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Infusion of docosahexaenoic acid protects against myocardial infarction
Prostaglandins, Leukotrienes and Essential Fatty Acids 90 (2014) 139–143
Contents lists available at ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Short communication
Infusion of docosahexaenoic acid protects against myocardial infarction$ D. Richard a,1, F. Oszust b,1, C. Guillaume c, H. Millart b, D. Laurent-Maquin c, C. Brou d, P. Bausero a, F. Visioli e,n a
Université Pierre et Marie Curie, Paris, France EA3801 HERVI (Hémostase et Remodelage Vasculaire post-Ischémique), Université de Reims Champagne-Ardenne, France c EA 4691 BiOs (Biomatériaux et inflammation en site Osseux), Université de Reims Champagne-Ardenne, France d Unité de Signalisation Moléculaire et Activation Cellulaire, Institut Pasteur, Paris, France e IMDEA-Food, CEI UAM þCSIC, C/Faraday 7, 28049 Madrid, Spain b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 December 2013 Received in revised form 1 January 2014 Accepted 2 January 2014
Most of the cardioprotective effects of long-chain omega 3 fatty acids, namely docosahexaenoic (DHA; 22:6n-3) and eicosapentaenoic (EPA; 20:5n-3), are due to their hypotriglyceridemic and antiinflammatory effects, which lower the risk for cardiovascular disease and myocardial infarction. Little is known on the direct preventive activities of DHA and EPA on heart function. In isolated hearts, we studied (1) whether infused DHA is able to protect the heart from ischemia/reperfusion damage and (2) the role played by Notch-mediated signal transduction pathways in myocardial infarction. Perfusion with DHA before and before/after induction of ischemia reperfusion significantly diminished cardiac damage and afforded antioxidant protection. Mechanistically, infusion of DHA before and before/after the induction of ischemia differentially modulated the expression of Notch2 and 3 target genes. In particular, DHA increased the expression of Hey1 when infused pre- and pre/post-ischemia; Jagged 1 and the Notch2 receptors increased with DHA pre-ischemia, but not pre/post; Notch2 and 3 receptors as well as Delta increased following DHA administration pre- and (especially) pre/postischemia. In conclusion, while the precise nature of the Notch-mediated protection from ischemia/reperfusion afforded by DHA is as yet to be fully elucidated, our data add to the growing body of literature that indicates how systemic administration of DHA provides cardiovascular protection. & 2014 Elsevier Ltd. All rights reserved.
Keywords: Docosahexaenoic acid Notch Ischemia Reperfusion Cardioprotection
1. Introduction Adequate dietary intakes of long-chain omega 3 fatty acids, namely docosahexaenoic (DHA; 22:6n-3) and eicosapentaenoic (EPA; 20:5n-3) acids, are associated with reduced cardiovascular mortality. Most of the putative cardioprotective effects of omega 3 fatty acids are due to their hypotriglyceridemic and antiinflammatory effects, which lower the risk for cardiovascular disease and myocardial infarction. Of note, the positive association between dietary intake and cardiovascular prognosis has not been confirmed by randomized clinical trials with pharmaceutical preparations [1]. Little is known on the direct preventive activities of DHA and EPA on heart function, although a seminal paper by Billman and co-workers [2] reported that infusion of omega 3 fatty $ n
In memory of Charlotte Schneider. Corresponding author. E-mail addresses: [email protected], [email protected] (F. Visioli). 1 These authors contributed equally to this work.
0952-3278/$ - see front matter & 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plefa.2014.01.001
acids to dogs before an exercise test plus myocardial ischemia significantly reduced the manifestation of ventricular tachycardia or fibrillation by about 75%. In humans, a paper by Schrepf et al. [3] showed rapid effects, namely reduction of sustained ventricular tachycardia, of omega 3 infusion to patients with implanted cardioverter defibrillators who were at high risk of sudden cardiac death. These acute anti-arrhythmic actions were recently confirmed by Heidt et al. [4] and led to the proposal to infuse omega 3 fatty acids pericardially to limit infarct size and arrhythmias [5]. However, it must be underscored that current accumulated evidence does not prove any anti-arrhythmic actions of DHA and EPA supplementation per os [6]. Therefore, the possibility exists that acute infusion and oral treatment have different effects on cardiac electrophysiology. Finally, a recent paper reported on the acute neuroprotective effects of DHA in a rat model of ischemia [7] and was subsequently confirmed [8]. The Notch signaling pathway governs cell fates between neighboring cells and, therefore, regulates the development of
140
D. Richard et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 90 (2014) 139–143
organisms [9]. Studies of Notch and its role in the cardiovascular system are rapidly growing, particularly in the field of angiogenesis and endothelial biology [10]. While the Notch signaling pathway is complex, it is noteworthy that Notch signals are based on the stimulation of Notch receptors by membrane-spanning ligands. The aim of this study was twofold: we addressed (1) the question of whether infused DHA is able to protect the heart from ischemia/reperfusion damage and (2) the role played by Notchmediated signal transduction pathways in myocardial infarction.
