- Email: [email protected]
Resolvin D1 decreases caspase-3 activation in the limbic system after myocardial infarction
PharmaNutrition 3 (2015) 78–82
Contents lists available at ScienceDirect
PharmaNutrition journal homepage: www.elsevier.com/locate/phanu
Resolvin D1 decreases caspase-3 activation in the limbic system after myocardial infarction Kim Gilberta,b , Mandy Malicka,b , Ness Madingoua,b , Roger Godbouta,c , Guy Rousseaua,b,* a b c
Centre de Biomédecine, Hôpital du Sacré-Cœur de Montréal, 5400, Boulevard Gouin Ouest, Montréal, Québec H4J 1C5, Canada Département de Pharmacologie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada Département de Psychiatrie, Université de Montréal, C.P. 6128 Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 December 2014 Received in revised form 21 May 2015 Accepted 26 May 2015 Available online 17 July 2015
Myocardial infarction (MI) induces an inflammatory process that is associated with increased apoptosis in the limbic system of rats and the development of post-MI depressive symptoms. Resolvin D1 (RvD1), an omega-3 fatty acid metabolite, is known for its pro-resolution properties; it reduces infarct size and attenuates post-MI depression-like symptoms. The present study was designed to determine if a single RvD1 dose could abate caspase-3 activation, a marker of apoptosis, in the limbic system. Male SpragueDawley rats underwent 40 min of myocardial ischemia, followed by 24-h reperfusion. Five min before MI, the animals received a single intra-cardiac RvD1 injection (0.01, 0.1 or 0.3 mg) or vehicle (saline). Infarct size was assessed and caspase-3 activity measured in the amygdala and hippocampus. Rats receiving 0.1 or 0.3 mg RvD1 showed significantly decreased infarct size. Caspase-3 activity was significantly attenuated in the lateral amygdala and dentate gyrus with 0.1 mg RvD1 and in the CA1 region of the hippocampus and medial amygdala with 0.3 mg RvD1. In conclusion, RvD1 could reduce infarct size and caspase-3 activity in the amygdala and hippocampus. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Resolvin D1 Myocardial infarction Reperfusion Caspase-3 Amygdala Hippocampus
1. Introduction There is evidence supporting the view that omega-3 fatty acids could be beneficial for cardiovascular health. Previous studies from our laboratory have disclosed that consumption of a high omega-3 fatty acid diet results in smaller myocardial infarction (MI) size [1]. Up to now, the mechanisms involved are elusive, with many hypotheses being proposed to explain this beneficial effect, including the insertion of omega-3 fatty acids in lipid membranes [2], reduction of inflammation [3], and activation of the phosphoinositol-3-kinase (PI3K)/Akt pathway [1]. Moreover, a recent meta-analysis has indicated that the beneficial effect of omega-3 fatty acids on secondary prevention of cardiovascular disease may not be significant in humans [4] see also [5,6]. The present research thus aimed at a better understanding of the effects of the metabolites of the omega-3 fatty acids on cardiovascular health.
* Corresponding author at: Centre de Biomédecine, Hôpital du Sacré-Cœur de Montréal, 5400, Boulevard Gouin Ouest, Montréal, Québec H4J 1C5, Canada. Tel.: +1 514 338 2222x3421; fax: +1 514 338 2694. E-mail address: [email protected] (G. Rousseau). http://dx.doi.org/10.1016/j.phanu.2015.05.003 2213-4344/ ã 2015 Elsevier B.V. All rights reserved.
Omega-3 fatty acids can be metabolized into different molecules, including resolvins [7,8]. Resolvins are metabolized from docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) by pathways comprising cyclooxygenase-2, lipoxygenase and P450 [9,10]. DHA-derived resolvin D1, administered before or with the onset of post-MI reperfusion, results in smaller infarct size [11]. These findings suggest that omega-3 fatty acid metabolism could be a potential mechanism mediating the beneficial effect of omega-3 fatty acids on cardiovascular health. Clinical signs of depression represent a major adverse event of MI in humans [12]. Depression can be detected in 65% of MI patients and about 20% of them will experience major depression for months after the episode [13]. The consequences of MI may even be dramatic, going beyond financial and familial outcomes, since mortality among post-MI depressed patients is 3–4 times higher than among post-MI patients without depression [14,15]. The therapeutic potential of omega-3 fatty acids in this context has been hypothesized for many years, and multiple clinical trials have been conducted, but the results are inconclusive [16–18]. In the last decade, we designed an experimental model to study the biochemical and behavioral consequences of MI in rats. On the one hand, we found increased caspase-3 activity as early as 3 days
K. Gilbert et al. / PharmaNutrition 3 (2015) 78–82
post-MI in the extended limbic system, including the hippocampus and the amygdala [19,20]. On the other hand, behavioral signs compatible with clinical depression were noted only 14 days postMI [21–24]. Still, we also confirmed the fact that treatment with antidepressant molecules, such as desipramine, sertraline and escitalopram, attenuates the biochemical as well as the behavioral consequences of MI in this model [19,21,22,24]. More recently, we showed that alternative therapeutic routes, including probiotics [25] and RvD1 [26], could be beneficial in our model, suggesting a link between the biochemical and behavioral components of MI, i.e., the biochemical response induced by MI could be responsible for its behavioral effects. In this context, however, we ignored if RvD1 could attenuate caspase-3 activation. The present study was therefore designed to ascertain if a single RvD1 dose could confirm the infarct size reduction observed in a previous study and determine if it could modulate caspase-3 activity in the limbic system. 2. Methods 2.1. Experimental design Twenty-five 3-month-old male Sprague-Dawley rats (Charles River, Canada, St.-Constant, QC, Canada), weighing 325–350 g at the start of the experiments, were handled in compliance with regulations of the local animal care committee and in accordance with guidelines of the Canadian Council on Animal Care. They were housed individually, under constant conditions (temperature 21– 22 C and humidity 40–50%), including a 12-h light-dark cycle beginning at 8 AM. Standard chow pellets and tap water were available ad libitum throughout the study. An acclimatization period of 3 days after delivery by the supplier was imposed before the rats were randomly assigned to 1 of 4 groups: 0.5 ml of vehicle (NaCl 0.9%) or 1 of 3 RvD1 doses (0.01, 0.1 or 0.3 mg) injected directly into the left ventricle (LV) chamber 5 min before ischemia. RvD1 (17(S)-RvD1) already dissolved in ethanol was procured from Cayman Chemical (Ann Arbor, MI, USA). Exact amount of RvD1 was added to saline (NaCl 0.9%) for a final volume of 0.5 ml. The animals were sacrificed 24 h post-MI to measure caspase-3 activity in the limbic system (see below). 2.2. Surgical procedure Anesthesia was induced by intraperitoneal ketamine/xylazine injection (60 and 10 mg/kg, respectively). Subsequently, the rats were intubated and anesthesia was maintained under isoflurane (1–2%) ventilation. Electrocardiogram and heart rate (HR) were monitored throughout the procedure with electrodes placed on the animals’ paws. Left thoracotomy at the 5th intercostal space enabled left anterior descending coronary artery occlusion with 4– 0 silk sutures (Syneture, Covidien, Mansfield, MA, USA) and plastic snare. Vehicle or RvD1 was injected into the left heart cavity 5 min before ischemia, which was confirmed by ST segment alterations and the presence of ventricular subepicardial cyanosis. Sutures were removed after 40 min of ischemia, permitting myocardial tissue reperfusion. The thorax was closed with 2–0, 3–0 and 4–0 silk sutures (Syneture, Covidien), and the animals were given a subcutaneous antibiotic injection of 15,000 IU penicillin G (Duplocillin LA, Intervet Canada Ltd., Whitby, ON, Canada) and a subcutaneous analgesic injection (2 mg/kg buprenorphine) before being returned to their respective cages. 2.3. Hemodynamic data HR and mean arterial pressure (MAP) were measured by the tail cuff technique (Kent Scientific Corporation, Torrington, CT, USA)
79
before occlusion, at 20 min of ischemia, at the beginning of the reperfusion period and after 10 min of reperfusion. 2.4. Measurement of infarct size After the 24-h reperfusion period, the rats were restrained in a cone bag and rapidly decapitated. Their hearts were removed immediately and placed in a dish kept on crushed ice. They were washed with saline 0.9% by retrograde perfusion of the aorta. The left anterior descending coronary artery was occluded at the same site as for MI induction (see above) to map the area at risk (AR) by Evans blue infusion (0.5%). The hearts were then frozen ( 80 C for 5 min), sliced into 4 transverse 2-mm sections and placed in 2,3,5triphenyltetrazolium chloride solution (1%, pH 7.4) at 37 C for 10 min to better distinguish the area of necrosis (I) from the AR. The different regions were carefully drawn on a glass plate, photocopied and cut. Thereafter, the complete infarct region, AR and LV were weighed separately to express MI as percentage of necrosis (I) of the AR ((I/AR) 100)) and AR as percentage of the LV area ((AR/ LV) 100). The brain was rapidly placed in a dish standing on crushed ice. Brain regions were identified according to the atlas of Paxinos and Watson [27]: the lateral and medial amygdala (LA, MA) and hippocampus (CA1, CA3 and dentate gyrus (DG) regions) were dissected out, snap-frozen in liquid nitrogen and maintained at 80 C until required. 2.5. Caspase-3 activity Caspase-3 activity was measured according to a previouslydescribed protocol [28]. Tissues were homogenized by sonication in lysis buffer and incubated for 30 min on ice. The tissue homogenates were centrifuged at 4 C for 10 min. Enzymatic reactions were undertaken in reaction buffer with 25 mg of protein (confirmed by the Bradford method) and fluorescent substrate (AcDEVD-AMC, 40 mM). They were studied after incubation in the dark for 3 h at 37 C and stopped by the addition of 0.4 M NaOH and 0.4 M glycine buffer. A negative control for each sample was performed in triplicate using Ac-DEVD-CHO (2 mM). Fluorescence was quantified by spectrofluorometry (Photon Technology International, Lawrenceville, NJ, USA) at 365 nm excitation wavelength and 465 nm emission wavelength. Activity was calculated from the difference between positive and negative controls. 2.6. Statistical analysis The data are reported as mean SEM. Statistical tests were performed with SPSS 20 (IBM Corp., Armonk, NY, USA). Infarct size and caspase-3 activity were compared by analysis of variance (ANOVA), followed by post hoc comparisons (Dunnett) when significant. If variances were heterogenous, Brown–Forsythe correction was followed by Games-Howell comparisons when applicable. Hemodynamic data were assessed by mixed ANOVA (intergroup analysis: between groups; intragroup comparison: by time). p < 0.05 was considered significant. 3. Results 3.1. Hemodynamic data (Table 1) Hemodynamic data analysis indicated that HR, MAP and pressure rate product (PRP) were similar among groups throughout the experiment. Intragroup comparisons disclosed significant differences in MAP (F(1,31, 10,51) = 15.27; p < 0.05) and PRP (F(1,56, 12,47) = 14.04; p < 0.05) during the experiment. Post hoc analysis revealed that MAP and PRP were reduced during ischemia and reperfusion compared to baseline (p < 0.05, Table 1).
