software resident on the ultrasound system at the end of the study.
Biochemical measurements
LV tissue was analyzed for phospholipid fatty acid composition by gas-liquid chromatography as described previously (13). Myocardial DNA binding activity of NF-κB in nuclear extracts was also measured by ELISA (Cayman Chemical, USA) and nuclear extract was isolated using the NE-PER Kit following the manufacturer’s instructions (Thermo Scientific, USA). Plasma concentration of adiponectin was measured by enzyme-linked immunosorbent assay (ELISA) (ALPCO Diagnostics, USA).
mRNA measurement
For assessment of mRNA expression, frozen LV tissue was homogenized using a Tissue Lyser (Qiagen, USA), RNA was isolated using the RNeasy Mini Kit following the manufacturer’s instructions (Qiagen), and Real time RT-PCR was performed as previously described (2). The following genes were analyzed, using TaqMan gene expression assays (Applied Biosystems, USA): atrial natriuretic peptide (Rn00561661_m1); myosin heavy chain α (Rn00568304_m1); myosin heavy chain β (Rn00568328_m1), TNFα (Rn_01473656_g1), IL-1β (Rn_99999009_m1), Bcl2 (Rn_99999125_m1), caspase 3 (Rn_00563902_m1), TGF-β1 (Rn_01475963_m1), and cyclophilin A (Rn00690933_m1). mRNA was normalized to fold increase to the standard diet sham group.
Statistical analysis
Mean values are presented ± S.E.M and a P < 0.05 level of significance was used. Comparisons were made using a two way ANOVA with the Bonferroni test for multiple comparisons.
Results
LV phospholipids composition
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