2.3. Biochemical indexes Creatine phosphokinase (CPK) was measured using a commercial kit from Boehringer Mannheim (Paris, France) [13]. Lipid peroxides were measured with the FOX assay, by using the Peroxoquant kit (Pierce, Rockford, USA) [14]. Adenosine triphosphate (ATP) was extracted with 0.4 N HClO4 from frozen biopsies obtained by freeze-clamping the ventricular apex with aluminum tongs [15] and was measured by bioluminescence (Sigma-Aldrich, Paris, France).
2. Materials and methods
2.4. Gene analyses
2.1. Isolated rat heart preparation
Hearts were fixed using 4% paraformaldehyde then were lysed with a β-protease from Omega Bio-tek (Paris, France). RNA was extracted with the ENZA kit, following the manufacturer0 s instructions. Following retrotranscription, qPCR was performed with a LightCycler LC480 (Roche Diagnostics, Paris, France). Primers were as follows (s, sense; as; antisense): HPRTs: AGG-ACC-TCT-CGAAGT-GT; HPRTas: ATC-CCT-GAA-GTG-CTC-ATT-ATA; Notch2s: GCAGGT-AGC-TCA-GAC-CA; Notch2as: CGA-GCA-TTT-GAG-GAG-GCATAA; Jagged1s: CAT-CGA-GAA-ACA-CGG-AGC; HEY1s: CGA-CGTGGG-GAG-CGA-GAA-CAA-T; Hey1as, GCC-AAG-AGC-ATG-GCGATC-AAA-GTA; Nox4s, TGG-TTT-GCA-GAC-TTG-CTC-TA; Nox4as: GTA-TCC-CAT-CTG-TTT-GCA-TGA-GGT-A; NKX2.5s: CTG-CAG-TCCCGC-CTA-CA; NKX2.5as: CGA-CGC-CAA-AGT-TCA-CGA.
This investigation conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Research Council (Eight Edition, 2010). Male Sprague-Dawley rats (275–325 g, Janvier Europe, St. Berthevin, France) were anesthetized with 40 mg/kg i.p. of sodium pentobarbital. After midline sternotomy, hearts were rapidly excised and immersed in ice-chilled physiological solution, and the aorta was cannulated. Retrograde perfusion was performed at 37 1C from a reservoir at a constant perfusion pressure of 7072 mm Hg. All hearts were perfused with a non-recirculating Krebs–Henseleit solution, which was prepared daily and contained NaCl 118.5 mmol/L, KCI 4.7 mmol/L, CaCI2 2.5 mmol/L, NaHCO3 25 mmol/L, MgSO4 1.2 mmol/L, KH2PO4 1.2 mmol/L, and glucose 11 mmol/L, pH 7.3–7.4. The perfusate was aerated with 95% O2 þ 5% CO2 [11]. Two side arms in the perfusion line proximal to the heart inflow cannula allowed for infusion of different solutions directly into the coronary inflow line [12]. 2.2. Assessment of heart function Coronary flow was measured by collecting the effluent from the right ventricular outflow tract in a graduated cylinder. The magnitude of reactive hyperemia was calculated by using postischemic changes of coronary flow. Left ventricular developed pressure (LVDevP) was determined by using a buffer-filled latex balloon-tipped polyethylene cannula (1.5 mm ID) connected to a pressure transducer, which was placed at the same height of the heart. The diastolic pressure was set at 10 mm Hg. Maximum rates of left ventricular development (LVdP/dtmax) and relaxation (LVdP/ dtmin), and heart rate were recorded. The time to asystolia after induction of ischemia, the time to restoration of contraction and the quantity of extrasystols during reperfusion were calculated from the recordings (which were done with a Spectramed P10EZ, connected to a Gould 8000S recorder). After a 15–20 min stabilization period, DHA 2. 5 μM (sodium salt, Sigma-Aldrich, Paris, France), was added to the perfusion buffer, and the hearts were subsequently (10 min) subjected to 20 min of global ischemia and 30 min of postischemic reperfusion. In a second set of experiments, DHA was added before (10 min) and immediately (10 min) after the ischemic period.
2.5. Immunostaining Paraffin-enclosed hearts were cut to 4 nm-thick slices by using an ultra-microtome. After removal of paraffin with toluene (1 h at 37 1C), re-hydration and treatment with pepsin (0.4% in HCl 0.01 M; 30 min at 37 1C), slices were incubated with primary antibodies against Nox4 (SantaCruz Biotechnology, Paris, France) or Notch2 (Abcam, Paris, France; dilutions 1:100). Following incubation with a peroxidase-conjugated antibody, slices were exposed to 3-amino-9-ethylcarbazole (AEC) substrate-chromogen (Dako, Paris, France). 2.6. Statistical analyses Data are shown as mean 7 SD, n ¼4. One-way ANOVA followed by the post-hoc Tukey0 s test (software: GraphPad Prism) was used to detect differences. A p o0.05 was considered as statistically significant.
3. Results Infusion with DHA markedly improved cardiac function after ischemia-reperfusion (Table 1), when administered either pre- or pre/post-induction of ischemia. Conversely, we did not observe any significant protective effect of DHA when administered postinduction of ischemia (data not shown), with the exception of lower creatine kinase release. Notably, DHA reduced oxygen and
Table 1 Main cardiac functional parameters.