80
K. Gilbert et al. / PharmaNutrition 3 (2015) 78–82
Table 1 Hemodynamic data (heart rate, mean arterial pressure and pressure rate product) for each RvD1 dose (mg). *p < 0.05 compared to baseline. Values are mean SEM. RvD1: Resolvin D1; HR: heart rate; PRP: pressure rate product. RvD1 (0.01 mg)
RvD1 (0.1 mg)
RvD1 (0.3 mg)
Baseline HR 253 13 MAP 98 3 PRP 249 16
265 13 85 6 225 26
244 2 88 12 215 30
236 12 94 8 221 13
20-min ischemia HR 254 7 MAP 67 8 PRP 170 18*
251 14 78 3 196 11*
244 5 73 4 178 10*
244 6 80 3 196 9*
Reperfusion HR 249 15 MAP 69 3* PRP 171 2*
255 13 70 2* 178 11*
249 7 69 4* 172 4*
256 9 73 3* 187 15*
10-min reperfusion HR 248 15 MAP 67 3* PRP 166 5*
256 13 70 1* 179 9*
258 6 66 1* 175 1*
264 8 77 2* 204 10*
Vehicle
HR: heart rate (beats/min); MAP: mean arterial pressure (mm Hg). PRP: HR MAP/ 100. * p < 0.05 versus baseline.
Fig. 2. Caspase-3 activity expressed in different areas of the brain as percentage of the vehicle group and assessed by in vitro spectrofluorescence (3–7 rats per group). RvD1: Resolvin D1; LA: lateral amygdala; MA: medial amygdala; DG: dentate gyrus; CA1: cornu ammonis area 1 of the hippocampus; CA3: cornu ammonis area 3 of the hippocampus. *Indicates significant difference between the experimental and vehicle groups; p < 0.05.
3.3.1. Correlation between caspase-3 activity and myocardial infarct size We tested the possibility of correlation between caspase-3 activity and MI. Correlation between caspase-3 activity in the LA and I/AR (r2 = 0.14, p = 0.087 (Fig. 3A) and CA1 (r2 = 0.17, p = 0.068, Fig. 3B) was almost significant. Correlation between MI and other regions was clearly non-significant. 4. Discussion
3.2. Infarct size Myocardial infarct size (I), expressed as percentage of AR, disclosed significant between group differences (F(3,21) = 9.58; p < 0.05). Post hoc analysis revealed significant differences between groups receiving 0.1 or 0.3 mg RvD1 compared to the control (vehicle) group. No difference was evident between the 0.01 mg and control groups. AR, expressed as percentage of LV, was similar between groups and represented around 70% of LV (Fig. 1). 3.3. Caspase-3 activity Caspase-3 activity in the amygdala and hippocampal areas presented significant differences between groups: LA F(3,19) = 7.11, p < 0.05; MA F(3,18) = 4.30; p < 0.05; DG F(3,12) = 5.14; p < 0.05; CA1 F(3,17) = 7.39; p < 0.05. Further analysis indicated significantly reduced caspase-3 activity in the 0.3 mg RvD1 group versus vehicle controls in the LA, MA, DG and CA1 areas (p < 0.05). Significant difference between the 0.1 mg RvD1 group versus the vehicle group was observed in the LA and DG areas (p < 0.05). No significant difference was apparent in the 0.01 RvD1 and vehicle groups. No difference was evident between groups in the CA3 area of the hippocampus (Fig. 2).
Fig. 1. Infarct size (I) expressed as percentage of the area at risk (AR), and AR as percentage of the left ventricle (LV). RvD1: Resolvin D1. *Indicates significant difference between the experimental and vehicle groups; p < 0.05.
The current study indicates that caspase-3 activity 24 h post-MI is significantly attenuated in the amygdala and hippocampus in the presence of RvD1. We also confirm our previous data according to which RvD1 significantly reduces infarct size without significant effects on hemodynamic parameters [26]. The decrease of caspase-3 activity in the amygdala and hippocampus by RvD1 is in line with our hypothesis. Indeed, we have already observed that caspase-3 activity increases after MI [28], with a peak at 3 days post-MI in different regions of the limbic system [20]. It must be noted that, at 7 days post-MI, we were unable to detect augmented caspase-3 activity in these regions, which suggests that the triggered signal is transient [20]. In an effort to ascertain the mechanism responsible for early caspase-3 activation, we administered various molecules, including antidepressants [19,21,22], the non-selective cytokine synthesis inhibitor pentoxifylline [23], probiotics [29] and a high-omega-3 fatty acid diet [1]. We proposed that caspase-3 activation is due, at least in part, to the inflammatory process seen in MI [24]. As a matter of fact, increases of inflammatory molecules, such as tumor necrosis factor-alpha [22,30] or interleukin-1beta [31], documented after MI, activate caspase-3 [32,33]. Other mechanisms, such as epicardial nerve activation, could also participate in augmenting caspase-3 activity. Indeed, Francis et al. [34] reported that increment of pro-inflammatory cytokines a few minutes after ischemia induction could be attenuated by the destruction of epicardial nerves. RvD1 is a molecule involved in the resolution phase of inflammation [35]. It has been reported that RvD1 can prevent the synthesis or effect of pro-inflammatory cytokines in different conditions and thus curb caspase-3 activity. However, it is also possible that reduction of infarct size itself by RvD1 may result in abatement of caspase-3 activity, as suggested by the correlation we observed with infarct size. Interestingly, we proposed previously that the post-MI apoptosis encountered in the CA1 region of the hippocampus is related to the ischemic damage induced in the myocardium [20], since this hippocampal region is more sensitive to ischemia than other regions [36]. With infarct size reduction evoked by RvD1, it is possible that smaller myocardial damage results in lower caspase-3 activation.
K. Gilbert et al. / PharmaNutrition 3 (2015) 78–82
81
Fig. 3. Positive correlation between myocardial infarct size (I/AR 100) and caspase-3 activity in the lateral amygdala (r2 = 0.13; p = 0.087, panel A) and CA1 (r2 = 0.17; p = 0.068, panel B).