I/R DHA preC DHA pre/post
Cardiac force (% of control)
ATP consumption (% of control)
O2 consumption (% of control)
Coronary flow (% of control)
84.2 7 2.3 91.3 7 1.2* 92.9 7 1.4*
82.4 7 0.94 807 0.8 76.6 7 1.2*
82.9 70.56 79.9 70.95* 73.5 71.2*
82.6 7 0.13 82.3 7 0.43 76.8 7 0.59*
Data are means 7 S.D.; n¼ 4; I/R, ischemia/reperfusion; DHA, docosahexaenoic acid. n
po 0.05
D. Richard et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 90 (2014) 139–143
141
Fig. 1. DHA infusion reduces myocardial infarct size and creatine kinase release. (A) Pictures of paraffin-embedded heart slices, obtained from organs removed at the indicated time points; (B) Variation of creatine kinase (CK) release following DHA treatment. CK release was determined in perfusion effluents from rat hearts at different time points and was expressed as percentage relative to the basal value in control. Data are means 7 SD, n¼ 4, np o 0.05.
Fig. 2. DHA limits oxidative stress but has no effect on Nox 4 expression. (A) Lipid peroxides, as evaluated by the FOX assay [14], were measured in perfusion effluents from rat hearts at different time points. (B) mRNA was extract from rat myocardium after ischemia/reperfusion and Nox 4 mRNA was quantified by real-time RT-PCR. mRNA expression was normalized to HPRT transcript level [23]. (C) Nox4 expression in infarcted myocardia as visualized by histochemistry.
ATP consumption, which was associated with stronger cardiac output. Also, provision of DHA remarkably reduced infarct size, pre- and pre/post-induction of ischemia (Fig. 1A) and, consequently, the release of creatine kinase (Fig. 1B). As a measure of oxidative stress (a key feature of ischemia/ reperfusion), we evaluated lipid peroxides by the FOX assay [14]. DHA administration significantly lowered the amount of lipid
peroxides produced by ischemia/reperfusion (Fig. 2A). The expression of Nox 4 – a major producer of free radicals in cardiovascular cells – increased did not significantly change between controls and DHA-perfused hearts (Fig. 2B), as also visualized by immunostaining (Fig. 2C). To gain some insight in the mechanism(s) responsible for the cardioprotective effects of DHA, we evaluated the expression of
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Table 2 DHA induces Notch signaling.
Controls I/R DHA pre DHA pre/post
Notch 2
Jagged 1
Jagged 2
Delta 1
Hey 1
Notch 3
0.53 7 0.03 0.217 0.02 0.75 7 0.03 0.667 0.02
0.75 70.02 0.51 70.02 1.3570.04 0.43 70.01
0.617 0.01 0.747 0.02 0.55 7 0.03 0.89 7 0.02
0.86 7 0.04 0.80 7 0.03 0.87 7 0.01 1.047 0.06
1.43 70.2 1.25 70.2 1.63 70.3 1.92 70.4
0.79 7 0.4 0.127 0.1 0.217 0.2 1.38 7 0.5
Data are means 7 SD of n¼ 4. Notch-2, Notch-3, Jagged-1, Jagged-2, Delta 1, Hey 1 mRNAs were quantified by real-time RT-PCR and normalized to HPRT transcript level.
Fig. 3. Notch 2 expression in infarct myocardium as visualized by histochemistry.
Notch2 and of some of its main effectors in myocardial tissues subjected to ischemia/reperfusion. As shown in Table 2, infusion of DHA before and before/after the induction of ischemia differentially modulates the expression of Notch2 and 3 target genes. In particular, DHA increases the expression of Hey1 when infused pre- and pre/post-ischemia; Jagged 1 and the Notch2 receptors increase with DHA pre-ischemia, but not pre/post; Notch2 and 3 receptors as well as Delta increase following DHA administration pre- and (especially) pre/post-ischemia. Immunostaining of myocardial tissues (Fig. 3) shows nuclear localization of Notch2, confirming qPCR data.
4. Discussion In this study, we demonstrate that systemic administration of DHA provides protection from ischemic damage in the isolated heart. DHA is an essential omega-3 fatty acid and is vital for proper heart functioning, where it contributes to signal transduction. Epidemiological and intervention studies confirm the cardioprotective properties of DHA and of long-chain omega 3 fatty acids. Indeed, there is accumulated evidence that diets rich in marine products are associated with better cardiovascular prognosis and scientific bodies suggest consumption of 500 mg/d of long-chain omega 3 fatty acids. Specific to myocardial infarction (MI), it is noteworthy that, in addition to epidemiological studies reporting lower incidence of MI in people who consume adequate amounts of EPA and DHA, Block et al. showed that blood omega 3 fatty acid concentrations are lower in MI patients than in matched controls. These findings were subsequently confirmed by Aarsetoey et al. [16]. However, little is known about the acute cardiac effects of DHA administration. As an example, Schrepf et al. [3] showed that the acute administration of DHA to 10 patients decrease sustained ventricular tachycardia in the majority of them. From a pharmanutrition viewpoint, it is worth noting that our investigation and those that show acute effects target the circulating free fatty acid pool. Therefore, the observed effects might not be replicated by dietary/supplementation studies that increase DHA concentrations in specific pools, namely phospholipids [17].