According to previous studies, the beneficial effect of RvD1 on infarct size is probably not related to diminution of proinflammatory cytokines. Indeed, in different experiments, we have observed that reduction of circulating pro-inflammatory cytokines does not correlate with a significant effect on infarct size [23,37]. We have reported earlier that the presence of RvD1 results in smaller infarct size with activation of the cardioprotective PI3K/ Akt signaling pathway [11]. It is known that when activated at the onset of reperfusion, the PI3K/Akt signaling pathway could diminish infarct size by inhibiting the opening of transient mitochondrial permeability pores [38]. The link between RvD1 and PI3K/Akt pathway activation is unknown, but it could be speculated that 1 of 2 G-protein-coupled receptors interacting with RvD1 is responsible for this activation [39], a hypothesis that needs to be confirmed in further studies. Since RvD1 is derived from DHA, it could also be possible that its effects are mediated by Notch receptors [40]. The reduction of caspase-3 activation by RvD1 after MI is interesting since we have demonstrated linkage between post-MI depression and caspase-3 activation. Independently of the interventions instituted [21,22], we observed that abatement of caspase-3 activation at 3 days post-MI resulted in significant diminution of clinical depression by 14 days post-MI. Since we have documented that clinical depression is attenuated with a single RvD1dose, these data argue for a role of apoptosis in post-MI depression. 4.1. Limitations One limitation of our study is the timing of RvD1 administration. Indeed, pre-ischemic administration is impossible in a clinical setting. However, since RvD1 is a metabolite of omega-3 fatty acids and synthetized in an inflammatory context, it could be present during the ischemic period with an omega-3-enriched diet. Another limitation could have to do with intraventricular RvD1 injection. Angioplasty, however, is an increasingly common reperfusion procedure [41–45], an easy way to access the LV chamber for drug injection via catheter. It adds ecological validity to our procedure. We have also chosen to measure caspase-3 activity selectively in the hippocampus and the amygdala. This choice was driven by the fact that we have previously demonstrated that, one day postMI, caspase-3 activity is significantly increased in the hippocampus and the amygdala and less so in other regions such as frontal and prefrontal cortices [20]. It still remains possible that MI and RvD1 could affect other regions of the brain but this has never been documented.
5. Conclusions In conclusion, RvD1 reduces infarct size as well as caspase-3 activity in the amygdala and hippocampus. Acknowledgments This work was supported by a grant from La Fondation des Maladies du Coeur du Canada. Kim Gilbert holds a studentship from Fonds de la recherche du Québec—Santé. On behalf of all authors, the corresponding author (GR) states that there is no conflict of interest. References [1] Rondeau I, Picard S, Bah TM, Roy L, Godbout R, Rousseau G. Effects of different dietary omega-6/3 polyunsaturated fatty acids ratios on infarct size and the limbic system after myocardial infarction. Can. J. Physiol. Pharmacol. 2011;89 (March (3)):169–76, doi:http://dx.doi.org/10.1139/Y11-007. 21423290. [2] Ander BP, Edel AL, McCullough R, Rodriguez-Leyva D, Rampersad P, Gilchrist JS, Lukas A, Pierce GN. Distribution of omega-3 fatty acids in tissues of rabbits fed a flaxseed-supplemented diet. Metab. Clin. Exp. 2010;59(5):620–7, doi:http:// dx.doi.org/10.1016/j.metabol.2009.09.005. 19913851. [3] Calder PC. n-3 Polyunsaturated fatty acids, inflammation, and inflammatory diseases. Am. J. Clin. Nutr. 2006;83(Suppl. 6):1505S–19S. 16841861. [4] Kwak SM, Myung SK, Lee YJ, Seo HG. Efficacy of omega-3 fatty acid supplements (eicosapentaenoic acid and docosahexaenoic acid) in the secondary prevention of cardiovascular disease: a meta-analysis of randomized, doubleblind, placebo-controlled trials. Arch. Intern. Med. 2012;172(May (9)):686–94, doi:http://dx.doi.org/10.1001/archinternmed.2012.262. 22493407. [5] Hu FB, Manson JE. Omega-3 fatty acids and secondary prevention of cardiovascular disease—is it just a fish tale? Comment on “efficacy of omega-3 fatty acid supplements (eicosapentaenoic acid and docosahexaenoic acid) in the secondary prevention of cardiovascular disease”. Arch. Intern. Med. 2012;172 (May (9)):694–6, doi:http://dx.doi.org/10.1001/archinternmed.2012.463. 22493410. [6] Messori A, Fadda V, Maratea D, Trippoli S. Omega-3 fatty acid supplements for secondary prevention of cardiovascular disease: from “no proof of effectiveness” to “proof of no effectiveness”. JAMA Intern. Med. 2013;173(August (15)):1466–8, doi:http://dx.doi.org/10.1001/jamainternmed.2013.6638. 23779264. [7] Serhan C, Hong S, Gronert K, Colgan S, Devchand P, Mirick G, Moussignac R. Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J. Exp. Med. 2002;196(8):1025–37, doi:http://dx.doi.org/10.1084/jem.20020760. 12391014. [8] Serhan CN, Arita M, Hong S, Gotlinger K. Resolvins, docosatrienes, and neuroprotectins, novel omega-3-derived mediators, and their endogenous aspirin-triggered epimers. Lipids 2004;39(November (11)):1125–32, doi:http://dx. doi.org/10.1007/s11745-004-1339-7. 15726828. [9] Dangi B, Obeng M, Nauroth JM, Chung G, Bailey-Hall E, Hallenbeck T, Arterburn LM. Metabolism and biological production of resolvins derived from docosapentaenoic acid (DPAn-6). Biochem. Pharmacol. 2010;79(January (2)):251– 60, doi:http://dx.doi.org/10.1016/j.bcp.2009.08.001. 19679107. [10] Sun YP, Oh SF, Uddin J, Yang R, Gotlinger K, Campbell E, Colgan SP, Petasis NA, Serhan CN. Resolvin D1 and its aspirin-triggered 17R epimer. Stereochemical assignments, anti-inflammatory properties, and enzymatic inactivation. J. Biol. Chem. 2007;282(March (13)):9323–34, doi:http://dx.doi.org/10.1074/jbc. M609212200. 17244615.
82
K. Gilbert et al. / PharmaNutrition 3 (2015) 78–82
[11] Tran QT, Gosselin A-A, Bourque-Riel V, Gilbert K, Charron T, Rousseau G. Effect of Resolvin D1 on experimental myocardial infarction. Exp. Clin. Cardiol. 2014;20(11):6704–12. [12] Frasure-Smith N, Lespérance F. Depression and other psychological risks following myocardial infarction. Arch. Gen. Psychiatry 2003;60(6):627–36, doi:http://dx.doi.org/10.1001/archpsyc.60.6.627. 12796226. [13] Guck TP, Kavan MG, Elsasser GN, Barone EJ. Assessment and treatment of depression following myocardial infarction. Am. Fam. Physician 2001;64 (August (4)):641–8. 11529263. [14] Frasure-Smith N, Lespérance F, Talajic M. Depression following myocardial infarction. Impact on 6-month survival. JAMA 1993;270(October (15)):1819– 25, doi:http://dx.doi.org/10.1001/jama.270.15.1819. 8411525. [15] Frasure-Smith N, Lespérance F, Talajic M. Depression and 18-month prognosis after myocardial infarction. Circulation 1995;91(February (4)):999–1005, doi: http://dx.doi.org/10.1161/01.CIR.91.4.999. 7531624. [16] Zimmer R, Riemer T, Rauch B, Schneider S, Schiele R, Gohlke H, Diller F, Steinbeck G, Katus H, Senges J. Effects of 1-year treatment with highly purified omega-3 fatty acids on depression after myocardial infarction: results from the Omega trial. J. Clin. Psychiatry 2013;74(November (11)):e1037–45, doi:http:// dx.doi.org/10.4088/JCP.13m08453. 24330904. [17] Siddiqui RA, Harvey KA. Dietary interventions with n-3 fatty acids or probiotics targeting post-myocardial infarction depression. Br. J. Nutr. 2013;109(January (1)):1–3, doi:http://dx.doi.org/10.1017/S0007114512004254. 23286323. [18] Giltay EJ, Geleijnse JM, Kromhout D. Effects of n-3 fatty acids on depressive symptoms and dispositional optimism after myocardial infarction. Am. J. Clin. Nutr. 2011;94(December (6)):1442–50, doi:http://dx.doi.org/10.3945/ ajcn.111.018259. 