In this investigation, we wanted to find out whether DHA, administered acutely either before or before and after the induction of heart ischemia, would protect the myocardium from damage. Moreover, we aimed at gaining partial insight into the mechanisms responsible for the observed effects. Indeed, both biochemical assays and immunohistochemistry analyses demonstrate significant protection afforded by DHA. The functional and biochemical indices were associated with an induction of the Notch signaling. Indeed, the effects of DHA on the Notch pathways have been previously reported in vitro (smooth muscle cells) by Delbosc et al. [18], who reported that inhibition of the Notch pathway participates in the transition of VSMCs toward a migratory phenotype. The antioxidant effect of DHA infusion confirms, in an ischemia/reperfusion setting, the results of several in vivo and in vitro investigations [19] that indicate how polyunsaturated fatty acids, namely EPA and DHA, exert antioxidant effects that might be relevant to cardioprotection [12] during acute injuries. As mentioned in the Introduction, acute infusions of DHA or of any fatty acid target the free fatty acid pool. In this study, we used 2.5 μM DHA, which approximates murine cardiomyocyte0 s concentrations [24]. A comparison with human physiology is difficult because (a) data on the absolute concentrations of DHA are scant (most data in the literature are reported as percentage of total fatty acids; (b) DHA preferentially incorporates into phospholipids [17]; and (c) the free fatty acid pool is of dubious physiological significance because ingested fatty acids are digested and incorporated into assorted lipoproteins for tissue delivery. Yet, Harper et al. [20] reported plasma (which includes esterified and free fatty acid) DHA levels of 80 μM, i.e. well below the efficacious concentrations of our intervention. We would like to reiterate that our results should be interpreted from a pharmanutrition viewpoint rather than from a merely dietary one. The major limitation of this study is the absence of another fatty acid as a comparator. Consequently, based on our data, we cannot claim that DHA is unique in its acute anti-ischemic effects. The issue of whether DHA only or other omega 3 polyunsaturated fatty acids such as EPA [21] and alpha-linolenic acid [22] putatively contribute to better prognosis is still unresolved. In conclusion, while the precise nature of the Notch-mediated protection from ischemia/reperfusion afforded by DHA is as yet to be fully elucidated, our data add to the growing body of literature
D. Richard et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 90 (2014) 139–143
that indicates how systemic administration of DHA affords cardiovascular protection. Future studies will eventually further clarify the molecular actions that underlie such effects. References [1] A. Grey, M. Bolland, Clinical trial evidence and use of fish oil supplements, JAMA Intern. Med. (2013) 1–4. [2] G.E. Billman, J.X. Kang, A. Leaf, Prevention of sudden cardiac death by dietary pure omega-3 polyunsaturated fatty acids in dogs, Circulation 99 (1999) 2452–2457. [3] R. Schrepf, T. Limmert, P. Claus Weber, K. Theisen, A. Sellmayer, Immediate effects of n-3 fatty acid infusion on the induction of sustained ventricular tachycardia, Lancet 363 (2004) 1441–1442. [4] M.C. Heidt, M. Vician, S.K. Stracke, T. Stadlbauer, M.T. Grebe, A. Boening, P.R. Vogt, A. Erdogan, Beneficial effects of intravenously administered n-3 fatty acids for the prevention of atrial fibrillation after coronary artery bypass surgery: a prospective randomized study, Thorac. Cardiovasc. Surg. 57 (2009) 276–280. [5] Y.F. Xiao, D.C. Sigg, M.R. Ujhelyi, J.J. Wilhelm, E.S. Richardson, P.A. Iaizzo, Pericardial delivery of omega-3 fatty acid: a novel approach to reducing myocardial infarct sizes and arrhythmias, Am. J. Physiol. Heart Circ. Physiol. 294 (2008) H2212–H2218. [6] D. Kromhout, J. de Goede, Update on cardiometabolic health effects of omega3 fatty acids, Curr. Opin. Lipidol. (2013). [7] L. Belayev, L. Khoutorova, K.D. Atkins, T.N. Eady, S. Hong, Y. Lu, A. Obenaus, N.G. Bazan, Docosahexaenoic acid therapy of experimental ischemic stroke, Transl. Stroke Res. 2 (2011) 33–41. [8] T.N. Eady, L. Khoutorova, D.V. Anzola, S.H. Hong, A. Obenaus, A. Mohd-Yusof, N.G. Bazan, L. Belayev, Acute treatment with docosahexaenoic Acid complexed to albumin reduces injury after a permanent focal cerebral ischemia in rats, PLoS ONE 8 (2013) e77237. [9] I. Dikic, M.H. Schmidt, Notch: implications of endogenous inhibitors for therapy, Bioessays 32 (2010) 481–487. [10] L.K. Phng, H. Gerhardt, Angiogenesis: a team effort coordinated by notch, Dev. Cell 16 (2009) 196–208. [11] B. Caillier, S. Pilote, D. Patoine, X. Levac, C. Couture, P. Daleau, C. Simard, B. Drolet, Metabolic syndrome potentiates the cardiac action potentialprolonging action of drugs: a possible ‘anti-proarrhythmic’ role for amlodipine, Pharmacol. Res. 65 (2012) 320–327.