22030221. [19] Wann BP, Bah TM, Boucher M, Courtemanche J, Le Marec N, Rousseau G, Godbout R. Vulnerability for apoptosis in the limbic system after myocardial infarction in rats: a possible model for human postinfarct major depression. J. Psychiatry Neurosci. 2007;32(January (1)):11–6. 17245469. [20] Kaloustian S, Wann BP, Bah TM, Girard SA, Apostolakis A, Ishak S, Mathieu S, Ryvlin P, Godbout R, Rousseau G. Apoptosis time course in the limbic system after myocardial infarction in the rat. Brain Res. 2008;1216(June):87–91, doi: http://dx.doi.org/10.1016/j.brainres.2008.04.019. 18495089. [21] Wann BP, Bah TM, Kaloustian S, Boucher M, Dufort AM, Le Marec N, Godbout R, Rousseau G. Behavioural signs of depression and apoptosis in the limbic system following myocardial infarction: effects of sertraline. J. Psychopharmacol. (Oxf., Engl.) 2009;23(June (4)):451–9, doi:http://dx.doi.org/ 10.1177/0269881108089820. 18562428. [22] Bah TM, Benderdour M, Kaloustian S, Karam R, Rousseau G, Godbout R. Escitalopram reduces circulating pro-inflammatory cytokines and improves depressive behavior without affecting sleep in a rat model of post-cardiac infarct depression. Behav. Brain Res. 2011;225(1):243–51, doi:http://dx.doi. org/10.1016/j.bbr.2011.07.039. 21824496. [23] Bah TM, Kaloustian S, Rousseau G, Godbout R. Pretreatment with pentoxifylline has antidepressant-like effects in a rat model of acute myocardial infarction. Behav. Pharmacol. 2011;22(December (8)):779–84, doi:http://dx. doi.org/10.1097/FBP.0b013e32834d1385. 21971020. [24] Rousseau G, Bah TM, Godbout R. Post-myocardial infarction depression. In: Lakshmanadoss U, editor. Novel Strategies in Ischemic Heart Disease. Rijeka, Croatia: InTech; 2012. p. 333–62. [25] Arseneault-Bréard J, Rondeau I, Gilbert K, Girard SA, Tompkins TA, Godbout R, Rousseau G. Combination of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 reduces post-myocardial infarction depression symptoms and restores intestinal permeability in a rat model. Br. J. Nutr. 2012;107(September (12)):1793–9, doi:http://dx.doi.org/10.1017/S0007114511005137. 21933458. [26] Gilbert K, Bernier J, Godbout R, Rousseau G. Resolvin d1, a metabolite of omega-3 polyunsaturated fatty acid, decreases post-myocardial infarct depression. Mar. Drugs 2014;12(11):5396–407, doi:http://dx.doi.org/10.3390/ md12115396. 25402828. [27] Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press; 1986. [28] Wann BP, Boucher M, Kaloustian S, Nim S, Godbout R, Rousseau G. Apoptosis detected in the amygdala following myocardial infarction in the rat. Biol. Psychiatry 2006;59(March (5)):430–3, doi:http://dx.doi.org/10.1016/j.biopsych.2005.07.018. 16202395. [29] Girard SA, Bah TM, Kaloustian S, Lada-Moldovan L, Rondeau I, Tompkins TA, Godbout R, Rousseau G. Lactobacillus helveticus and Bifidobacterium longum taken in combination reduce the apoptosis propensity in the limbic system after myocardial infarction in a rat model. Br. J. Nutr. 2009;102(June (10)):1420–5, doi:http://dx.doi.org/10.1017/S0007114509990766. 19563693.
[30] Nossuli TO, Lakshminarayanan V, Baumgarten G, Taffet GE, Ballantyne CM, Michael LH, Entman ML. A chronic mouse model of myocardial ischemia– reperfusion: essential in cytokine studies. Am. J. Physiol. Heart Circ. Physiol. 2000;278(April (4)):H1049–55. 10749697. [31] Deten A, Volz HC, Briest W, Zimmer HG. Cardiac cytokine expression is upregulated in the acute phase after myocardial infarction. Experimental studies in rats. Cardiovasc. Res. 2002;55(August (2)):329–40, doi:http://dx. doi.org/10.1016/S0008-6363(02)00413-3. 12123772. [32] Lopez-Ramirez MA, Fischer R, Torres-Badillo CC, Davies HA, Logan K, Pfizenmaier K, Male DK, Sharrack B, Romero IA. Role of caspases in cytokineinduced barrier breakdown in human brain endothelial cells. J. Immunol. (Baltim., Md.: 1950) 2012;189(September (6)):3130–9, doi:http://dx.doi.org/ 10.4049/jimmunol.1103460. 22896632. [33] Augstein P, Bahr J, Wachlin G, Heinke P, Berg S, Salzsieder E, Harrison LC. Cytokines activate caspase-3 in insulinoma cells of diabetes-prone NOD mice directly and via upregulation of Fas. J. Autoimmun. 2004;23(December (4)):301–9, doi:http://dx.doi.org/10.1016/j.jaut.2004.09.006. 15571924. [34] Francis J, Zhang Z-H, Weiss RM, Felder RB. Neural regulation of the proinflammatory cytokine response to acute myocardial infarction. Am. J. Physiol. Heart Circ. Physiol. 2004;287(2):H791–7, doi:http://dx.doi.org/10.1152/ ajpheart.00099.2004. 15277202. [35] Serhan CN, Savill J. Resolution of inflammation: the beginning programs the end. Nat. Immunol. 2005;6(December (12)):1191–7, doi:http://dx.doi.org/ 10.1038/ni1276. 16369558. [36] Sugawara T, Fujimura M, Morita-Fujimura Y, Kawase M, Chan PH. Mitochondrial release of cytochrome c corresponds to the selective vulnerability of hippocampal CA1 neurons in rats after transient global cerebral ischemia. J. Neurosci. 1999;19(November (22)):RC39. 10559429. [37] Kaloustian S, Bah TM, Rondeau I, Mathieu S, Lada-Moldovan L, Ryvlin P, Godbout R, Rousseau G. Tumor necrosis factor-alpha participates in apoptosis in the limbic system after myocardial infarction. Apoptosis 2009;14(August (11)):1308–16, doi:http://dx.doi.org/10.1007/s10495-009-0395-x. 19728097. [38] Hausenloy DJ, Yellon DM. Preconditioning and postconditioning: united at reperfusion. Pharmacol. Ther. 2007;116(November (2)):173–91, doi:http://dx. doi.org/10.1016/j.pharmthera.2007.06.005. [39] Krishnamoorthy S, Recchiuti A, Chiang N, Yacoubian S, Lee CH, Yang R, Petasis NA, Serhan CN. Resolvin D1 binds human phagocytes with evidence for proresolving receptors. Proc. Natl. Acad. Sci. U. S. A 2010;107(January (4)):1660–5, doi:http://dx.doi.org/10.1073/pnas.0907342107. 20080636. [40] Richard D, Oszust F, Guillaume C, Millart H, Laurent-Maquin D, Brou C, Bausero P, Visioli F. Infusion of docosahexaenoic acid protects against myocardial infarction. Prostaglandins Leukot. Essent. Fatty Acids 2014;90(April (4)):139– 43, doi:http://dx.doi.org/10.1016/j.plefa.2014.01.001. 24480264. [41] Al-Zakwani I, Zubaid M, Al-Riyami A, Alanbaei M, Sulaiman K, Almahmeed W, Al-Motarreb A, Al Suwaidi J. Primary coronary intervention versus thrombolytic therapy in myocardial infarction patients in the Middle East. Int. J. Clin. Pharm. 2012;34(June (3)):445–51, doi:http://dx.doi.org/10.1007/s11096-0129627-1. 22477207. _ [42] Rymuza H, Kowalik I, Drzewiecki A, Krzyzanowski W, Olszewski M, Da˛browski R, Je˛drzejczyk B, Wo zniak J, Sosnowski C, Szwed H. Successful primary coronary angioplasty improves early and long-term outcomes in ST segment elevation acute coronary syndromes in patients above 80 years of age. Kardiol. Pol. 2011;69(4):346–54. 21523668. [43] Westerhout CM, Bonnefoy E, Welsh RC, Steg PG, Boutitie F, Armstrong PW. The influence of time from symptom onset and reperfusion strategy on 1-year survival in ST-elevation myocardial infarction: a pooled analysis of an early fibrinolytic strategy versus primary percutaneous coronary intervention from CAPTIM and WEST. Am. Heart J. 2011;161(February (2)):283–90, doi:http://dx. doi.org/10.1016/j.ahj.2010.10.033. 21315210. [44] Gao RL, Han YL, Yang XC, Mao JM, Fang WY, Wang L, Shen WF, Li ZQ, Jia GL, Lü SZ, Wei M, Zeng DY, Chen JL, Qin XW, Xu B, Du CH. Thorombolytic therapy with rescue percutaneous coronary intervention versus primary percutaneous coronary intervention in patients with acute myocardial infarction: a multicenter randomized clinical trial. Chin. Med. J. 2010;123(June (11)):1365–72. 20819587. [45] Borgia F, Goodman SG, Halvorsen S, Cantor WJ, Piscione F, Le May MR, Fernández-Avilés F, Sánchez PL, Dimopoulos K, Scheller B, Armstrong PW, Di Mario C. Early routine percutaneous coronary intervention after fibrinolysis vs. standard therapy in ST-segment elevation myocardial infarction: a metaanalysis. Eur. Heart J. 2010;31(September (17)):2156–69, doi:http://dx.doi.org/ 10.1093/eurheartj/ehq204. 20601393.