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[12] V.F. Sagach, M. Scrosati, J. Fielding, G. Rossoni, C. Galli, F. Visioli, The watersoluble vitamin E analogue Trolox protects against ischaemia/reperfusion damage in vitro and ex vivo. A comparison with vitamin E, Pharmacol. Res. 45 (2002) 435–439. [13] E. Pasini, R. Solfrini, T. Bachetti, M. Marino, P. Bernocchi, F. Visioli, R. Ferrari, The blood perfused isolated heart: characterization of the model, Basic Res. Cardiol. 94 (1999) 215–222. [14] K. Fukuzawa, A. Fujisaki, K. Akai, A. Tokumura, J. Terao, J.M. Gebicki, Measurement of phosphatidylcholine hydroperoxides in solution and in intact membranes by the ferric-xylenol orange assay, Anal. Biochem. 359 (2006) 18–25. [15] C. Ceconi, A. Cargnoni, G. Francolini, G. Parinello, R. Ferrari, Heart rate reduction with ivabradine improves energy metabolism and mechanical function of isolated ischaemic rabbit heart, Cardiovasc. Res. 84 (2009) 72–82. [16] H. Aarsetoey, R. Aarsetoey, T. Lindner, H. Staines, W.S. Harris, D.W. Nilsen, Low levels of the omega-3 index are associated with sudden cardiac arrest and remain stable in survivors in the subacute phase, Lipids 46 (2011) 151–161. [17] P. Rise, S. Eligini, S. Ghezzi, S. Colli, C. Galli, Fatty acid composition of plasma, blood cells and whole blood: relevance for the assessment of the fatty acid status in humans, Prostaglandins Leukot. Essent. Fatty Acids 76 (2007) 363–369. [18] S. Delbosc, M. Glorian, A.S. Le Port, G. Bereziat, M. Andreani, I. Limon, The benefit of docosahexanoic acid on the migration of vascular smooth muscle cells is partially dependent on Notch regulation of MMP-2/-9, Am. J. Pathol 172 (2008) 1430–1440. [19] E. Giordano, F. Visioli, Long-chain omega 3 fatty acids: molecular bases of potential antioxidant actions, Prostaglandins Leukot. Essent. Fatty Acids 90 (2014) 1–4. [20] C.R. Harper, M.J. Edwards, A.P. DeFilippis, T.A. Jacobson, Flaxseed oil increases the plasma concentrations of cardioprotective (n-3) fatty acids in humans, J. Nutr. 136 (2006) 83–87. [21] W.S. Harris, T.D. Dayspring, T.J. Moran, Omega-3 fatty acids and cardiovascular disease: new developments and applications, Postgrad. Med. 125 (2013) 100–113. [22] M.S. Vedtofte, M.U. Jakobsen, L. Lauritzen, B.L. Heitmann, Dietary alphalinolenic acid, linoleic acid, and n-3 long-chain PUFA and risk of ischemic heart disease, Am. J. Clin. Nutr. 94 (2011) 1097–1103. [23] D. Richard, C. Wolf, U. Barbe, K. Kefi, P. Bausero, F. Visioli, Docosahexaenoic acid down-regulates endothelial Nox 4 through a sPLA2 signaling pathway, Biochem. Biophys. Res. Commun. 389 (2009) 516–522. [24] Richard et al., in preparation.
Contents lists available at ScienceDirect
Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Short communication
Infusion of docosahexaenoic acid protects against myocardial infarction$ D. Richard a,1, F. Oszust b,1, C. Guillaume c, H. Millart b, D. Laurent-Maquin c, C. Brou d, P. Bausero a, F. Visioli e,n a
Université Pierre et Marie Curie, Paris, France EA3801 HERVI (Hémostase et Remodelage Vasculaire post-Ischémique), Université de Reims Champagne-Ardenne, France c EA 4691 BiOs (Biomatériaux et inflammation en site Osseux), Université de Reims Champagne-Ardenne, France d Unité de Signalisation Moléculaire et Activation Cellulaire, Institut Pasteur, Paris, France e IMDEA-Food, CEI UAM þCSIC, C/Faraday 7, 28049 Madrid, Spain b
art ic l e i nf o
a b s t r a c t
Article history: Received 2 December 2013 Received in revised form 1 January 2014 Accepted 2 January 2014
Most of the cardioprotective effects of long-chain omega 3 fatty acids, namely docosahexaenoic (DHA; 22:6n-3) and eicosapentaenoic (EPA; 20:5n-3), are due to their hypotriglyceridemic and antiinflammatory effects, which lower the risk for cardiovascular disease and myocardial infarction. Little is known on the direct preventive activities of DHA and EPA on heart function. In isolated hearts, we studied (1) whether infused DHA is able to protect the heart from ischemia/reperfusion damage and (2) the role played by Notch-mediated signal transduction pathways in myocardial infarction. Perfusion with DHA before and before/after induction of ischemia reperfusion significantly diminished cardiac damage and afforded antioxidant protection. Mechanistically, infusion of DHA before and before/after the induction of ischemia differentially modulated the expression of Notch2 and 3 target genes. In particular, DHA increased the expression of Hey1 when infused pre- and pre/post-ischemia; Jagged 1 and the Notch2 receptors increased with DHA pre-ischemia, but not pre/post; Notch2 and 3 receptors as well as Delta increased following DHA administration pre- and (especially) pre/postischemia. In conclusion, while the precise nature of the Notch-mediated protection from ischemia/reperfusion afforded by DHA is as yet to be fully elucidated, our data add to the growing body of literature that indicates how systemic administration of DHA provides cardiovascular protection. & 2014 Elsevier Ltd. All rights reserved.
Keywords: Docosahexaenoic acid Notch Ischemia Reperfusion Cardioprotection
1. Introduction Adequate dietary intakes of long-chain omega 3 fatty acids, namely docosahexaenoic (DHA; 22:6n-3) and eicosapentaenoic (EPA; 20:5n-3) acids, are associated with reduced cardiovascular mortality. Most of the putative cardioprotective effects of omega 3 fatty acids are due to their hypotriglyceridemic and antiinflammatory effects, which lower the risk for cardiovascular disease and myocardial infarction. Of note, the positive association between dietary intake and cardiovascular prognosis has not been confirmed by randomized clinical trials with pharmaceutical preparations [1]. Little is known on the direct preventive activities of DHA and EPA on heart function, although a seminal paper by Billman and co-workers [2] reported that infusion of omega 3 fatty $ n
In memory of Charlotte Schneider. Corresponding author. E-mail addresses: [email protected], [email protected] (F. Visioli). 1 These authors contributed equally to this work.
0952-3278/$ - see front matter & 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plefa.2014.01.001
acids to dogs before an exercise test plus myocardial ischemia significantly reduced the manifestation of ventricular tachycardia or fibrillation by about 75%. In humans, a paper by Schrepf et al. [3] showed rapid effects, namely reduction of sustained ventricular tachycardia, of omega 3 infusion to patients with implanted cardioverter defibrillators who were at high risk of sudden cardiac death. These acute anti-arrhythmic actions were recently confirmed by Heidt et al. [4] and led to the proposal to infuse omega 3 fatty acids pericardially to limit infarct size and arrhythmias [5]. However, it must be underscored that current accumulated evidence does not prove any anti-arrhythmic actions of DHA and EPA supplementation per os [6]. Therefore, the possibility exists that acute infusion and oral treatment have different effects on cardiac electrophysiology. Finally, a recent paper reported on the acute neuroprotective effects of DHA in a rat model of ischemia [7] and was subsequently confirmed [8]. The Notch signaling pathway governs cell fates between neighboring cells and, therefore, regulates the development of
140
D. Richard et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 90 (2014) 139–143
organisms [9]. Studies of Notch and its role in the cardiovascular system are rapidly growing, particularly in the field of angiogenesis and endothelial biology [10]. While the Notch signaling pathway is complex, it is noteworthy that Notch signals are based on the stimulation of Notch receptors by membrane-spanning ligands. The aim of this study was twofold: we addressed (1) the question of whether infused DHA is able to protect the heart from ischemia/reperfusion damage and (2) the role played by Notchmediated signal transduction pathways in myocardial infarction.
2.3. Biochemical indexes Creatine phosphokinase (CPK) was measured using a commercial kit from Boehringer Mannheim (Paris, France) [13]. Lipid peroxides were measured with the FOX assay, by using the Peroxoquant kit (Pierce, Rockford, USA) [14]. Adenosine triphosphate (ATP) was extracted with 0.4 N HClO4 from frozen biopsies obtained by freeze-clamping the ventricular apex with aluminum tongs [15] and was measured by bioluminescence (Sigma-Aldrich, Paris, France).
2. Materials and methods
2.4. Gene analyses
2.1. Isolated rat heart preparation
Hearts were fixed using 4% paraformaldehyde then were lysed with a β-protease from Omega Bio-tek (Paris, France). RNA was extracted with the ENZA kit, following the manufacturer0 s instructions. Following retrotranscription, qPCR was performed with a LightCycler LC480 (Roche Diagnostics, Paris, France). Primers were as follows (s, sense; as; antisense): HPRTs: AGG-ACC-TCT-CGAAGT-GT; HPRTas: ATC-CCT-GAA-GTG-CTC-ATT-ATA; Notch2s: GCAGGT-AGC-TCA-GAC-CA; Notch2as: CGA-GCA-TTT-GAG-GAG-GCATAA; Jagged1s: CAT-CGA-GAA-ACA-CGG-AGC; HEY1s: CGA-CGTGGG-GAG-CGA-GAA-CAA-T; Hey1as, GCC-AAG-AGC-ATG-GCGATC-AAA-GTA; Nox4s, TGG-TTT-GCA-GAC-TTG-CTC-TA; Nox4as: GTA-TCC-CAT-CTG-TTT-GCA-TGA-GGT-A; NKX2.5s: CTG-CAG-TCCCGC-CTA-CA; NKX2.5as: CGA-CGC-CAA-AGT-TCA-CGA.