Contents lists available at ScienceDirect
PharmaNutrition journal homepage: www.elsevier.com/locate/phanu
Resolvin D1 decreases caspase-3 activation in the limbic system after myocardial infarction Kim Gilberta,b , Mandy Malicka,b , Ness Madingoua,b , Roger Godbouta,c , Guy Rousseaua,b,* a b c
Centre de Biomédecine, Hôpital du Sacré-Cœur de Montréal, 5400, Boulevard Gouin Ouest, Montréal, Québec H4J 1C5, Canada Département de Pharmacologie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada Département de Psychiatrie, Université de Montréal, C.P. 6128 Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 December 2014 Received in revised form 21 May 2015 Accepted 26 May 2015 Available online 17 July 2015
Myocardial infarction (MI) induces an inflammatory process that is associated with increased apoptosis in the limbic system of rats and the development of post-MI depressive symptoms. Resolvin D1 (RvD1), an omega-3 fatty acid metabolite, is known for its pro-resolution properties; it reduces infarct size and attenuates post-MI depression-like symptoms. The present study was designed to determine if a single RvD1 dose could abate caspase-3 activation, a marker of apoptosis, in the limbic system. Male SpragueDawley rats underwent 40 min of myocardial ischemia, followed by 24-h reperfusion. Five min before MI, the animals received a single intra-cardiac RvD1 injection (0.01, 0.1 or 0.3 mg) or vehicle (saline). Infarct size was assessed and caspase-3 activity measured in the amygdala and hippocampus. Rats receiving 0.1 or 0.3 mg RvD1 showed significantly decreased infarct size. Caspase-3 activity was significantly attenuated in the lateral amygdala and dentate gyrus with 0.1 mg RvD1 and in the CA1 region of the hippocampus and medial amygdala with 0.3 mg RvD1. In conclusion, RvD1 could reduce infarct size and caspase-3 activity in the amygdala and hippocampus. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Resolvin D1 Myocardial infarction Reperfusion Caspase-3 Amygdala Hippocampus
1. Introduction There is evidence supporting the view that omega-3 fatty acids could be beneficial for cardiovascular health. Previous studies from our laboratory have disclosed that consumption of a high omega-3 fatty acid diet results in smaller myocardial infarction (MI) size [1]. Up to now, the mechanisms involved are elusive, with many hypotheses being proposed to explain this beneficial effect, including the insertion of omega-3 fatty acids in lipid membranes [2], reduction of inflammation [3], and activation of the phosphoinositol-3-kinase (PI3K)/Akt pathway [1]. Moreover, a recent meta-analysis has indicated that the beneficial effect of omega-3 fatty acids on secondary prevention of cardiovascular disease may not be significant in humans [4] see also [5,6]. The present research thus aimed at a better understanding of the effects of the metabolites of the omega-3 fatty acids on cardiovascular health.
* Corresponding author at: Centre de Biomédecine, Hôpital du Sacré-Cœur de Montréal, 5400, Boulevard Gouin Ouest, Montréal, Québec H4J 1C5, Canada. Tel.: +1 514 338 2222x3421; fax: +1 514 338 2694. E-mail address: [email protected] (G. Rousseau). http://dx.doi.org/10.1016/j.phanu.2015.05.003 2213-4344/ ã 2015 Elsevier B.V. All rights reserved.
Omega-3 fatty acids can be metabolized into different molecules, including resolvins [7,8]. Resolvins are metabolized from docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) by pathways comprising cyclooxygenase-2, lipoxygenase and P450 [9,10]. DHA-derived resolvin D1, administered before or with the onset of post-MI reperfusion, results in smaller infarct size [11]. These findings suggest that omega-3 fatty acid metabolism could be a potential mechanism mediating the beneficial effect of omega-3 fatty acids on cardiovascular health. Clinical signs of depression represent a major adverse event of MI in humans [12]. Depression can be detected in 65% of MI patients and about 20% of them will experience major depression for months after the episode [13]. The consequences of MI may even be dramatic, going beyond financial and familial outcomes, since mortality among post-MI depressed patients is 3–4 times higher than among post-MI patients without depression [14,15]. The therapeutic potential of omega-3 fatty acids in this context has been hypothesized for many years, and multiple clinical trials have been conducted, but the results are inconclusive [16–18]. In the last decade, we designed an experimental model to study the biochemical and behavioral consequences of MI in rats. On the one hand, we found increased caspase-3 activity as early as 3 days
K. Gilbert et al. / PharmaNutrition 3 (2015) 78–82
post-MI in the extended limbic system, including the hippocampus and the amygdala [19,20]. On the other hand, behavioral signs compatible with clinical depression were noted only 14 days postMI [21–24]. Still, we also confirmed the fact that treatment with antidepressant molecules, such as desipramine, sertraline and escitalopram, attenuates the biochemical as well as the behavioral consequences of MI in this model [19,21,22,24]. More recently, we showed that alternative therapeutic routes, including probiotics [25] and RvD1 [26], could be beneficial in our model, suggesting a link between the biochemical and behavioral components of MI, i.e., the biochemical response induced by MI could be responsible for its behavioral effects. In this context, however, we ignored if RvD1 could attenuate caspase-3 activation. The present study was therefore designed to ascertain if a single RvD1 dose could confirm the infarct size reduction observed in a previous study and determine if it could modulate caspase-3 activity in the limbic system. 2. Methods 2.1. Experimental design Twenty-five 3-month-old male Sprague-Dawley rats (Charles River, Canada, St.-Constant, QC, Canada), weighing 325–350 g at the start of the experiments, were handled in compliance with regulations of the local animal care committee and in accordance with guidelines of the Canadian Council on Animal Care. They were housed individually, under constant conditions (temperature 21– 22 C and humidity 40–50%), including a 12-h light-dark cycle beginning at 8 AM. Standard chow pellets and tap water were available ad libitum throughout the study. An acclimatization period of 3 days after delivery by the supplier was imposed before the rats were randomly assigned to 1 of 4 groups: 0.5 ml of vehicle (NaCl 0.9%) or 1 of 3 RvD1 doses (0.01, 0.1 or 0.3 mg) injected directly into the left ventricle (LV) chamber 5 min before ischemia. RvD1 (17(S)-RvD1) already dissolved in ethanol was procured from Cayman Chemical (Ann Arbor, MI, USA). Exact amount of RvD1 was added to saline (NaCl 0.9%) for a final volume of 0.5 ml. The animals were sacrificed 24 h post-MI to measure caspase-3 activity in the limbic system (see below). 2.2. Surgical procedure Anesthesia was induced by intraperitoneal ketamine/xylazine injection (60 and 10 mg/kg, respectively). Subsequently, the rats were intubated and anesthesia was maintained under isoflurane (1–2%) ventilation. Electrocardiogram and heart rate (HR) were monitored throughout the procedure with electrodes placed on the animals’ paws. Left thoracotomy at the 5th intercostal space enabled left anterior descending coronary artery occlusion with 4– 0 silk sutures (Syneture, Covidien, Mansfield, MA, USA) and plastic snare. Vehicle or RvD1 was injected into the left heart cavity 5 min before ischemia, which was confirmed by ST segment alterations and the presence of ventricular subepicardial cyanosis. Sutures were removed after 40 min of ischemia, permitting myocardial tissue reperfusion. The thorax was closed with 2–0, 3–0 and 4–0 silk sutures (Syneture, Covidien), and the animals were given a subcutaneous antibiotic injection of 15,000 IU penicillin G (Duplocillin LA, Intervet Canada Ltd., Whitby, ON, Canada) and a subcutaneous analgesic injection (2 mg/kg buprenorphine) before being returned to their respective cages. 2.3. Hemodynamic data HR and mean arterial pressure (MAP) were measured by the tail cuff technique (Kent Scientific Corporation, Torrington, CT, USA)
79
before occlusion, at 20 min of ischemia, at the beginning of the reperfusion period and after 10 min of reperfusion. 2.4. Measurement of infarct size After the 24-h reperfusion period, the rats were restrained in a cone bag and rapidly decapitated. Their hearts were removed immediately and placed in a dish kept on crushed ice. They were washed with saline 0.9% by retrograde perfusion of the aorta. The left anterior descending coronary artery was occluded at the same site as for MI induction (see above) to map the area at risk (AR) by Evans blue infusion (0.5%). The hearts were then frozen ( 80 C for 5 min), sliced into 4 transverse 2-mm sections and placed in 2,3,5triphenyltetrazolium chloride solution (1%, pH 7.4) at 37 C for 10 min to better distinguish the area of necrosis (I) from the AR. The different regions were carefully drawn on a glass plate, photocopied and cut. Thereafter, the complete infarct region, AR and LV were weighed separately to express MI as percentage of necrosis (I) of the AR ((I/AR) 100)) and AR as percentage of the LV area ((AR/ LV) 100). The brain was rapidly placed in a dish standing on crushed ice. Brain regions were identified according to the atlas of Paxinos and Watson [27]: the lateral and medial amygdala (LA, MA) and hippocampus (CA1, CA3 and dentate gyrus (DG) regions) were dissected out, snap-frozen in liquid nitrogen and maintained at 80 C until required. 2.5. Caspase-3 activity Caspase-3 activity was measured according to a previouslydescribed protocol [28]. Tissues were homogenized by sonication in lysis buffer and incubated for 30 min on ice. The tissue homogenates were centrifuged at 4 C for 10 min. Enzymatic reactions were undertaken in reaction buffer with 25 mg of protein (confirmed by the Bradford method) and fluorescent substrate (AcDEVD-AMC, 40 mM). They were studied after incubation in the dark for 3 h at 37 C and stopped by the addition of 0.4 M NaOH and 0.4 M glycine buffer. A negative control for each sample was performed in triplicate using Ac-DEVD-CHO (2 mM). Fluorescence was quantified by spectrofluorometry (Photon Technology International, Lawrenceville, NJ, USA) at 365 nm excitation wavelength and 465 nm emission wavelength. Activity was calculated from the difference between positive and negative controls. 2.6. Statistical analysis The data are reported as mean SEM. Statistical tests were performed with SPSS 20 (IBM Corp., Armonk, NY, USA). Infarct size and caspase-3 activity were compared by analysis of variance (ANOVA), followed by post hoc comparisons (Dunnett) when significant. If variances were heterogenous, Brown–Forsythe correction was followed by Games-Howell comparisons when applicable. Hemodynamic data were assessed by mixed ANOVA (intergroup analysis: between groups; intragroup comparison: by time). p < 0.05 was considered significant. 3. Results 3.1. Hemodynamic data (Table 1) Hemodynamic data analysis indicated that HR, MAP and pressure rate product (PRP) were similar among groups throughout the experiment. Intragroup comparisons disclosed significant differences in MAP (F(1,31, 10,51) = 15.27; p < 0.05) and PRP (F(1,56, 12,47) = 14.04; p < 0.05) during the experiment. Post hoc analysis revealed that MAP and PRP were reduced during ischemia and reperfusion compared to baseline (p < 0.05, Table 1).
80
K. Gilbert et al. / PharmaNutrition 3 (2015) 78–82
Table 1 Hemodynamic data (heart rate, mean arterial pressure and pressure rate product) for each RvD1 dose (mg). *p < 0.05 compared to baseline. Values are mean SEM. RvD1: Resolvin D1; HR: heart rate; PRP: pressure rate product. RvD1 (0.01 mg)
RvD1 (0.1 mg)
RvD1 (0.3 mg)
Baseline HR 253 13 MAP 98 3 PRP 249 16
265 13 85 6 225 26
244 2 88 12 215 30
236 12 94 8 221 13
20-min ischemia HR 254 7 MAP 67 8 PRP 170 18*
251 14 78 3 196 11*
244 5 73 4 178 10*
244 6 80 3 196 9*
Reperfusion HR 249 15 MAP 69 3* PRP 171 2*
255 13 70 2* 178 11*
249 7 69 4* 172 4*
256 9 73 3* 187 15*
10-min reperfusion HR 248 15 MAP 67 3* PRP 166 5*
256 13 70 1* 179 9*
258 6 66 1* 175 1*
264 8 77 2* 204 10*
Vehicle
HR: heart rate (beats/min); MAP: mean arterial pressure (mm Hg). PRP: HR MAP/ 100. * p < 0.05 versus baseline.
Fig. 2. Caspase-3 activity expressed in different areas of the brain as percentage of the vehicle group and assessed by in vitro spectrofluorescence (3–7 rats per group). RvD1: Resolvin D1; LA: lateral amygdala; MA: medial amygdala; DG: dentate gyrus; CA1: cornu ammonis area 1 of the hippocampus; CA3: cornu ammonis area 3 of the hippocampus. *Indicates significant difference between the experimental and vehicle groups; p < 0.05.
3.3.1. Correlation between caspase-3 activity and myocardial infarct size We tested the possibility of correlation between caspase-3 activity and MI. Correlation between caspase-3 activity in the LA and I/AR (r2 = 0.14, p = 0.087 (Fig. 3A) and CA1 (r2 = 0.17, p = 0.068, Fig. 3B) was almost significant. Correlation between MI and other regions was clearly non-significant. 4. Discussion
3.2. Infarct size Myocardial infarct size (I), expressed as percentage of AR, disclosed significant between group differences (F(3,21) = 9.58; p < 0.05). Post hoc analysis revealed significant differences between groups receiving 0.1 or 0.3 mg RvD1 compared to the control (vehicle) group. No difference was evident between the 0.01 mg and control groups. AR, expressed as percentage of LV, was similar between groups and represented around 70% of LV (Fig. 1). 3.3. Caspase-3 activity Caspase-3 activity in the amygdala and hippocampal areas presented significant differences between groups: LA F(3,19) = 7.11, p < 0.05; MA F(3,18) = 4.30; p < 0.05; DG F(3,12) = 5.14; p < 0.05; CA1 F(3,17) = 7.39; p < 0.05. Further analysis indicated significantly reduced caspase-3 activity in the 0.3 mg RvD1 group versus vehicle controls in the LA, MA, DG and CA1 areas (p < 0.05). Significant difference between the 0.1 mg RvD1 group versus the vehicle group was observed in the LA and DG areas (p < 0.05). No significant difference was apparent in the 0.01 RvD1 and vehicle groups. No difference was evident between groups in the CA3 area of the hippocampus (Fig. 2).
Fig. 1. Infarct size (I) expressed as percentage of the area at risk (AR), and AR as percentage of the left ventricle (LV). RvD1: Resolvin D1. *Indicates significant difference between the experimental and vehicle groups; p < 0.05.
The current study indicates that caspase-3 activity 24 h post-MI is significantly attenuated in the amygdala and hippocampus in the presence of RvD1. We also confirm our previous data according to which RvD1 significantly reduces infarct size without significant effects on hemodynamic parameters [26]. The decrease of caspase-3 activity in the amygdala and hippocampus by RvD1 is in line with our hypothesis. Indeed, we have already observed that caspase-3 activity increases after MI [28], with a peak at 3 days post-MI in different regions of the limbic system [20]. It must be noted that, at 7 days post-MI, we were unable to detect augmented caspase-3 activity in these regions, which suggests that the triggered signal is transient [20]. In an effort to ascertain the mechanism responsible for early caspase-3 activation, we administered various molecules, including antidepressants [19,21,22], the non-selective cytokine synthesis inhibitor pentoxifylline [23], probiotics [29] and a high-omega-3 fatty acid diet [1]. We proposed that caspase-3 activation is due, at least in part, to the inflammatory process seen in MI [24]. As a matter of fact, increases of inflammatory molecules, such as tumor necrosis factor-alpha [22,30] or interleukin-1beta [31], documented after MI, activate caspase-3 [32,33]. Other mechanisms, such as epicardial nerve activation, could also participate in augmenting caspase-3 activity. Indeed, Francis et al. [34] reported that increment of pro-inflammatory cytokines a few minutes after ischemia induction could be attenuated by the destruction of epicardial nerves. RvD1 is a molecule involved in the resolution phase of inflammation [35]. It has been reported that RvD1 can prevent the synthesis or effect of pro-inflammatory cytokines in different conditions and thus curb caspase-3 activity. However, it is also possible that reduction of infarct size itself by RvD1 may result in abatement of caspase-3 activity, as suggested by the correlation we observed with infarct size. Interestingly, we proposed previously that the post-MI apoptosis encountered in the CA1 region of the hippocampus is related to the ischemic damage induced in the myocardium [20], since this hippocampal region is more sensitive to ischemia than other regions [36]. With infarct size reduction evoked by RvD1, it is possible that smaller myocardial damage results in lower caspase-3 activation.
K. Gilbert et al. / PharmaNutrition 3 (2015) 78–82
81
Fig. 3. Positive correlation between myocardial infarct size (I/AR 100) and caspase-3 activity in the lateral amygdala (r2 = 0.13; p = 0.087, panel A) and CA1 (r2 = 0.17; p = 0.068, panel B).