This investigation conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Research Council (Eight Edition, 2010). Male Sprague-Dawley rats (275–325 g, Janvier Europe, St. Berthevin, France) were anesthetized with 40 mg/kg i.p. of sodium pentobarbital. After midline sternotomy, hearts were rapidly excised and immersed in ice-chilled physiological solution, and the aorta was cannulated. Retrograde perfusion was performed at 37 1C from a reservoir at a constant perfusion pressure of 7072 mm Hg. All hearts were perfused with a non-recirculating Krebs–Henseleit solution, which was prepared daily and contained NaCl 118.5 mmol/L, KCI 4.7 mmol/L, CaCI2 2.5 mmol/L, NaHCO3 25 mmol/L, MgSO4 1.2 mmol/L, KH2PO4 1.2 mmol/L, and glucose 11 mmol/L, pH 7.3–7.4. The perfusate was aerated with 95% O2 þ 5% CO2 [11]. Two side arms in the perfusion line proximal to the heart inflow cannula allowed for infusion of different solutions directly into the coronary inflow line [12]. 2.2. Assessment of heart function Coronary flow was measured by collecting the effluent from the right ventricular outflow tract in a graduated cylinder. The magnitude of reactive hyperemia was calculated by using postischemic changes of coronary flow. Left ventricular developed pressure (LVDevP) was determined by using a buffer-filled latex balloon-tipped polyethylene cannula (1.5 mm ID) connected to a pressure transducer, which was placed at the same height of the heart. The diastolic pressure was set at 10 mm Hg. Maximum rates of left ventricular development (LVdP/dtmax) and relaxation (LVdP/ dtmin), and heart rate were recorded. The time to asystolia after induction of ischemia, the time to restoration of contraction and the quantity of extrasystols during reperfusion were calculated from the recordings (which were done with a Spectramed P10EZ, connected to a Gould 8000S recorder). After a 15–20 min stabilization period, DHA 2. 5 μM (sodium salt, Sigma-Aldrich, Paris, France), was added to the perfusion buffer, and the hearts were subsequently (10 min) subjected to 20 min of global ischemia and 30 min of postischemic reperfusion. In a second set of experiments, DHA was added before (10 min) and immediately (10 min) after the ischemic period.
2.5. Immunostaining Paraffin-enclosed hearts were cut to 4 nm-thick slices by using an ultra-microtome. After removal of paraffin with toluene (1 h at 37 1C), re-hydration and treatment with pepsin (0.4% in HCl 0.01 M; 30 min at 37 1C), slices were incubated with primary antibodies against Nox4 (SantaCruz Biotechnology, Paris, France) or Notch2 (Abcam, Paris, France; dilutions 1:100). Following incubation with a peroxidase-conjugated antibody, slices were exposed to 3-amino-9-ethylcarbazole (AEC) substrate-chromogen (Dako, Paris, France). 2.6. Statistical analyses Data are shown as mean 7 SD, n ¼4. One-way ANOVA followed by the post-hoc Tukey0 s test (software: GraphPad Prism) was used to detect differences. A p o0.05 was considered as statistically significant.
3. Results Infusion with DHA markedly improved cardiac function after ischemia-reperfusion (Table 1), when administered either pre- or pre/post-induction of ischemia. Conversely, we did not observe any significant protective effect of DHA when administered postinduction of ischemia (data not shown), with the exception of lower creatine kinase release. Notably, DHA reduced oxygen and
Table 1 Main cardiac functional parameters.
I/R DHA preC DHA pre/post
Cardiac force (% of control)
ATP consumption (% of control)
O2 consumption (% of control)
Coronary flow (% of control)
84.2 7 2.3 91.3 7 1.2* 92.9 7 1.4*
82.4 7 0.94 807 0.8 76.6 7 1.2*
82.9 70.56 79.9 70.95* 73.5 71.2*
82.6 7 0.13 82.3 7 0.43 76.8 7 0.59*
Data are means 7 S.D.; n¼ 4; I/R, ischemia/reperfusion; DHA, docosahexaenoic acid. n
po 0.05
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Fig. 1. DHA infusion reduces myocardial infarct size and creatine kinase release. (A) Pictures of paraffin-embedded heart slices, obtained from organs removed at the indicated time points; (B) Variation of creatine kinase (CK) release following DHA treatment. CK release was determined in perfusion effluents from rat hearts at different time points and was expressed as percentage relative to the basal value in control. Data are means 7 SD, n¼ 4, np o 0.05.
Fig. 2. DHA limits oxidative stress but has no effect on Nox 4 expression. (A) Lipid peroxides, as evaluated by the FOX assay [14], were measured in perfusion effluents from rat hearts at different time points. (B) mRNA was extract from rat myocardium after ischemia/reperfusion and Nox 4 mRNA was quantified by real-time RT-PCR. mRNA expression was normalized to HPRT transcript level [23]. (C) Nox4 expression in infarcted myocardia as visualized by histochemistry.
ATP consumption, which was associated with stronger cardiac output. Also, provision of DHA remarkably reduced infarct size, pre- and pre/post-induction of ischemia (Fig. 1A) and, consequently, the release of creatine kinase (Fig. 1B). As a measure of oxidative stress (a key feature of ischemia/ reperfusion), we evaluated lipid peroxides by the FOX assay [14]. DHA administration significantly lowered the amount of lipid
peroxides produced by ischemia/reperfusion (Fig. 2A). The expression of Nox 4 – a major producer of free radicals in cardiovascular cells – increased did not significantly change between controls and DHA-perfused hearts (Fig. 2B), as also visualized by immunostaining (Fig. 2C). To gain some insight in the mechanism(s) responsible for the cardioprotective effects of DHA, we evaluated the expression of
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Table 2 DHA induces Notch signaling.