According to previous studies, the beneficial effect of RvD1 on infarct size is probably not related to diminution of proinflammatory cytokines. Indeed, in different experiments, we have observed that reduction of circulating pro-inflammatory cytokines does not correlate with a significant effect on infarct size [23,37]. We have reported earlier that the presence of RvD1 results in smaller infarct size with activation of the cardioprotective PI3K/ Akt signaling pathway [11]. It is known that when activated at the onset of reperfusion, the PI3K/Akt signaling pathway could diminish infarct size by inhibiting the opening of transient mitochondrial permeability pores [38]. The link between RvD1 and PI3K/Akt pathway activation is unknown, but it could be speculated that 1 of 2 G-protein-coupled receptors interacting with RvD1 is responsible for this activation [39], a hypothesis that needs to be confirmed in further studies. Since RvD1 is derived from DHA, it could also be possible that its effects are mediated by Notch receptors [40]. The reduction of caspase-3 activation by RvD1 after MI is interesting since we have demonstrated linkage between post-MI depression and caspase-3 activation. Independently of the interventions instituted [21,22], we observed that abatement of caspase-3 activation at 3 days post-MI resulted in significant diminution of clinical depression by 14 days post-MI. Since we have documented that clinical depression is attenuated with a single RvD1dose, these data argue for a role of apoptosis in post-MI depression. 4.1. Limitations One limitation of our study is the timing of RvD1 administration. Indeed, pre-ischemic administration is impossible in a clinical setting. However, since RvD1 is a metabolite of omega-3 fatty acids and synthetized in an inflammatory context, it could be present during the ischemic period with an omega-3-enriched diet. Another limitation could have to do with intraventricular RvD1 injection. Angioplasty, however, is an increasingly common reperfusion procedure [41–45], an easy way to access the LV chamber for drug injection via catheter. It adds ecological validity to our procedure. We have also chosen to measure caspase-3 activity selectively in the hippocampus and the amygdala. This choice was driven by the fact that we have previously demonstrated that, one day postMI, caspase-3 activity is significantly increased in the hippocampus and the amygdala and less so in other regions such as frontal and prefrontal cortices [20]. It still remains possible that MI and RvD1 could affect other regions of the brain but this has never been documented.
5. Conclusions In conclusion, RvD1 reduces infarct size as well as caspase-3 activity in the amygdala and hippocampus. Acknowledgments This work was supported by a grant from La Fondation des Maladies du Coeur du Canada. Kim Gilbert holds a studentship from Fonds de la recherche du Québec—Santé. On behalf of all authors, the corresponding author (GR) states that there is no conflict of interest. References [1] Rondeau I, Picard S, Bah TM, Roy L, Godbout R, Rousseau G. Effects of different dietary omega-6/3 polyunsaturated fatty acids ratios on infarct size and the limbic system after myocardial infarction. Can. J. Physiol. Pharmacol. 2011;89 (March (3)):169–76, doi:http://dx.doi.org/10.1139/Y11-007. 21423290. [2] Ander BP, Edel AL, McCullough R, Rodriguez-Leyva D, Rampersad P, Gilchrist JS, Lukas A, Pierce GN. Distribution of omega-3 fatty acids in tissues of rabbits fed a flaxseed-supplemented diet. Metab. Clin. Exp. 2010;59(5):620–7, doi:http:// dx.doi.org/10.1016/j.metabol.2009.09.005. 19913851. [3] Calder PC. n-3 Polyunsaturated fatty acids, inflammation, and inflammatory diseases. Am. J. Clin. Nutr. 2006;83(Suppl. 6):1505S–19S. 16841861. [4] Kwak SM, Myung SK, Lee YJ, Seo HG. Efficacy of omega-3 fatty acid supplements (eicosapentaenoic acid and docosahexaenoic acid) in the secondary prevention of cardiovascular disease: a meta-analysis of randomized, doubleblind, placebo-controlled trials. Arch. Intern. Med. 2012;172(May (9)):686–94, doi:http://dx.doi.org/10.1001/archinternmed.2012.262. 22493407. [5] Hu FB, Manson JE. Omega-3 fatty acids and secondary prevention of cardiovascular disease—is it just a fish tale? Comment on “efficacy of omega-3 fatty acid supplements (eicosapentaenoic acid and docosahexaenoic acid) in the secondary prevention of cardiovascular disease”. Arch. Intern. Med. 2012;172 (May (9)):694–6, doi:http://dx.doi.org/10.1001/archinternmed.2012.463. 22493410. [6] Messori A, Fadda V, Maratea D, Trippoli S. Omega-3 fatty acid supplements for secondary prevention of cardiovascular disease: from “no proof of effectiveness” to “proof of no effectiveness”. JAMA Intern. Med. 2013;173(August (15)):1466–8, doi:http://dx.doi.org/10.1001/jamainternmed.2013.6638. 23779264. [7] Serhan C, Hong S, Gronert K, Colgan S, Devchand P, Mirick G, Moussignac R. Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J. Exp. Med. 2002;196(8):1025–37, doi:http://dx.doi.org/10.1084/jem.20020760. 12391014. [8] Serhan CN, Arita M, Hong S, Gotlinger K. Resolvins, docosatrienes, and neuroprotectins, novel omega-3-derived mediators, and their endogenous aspirin-triggered epimers. Lipids 2004;39(November (11)):1125–32, doi:http://dx. doi.org/10.1007/s11745-004-1339-7. 15726828. [9] Dangi B, Obeng M, Nauroth JM, Chung G, Bailey-Hall E, Hallenbeck T, Arterburn LM. Metabolism and biological production of resolvins derived from docosapentaenoic acid (DPAn-6). Biochem. Pharmacol. 2010;79(January (2)):251– 60, doi:http://dx.doi.org/10.1016/j.bcp.2009.08.001. 19679107. [10] Sun YP, Oh SF, Uddin J, Yang R, Gotlinger K, Campbell E, Colgan SP, Petasis NA, Serhan CN. Resolvin D1 and its aspirin-triggered 17R epimer. Stereochemical assignments, anti-inflammatory properties, and enzymatic inactivation. J. Biol. Chem. 2007;282(March (13)):9323–34, doi:http://dx.doi.org/10.1074/jbc. M609212200. 17244615.
82
K. Gilbert et al. / PharmaNutrition 3 (2015) 78–82
[11] Tran QT, Gosselin A-A, Bourque-Riel V, Gilbert K, Charron T, Rousseau G. Effect of Resolvin D1 on experimental myocardial infarction. Exp. Clin. Cardiol. 2014;20(11):6704–12. [12] Frasure-Smith N, Lespérance F. Depression and other psychological risks following myocardial infarction. Arch. Gen. Psychiatry 2003;60(6):627–36, doi:http://dx.doi.org/10.1001/archpsyc.60.6.627. 12796226. [13] Guck TP, Kavan MG, Elsasser GN, Barone EJ. Assessment and treatment of depression following myocardial infarction. Am. Fam. Physician 2001;64 (August (4)):641–8. 11529263. [14] Frasure-Smith N, Lespérance F, Talajic M. Depression following myocardial infarction. Impact on 6-month survival. JAMA 1993;270(October (15)):1819– 25, doi:http://dx.doi.org/10.1001/jama.270.15.1819. 8411525. [15] Frasure-Smith N, Lespérance F, Talajic M. Depression and 18-month prognosis after myocardial infarction. Circulation 1995;91(February (4)):999–1005, doi: http://dx.doi.org/10.1161/01.CIR.91.4.999. 7531624. [16] Zimmer R, Riemer T, Rauch B, Schneider S, Schiele R, Gohlke H, Diller F, Steinbeck G, Katus H, Senges J. Effects of 1-year treatment with highly purified omega-3 fatty acids on depression after myocardial infarction: results from the Omega trial. J. Clin. Psychiatry 2013;74(November (11)):e1037–45, doi:http:// dx.doi.org/10.4088/JCP.13m08453. 24330904. [17] Siddiqui RA, Harvey KA. Dietary interventions with n-3 fatty acids or probiotics targeting post-myocardial infarction depression. Br. J. Nutr. 2013;109(January (1)):1–3, doi:http://dx.doi.org/10.1017/S0007114512004254. 23286323. [18] Giltay EJ, Geleijnse JM, Kromhout D. Effects of n-3 fatty acids on depressive symptoms and dispositional optimism after myocardial infarction. Am. J. Clin. Nutr. 2011;94(December (6)):1442–50, doi:http://dx.doi.org/10.3945/ ajcn.111.018259. 22030221. [19] Wann BP, Bah TM, Boucher M, Courtemanche J, Le Marec N, Rousseau G, Godbout R. Vulnerability for apoptosis in the limbic system after myocardial infarction in rats: a possible model for human postinfarct major depression. J. Psychiatry Neurosci. 2007;32(January (1)):11–6. 17245469. [20] Kaloustian S, Wann BP, Bah TM, Girard SA, Apostolakis A, Ishak S, Mathieu S, Ryvlin P, Godbout R, Rousseau G. Apoptosis time course in the limbic system after myocardial infarction in the rat. Brain Res. 2008;1216(June):87–91, doi: http://dx.doi.org/10.1016/j.brainres.2008.04.019. 18495089. [21] Wann BP, Bah TM, Kaloustian S, Boucher M, Dufort AM, Le Marec N, Godbout R, Rousseau G. Behavioural signs of depression and apoptosis in the limbic system following myocardial infarction: effects of sertraline. J. Psychopharmacol. (Oxf., Engl.) 2009;23(June (4)):451–9, doi:http://dx.doi.org/ 10.1177/0269881108089820. 18562428. [22] Bah TM, Benderdour M, Kaloustian S, Karam R, Rousseau G, Godbout R. Escitalopram reduces circulating pro-inflammatory cytokines and improves depressive behavior without affecting sleep in a rat model of post-cardiac infarct depression. Behav. Brain Res. 2011;225(1):243–51, doi:http://dx.doi. org/10.1016/j.bbr.2011.07.039. 21824496. [23] Bah TM, Kaloustian S, Rousseau G, Godbout R. Pretreatment with pentoxifylline has antidepressant-like effects in a rat model of acute myocardial infarction. Behav. Pharmacol. 2011;22(December (8)):779–84, doi:http://dx. doi.org/10.1097/FBP.0b013e32834d1385. 21971020. [24] Rousseau G, Bah TM, Godbout R. Post-myocardial infarction depression. In: Lakshmanadoss U, editor. Novel Strategies in Ischemic Heart Disease. Rijeka, Croatia: InTech; 2012. p. 333–62. [25] Arseneault-Bréard J, Rondeau I, Gilbert K, Girard SA, Tompkins TA, Godbout R, Rousseau G. Combination of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 reduces post-myocardial infarction depression symptoms and restores intestinal permeability in a rat model. Br. J. Nutr. 2012;107(September (12)):1793–9, doi:http://dx.doi.org/10.1017/S0007114511005137. 21933458. [26] Gilbert K, Bernier J, Godbout R, Rousseau G. Resolvin d1, a metabolite of omega-3 polyunsaturated fatty acid, decreases post-myocardial infarct depression. Mar. Drugs 2014;12(11):5396–407, doi:http://dx.doi.org/10.3390/ md12115396. 25402828. [27] Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. San Diego: Academic Press; 1986. [28] Wann BP, Boucher M, Kaloustian S, Nim S, Godbout R, Rousseau G. Apoptosis detected in the amygdala following myocardial infarction in the rat. Biol. Psychiatry 2006;59(March (5)):430–3, doi:http://dx.doi.org/10.1016/j.biopsych.2005.07.018. 16202395. [29] Girard SA, Bah TM, Kaloustian S, Lada-Moldovan L, Rondeau I, Tompkins TA, Godbout R, Rousseau G. Lactobacillus helveticus and Bifidobacterium longum taken in combination reduce the apoptosis propensity in the limbic system after myocardial infarction in a rat model. Br. J. Nutr. 2009;102(June (10)):1420–5, doi:http://dx.doi.org/10.1017/S0007114509990766. 19563693.
[30] Nossuli TO, Lakshminarayanan V, Baumgarten G, Taffet GE, Ballantyne CM, Michael LH, Entman ML. A chronic mouse model of myocardial ischemia– reperfusion: essential in cytokine studies. Am. J. Physiol. Heart Circ. Physiol. 2000;278(April (4)):H1049–55. 10749697. [31] Deten A, Volz HC, Briest W, Zimmer HG. Cardiac cytokine expression is upregulated in the acute phase after myocardial infarction. Experimental studies in rats. Cardiovasc. Res. 2002;55(August (2)):329–40, doi:http://dx. doi.org/10.1016/S0008-6363(02)00413-3. 12123772. [32] Lopez-Ramirez MA, Fischer R, Torres-Badillo CC, Davies HA, Logan K, Pfizenmaier K, Male DK, Sharrack B, Romero IA. Role of caspases in cytokineinduced barrier breakdown in human brain endothelial cells. J. Immunol. (Baltim., Md.: 1950) 2012;189(September (6)):3130–9, doi:http://dx.doi.org/ 10.4049/jimmunol.1103460. 22896632. [33] Augstein P, Bahr J, Wachlin G, Heinke P, Berg S, Salzsieder E, Harrison LC. Cytokines activate caspase-3 in insulinoma cells of diabetes-prone NOD mice directly and via upregulation of Fas. J. Autoimmun. 2004;23(December (4)):301–9, doi:http://dx.doi.org/10.1016/j.jaut.2004.09.006. 15571924. [34] Francis J, Zhang Z-H, Weiss RM, Felder RB. Neural regulation of the proinflammatory cytokine response to acute myocardial infarction. Am. J. Physiol. Heart Circ. Physiol. 2004;287(2):H791–7, doi:http://dx.doi.org/10.1152/ ajpheart.00099.2004. 15277202. [35] Serhan CN, Savill J. Resolution of inflammation: the beginning programs the end. Nat. Immunol. 2005;6(December (12)):1191–7, doi:http://dx.doi.org/ 10.1038/ni1276. 16369558. [36] Sugawara T, Fujimura M, Morita-Fujimura Y, Kawase M, Chan PH. Mitochondrial release of cytochrome c corresponds to the selective vulnerability of hippocampal CA1 neurons in rats after transient global cerebral ischemia. J. Neurosci. 1999;19(November (22)):RC39. 10559429. [37] Kaloustian S, Bah TM, Rondeau I, Mathieu S, Lada-Moldovan L, Ryvlin P, Godbout R, Rousseau G. Tumor necrosis factor-alpha participates in apoptosis in the limbic system after myocardial infarction. Apoptosis 2009;14(August (11)):1308–16, doi:http://dx.doi.org/10.1007/s10495-009-0395-x. 19728097. [38] Hausenloy DJ, Yellon DM. Preconditioning and postconditioning: united at reperfusion. Pharmacol. Ther. 2007;116(November (2)):173–91, doi:http://dx. doi.org/10.1016/j.pharmthera.2007.06.005. [39] Krishnamoorthy S, Recchiuti A, Chiang N, Yacoubian S, Lee CH, Yang R, Petasis NA, Serhan CN. Resolvin D1 binds human phagocytes with evidence for proresolving receptors. Proc. Natl. Acad. Sci. U. S. A 2010;107(January (4)):1660–5, doi:http://dx.doi.org/10.1073/pnas.0907342107. 20080636. [40] Richard D, Oszust F, Guillaume C, Millart H, Laurent-Maquin D, Brou C, Bausero P, Visioli F. Infusion of docosahexaenoic acid protects against myocardial infarction. Prostaglandins Leukot. Essent. Fatty Acids 2014;90(April (4)):139– 43, doi:http://dx.doi.org/10.1016/j.plefa.2014.01.001. 24480264. [41] Al-Zakwani I, Zubaid M, Al-Riyami A, Alanbaei M, Sulaiman K, Almahmeed W, Al-Motarreb A, Al Suwaidi J. Primary coronary intervention versus thrombolytic therapy in myocardial infarction patients in the Middle East. Int. J. Clin. Pharm. 2012;34(June (3)):445–51, doi:http://dx.doi.org/10.1007/s11096-0129627-1. 22477207. _ [42] Rymuza H, Kowalik I, Drzewiecki A, Krzyzanowski W, Olszewski M, Da˛browski R, Je˛drzejczyk B, Wo zniak J, Sosnowski C, Szwed H. Successful primary coronary angioplasty improves early and long-term outcomes in ST segment elevation acute coronary syndromes in patients above 80 years of age. Kardiol. Pol. 2011;69(4):346–54. 21523668. [43] Westerhout CM, Bonnefoy E, Welsh RC, Steg PG, Boutitie F, Armstrong PW. The influence of time from symptom onset and reperfusion strategy on 1-year survival in ST-elevation myocardial infarction: a pooled analysis of an early fibrinolytic strategy versus primary percutaneous coronary intervention from CAPTIM and WEST. Am. Heart J. 2011;161(February (2)):283–90, doi:http://dx. doi.org/10.1016/j.ahj.2010.10.033. 21315210. [44] Gao RL, Han YL, Yang XC, Mao JM, Fang WY, Wang L, Shen WF, Li ZQ, Jia GL, Lü SZ, Wei M, Zeng DY, Chen JL, Qin XW, Xu B, Du CH. Thorombolytic therapy with rescue percutaneous coronary intervention versus primary percutaneous coronary intervention in patients with acute myocardial infarction: a multicenter randomized clinical trial. Chin. Med. J. 2010;123(June (11)):1365–72. 20819587. [45] Borgia F, Goodman SG, Halvorsen S, Cantor WJ, Piscione F, Le May MR, Fernández-Avilés F, Sánchez PL, Dimopoulos K, Scheller B, Armstrong PW, Di Mario C. Early routine percutaneous coronary intervention after fibrinolysis vs. standard therapy in ST-segment elevation myocardial infarction: a metaanalysis. Eur. Heart J. 2010;31(September (17)):2156–69, doi:http://dx.doi.org/ 10.1093/eurheartj/ehq204. 20601393.