Controls I/R DHA pre DHA pre/post
Notch 2
Jagged 1
Jagged 2
Delta 1
Hey 1
Notch 3
0.53 7 0.03 0.217 0.02 0.75 7 0.03 0.667 0.02
0.75 70.02 0.51 70.02 1.3570.04 0.43 70.01
0.617 0.01 0.747 0.02 0.55 7 0.03 0.89 7 0.02
0.86 7 0.04 0.80 7 0.03 0.87 7 0.01 1.047 0.06
1.43 70.2 1.25 70.2 1.63 70.3 1.92 70.4
0.79 7 0.4 0.127 0.1 0.217 0.2 1.38 7 0.5
Data are means 7 SD of n¼ 4. Notch-2, Notch-3, Jagged-1, Jagged-2, Delta 1, Hey 1 mRNAs were quantified by real-time RT-PCR and normalized to HPRT transcript level.
Fig. 3. Notch 2 expression in infarct myocardium as visualized by histochemistry.
Notch2 and of some of its main effectors in myocardial tissues subjected to ischemia/reperfusion. As shown in Table 2, infusion of DHA before and before/after the induction of ischemia differentially modulates the expression of Notch2 and 3 target genes. In particular, DHA increases the expression of Hey1 when infused pre- and pre/post-ischemia; Jagged 1 and the Notch2 receptors increase with DHA pre-ischemia, but not pre/post; Notch2 and 3 receptors as well as Delta increase following DHA administration pre- and (especially) pre/post-ischemia. Immunostaining of myocardial tissues (Fig. 3) shows nuclear localization of Notch2, confirming qPCR data.
4. Discussion In this study, we demonstrate that systemic administration of DHA provides protection from ischemic damage in the isolated heart. DHA is an essential omega-3 fatty acid and is vital for proper heart functioning, where it contributes to signal transduction. Epidemiological and intervention studies confirm the cardioprotective properties of DHA and of long-chain omega 3 fatty acids. Indeed, there is accumulated evidence that diets rich in marine products are associated with better cardiovascular prognosis and scientific bodies suggest consumption of 500 mg/d of long-chain omega 3 fatty acids. Specific to myocardial infarction (MI), it is noteworthy that, in addition to epidemiological studies reporting lower incidence of MI in people who consume adequate amounts of EPA and DHA, Block et al. showed that blood omega 3 fatty acid concentrations are lower in MI patients than in matched controls. These findings were subsequently confirmed by Aarsetoey et al. [16]. However, little is known about the acute cardiac effects of DHA administration. As an example, Schrepf et al. [3] showed that the acute administration of DHA to 10 patients decrease sustained ventricular tachycardia in the majority of them. From a pharmanutrition viewpoint, it is worth noting that our investigation and those that show acute effects target the circulating free fatty acid pool. Therefore, the observed effects might not be replicated by dietary/supplementation studies that increase DHA concentrations in specific pools, namely phospholipids [17].
In this investigation, we wanted to find out whether DHA, administered acutely either before or before and after the induction of heart ischemia, would protect the myocardium from damage. Moreover, we aimed at gaining partial insight into the mechanisms responsible for the observed effects. Indeed, both biochemical assays and immunohistochemistry analyses demonstrate significant protection afforded by DHA. The functional and biochemical indices were associated with an induction of the Notch signaling. Indeed, the effects of DHA on the Notch pathways have been previously reported in vitro (smooth muscle cells) by Delbosc et al. [18], who reported that inhibition of the Notch pathway participates in the transition of VSMCs toward a migratory phenotype. The antioxidant effect of DHA infusion confirms, in an ischemia/reperfusion setting, the results of several in vivo and in vitro investigations [19] that indicate how polyunsaturated fatty acids, namely EPA and DHA, exert antioxidant effects that might be relevant to cardioprotection [12] during acute injuries. As mentioned in the Introduction, acute infusions of DHA or of any fatty acid target the free fatty acid pool. In this study, we used 2.5 μM DHA, which approximates murine cardiomyocyte0 s concentrations [24]. A comparison with human physiology is difficult because (a) data on the absolute concentrations of DHA are scant (most data in the literature are reported as percentage of total fatty acids; (b) DHA preferentially incorporates into phospholipids [17]; and (c) the free fatty acid pool is of dubious physiological significance because ingested fatty acids are digested and incorporated into assorted lipoproteins for tissue delivery. Yet, Harper et al. [20] reported plasma (which includes esterified and free fatty acid) DHA levels of 80 μM, i.e. well below the efficacious concentrations of our intervention. We would like to reiterate that our results should be interpreted from a pharmanutrition viewpoint rather than from a merely dietary one. The major limitation of this study is the absence of another fatty acid as a comparator. Consequently, based on our data, we cannot claim that DHA is unique in its acute anti-ischemic effects. The issue of whether DHA only or other omega 3 polyunsaturated fatty acids such as EPA [21] and alpha-linolenic acid [22] putatively contribute to better prognosis is still unresolved. In conclusion, while the precise nature of the Notch-mediated protection from ischemia/reperfusion afforded by DHA is as yet to be fully elucidated, our data add to the growing body of literature
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