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EP 80317, a selective CD36 ligand, shows cardioprotective effects against post-ischaemic myocardial damage in mice

Valérie L. Bessi, Sébastien M. Labbé, David N. Huynh, Liliane Ménard, Christian Jossart, Maria Febbraio, Brigitte Guérin, M'Hamed Bentourkia, Roger Lecomte, André C. Carpentier, Huy Ong, Sylvie Marleau
DOI: http://dx.doi.org/10.1093/cvr/cvs225 99-108 First published online: 11 July 2012

Abstract

Aims The CD36 receptor plays an important role in facilitating fatty acid transport to the heart. The present study aimed to assess whether EP 80317, a selective synthetic peptide ligand of CD36, is cardioprotective in a murine model of myocardial ischaemia and reperfusion (MI/R) injury.

Methods and results Mice were pretreated with daily subcutaneous injections of EP 80317 for 14 days before being subjected to a 30 min ligation of the left anterior descending coronary artery. The treatment reduced the infarct area and improved myocardial haemodynamics and function, as shown by an increase in cardiac output, ejection fraction and stroke work, and a reduced total peripheral resistance. In contrast, administration of EP 51389, a tripeptide analogue devoid of binding affinity to CD36, did not protect against myocardial injury. Six hours after myocardial reperfusion, EP 80317-treated mice showed reduced myocardial fatty acid uptake, as assessed by micro-positron emission tomography, in agreement with reduced levels of circulating non-esterified fatty acids. Studies using [14C]-palmitate infusion revealed reduced lipolysis, although no significant change in insulin or catecholamine plasma levels were observed. Increased expression levels of adipogenic and anti-lipolytic genes further supported an effect of EP 80317 in preventing fatty acid mobilization from adipose tissue. No effect of the treatment was observed in CD36−/− mice.

Conclusion Our results show that pretreatment with EP 80317 protected the heart against damage and dysfunction elicited by MI/R, along with a transient reduction in peripheral lipolysis. Our findings support CD36 as a novel target for the treatment of ischaemic cardiopathy.

  • CD36
  • Myocardial I/R
  • AMPK
  • Micro-PET

1. Introduction

CD36 has been shown to play a pivotal role in cellular fatty acid uptake, along with plasma membrane associated fatty acid binding and transport proteins.1 CD36 function is closely related to its plasmalemmal localization, following its trafficking from an intracellular membrane compartment to lipid rafts, in a ubiquitin-regulated manner.2 In agreement with its role in facilitating myocardial fatty acid uptake, CD36 overexpression was associated with intramyocardial lipid accumulation as well as energetically and functionally compromised hearts in aged mice,3 whereas CD36 deficiency was shown to rescue a lipotoxic form of cardiomyopathy through the preferential use of glucose as a source of energy.4 Together, these observations suggested that targeting CD36 may constitute a novel therapeutic avenue to reduce myocardial damage associated with ischaemia and reperfusion.

Early studies investigating the cardiovascular effect of hexarelin, a synthetic growth hormone-releasing peptide (GHRP) modelled from the structure of Met-enkephalin, revealed protectant activity against cardiac ischaemia that was independent of GH secretion.57 Along this line, CD36 was identified as a putative receptor for hexarelin binding to myocardial membranes.8 Yet, the dual binding of growth hormone secretagogue receptor-1a (GHS-R1a)-, and CD36-pathways by hexarelin prevented delineation of the role of CD36 in mediating the cardioprotective effect of the drug against myocardial ischaemia/reperfusion (MI/R).

The present study aimed to investigate the potentially cardioprotective effect of EP 80317, a GHRP analogue devoid of binding affinity towards GHS-R1a,9 on myocardial injury, oxidative metabolism, and left ventricular (LV) function in a mouse model of MI/R. Micro-positron emission tomography (µPET) imaging of [18F]-labelled fluoro-6-thia-heptadecanoic acid ([18F]-FTHA) and [18F]-labelled 2-fluorodeoxyglucose ([18F]-FDG), and real-time LV pressure–volume relations were used for the investigation of cardiac metabolism and function in mice pretreated with EP 80317 or vehicle (0.9% NaCl) for 14 days prior to undergoing a transient occlusion of the left anterior descending (LAD) coronary artery. Our results show that EP 80317 exerts a cardioprotective effect following transient LV ischaemia in mice.

2. Methods

2.1 Animals

All animal experimental procedures were approved by the Institutional Animal Ethics Committee (Comité de déontologie de l'expérimentation sur les animaux de l'Université de Montréal), in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (no: 85-23, revised 1996). CD36-deficient (CD36−/−) mice and their control littermates (CD36+/+) were generated as described previously.10 Mice were sorted into three main study protocols: (i) the determination of the myocardial infarct size after 48 h reperfusion; (ii) myocardial metabolic studies during the early reperfusion stage (6 h); and (iii) myocardial functional recovery at 6 and 48 h of reperfusion.

2.2 Ischaemia-reperfusion of the left descending coronary artery bed

Myocardial I/R was performed according to Tarnavski et al.11 as detailed in Supplementary material online, Methods. Briefly, 3–4-month-old male mice were injected subcutaneously (sc) with EP 80317 (289 nmoL/kg) or vehicle (0.9% NaCl) daily during 14 days and treated 30 min before being subjected to acute (30 min) coronary artery occlusion and on the following day. Prior to surgery, mice were injected intraperitoneally (ip) with buprenorphine (0.05 mg/kg), intubated and artificially ventilated. Anaesthesia was maintained with 2% isoflurane and the temperature of the animals was maintained using a heating pad. The adequacy of anaesthesia was monitored for the disappearance of pedal withdrawal reflex. After 6 or 48 h of reperfusion, animals were anaesthetized with isoflurane and euthanized by exsanguination. Sham-operated mice underwent the identical procedure without placement of the ligature.

2.3 Determination of the area at risk and myocardial infarct size

CD36+/+ and CD36−/− mice were divided into two groups of seven mice each, and assigned to vehicle or EP 80317 treatment. After 48 h of reperfusion, mice underwent anaesthesia, surgical preparation, and a suture was tied at the previous ligation site. Evans blue 2% dye was injected in the aorta to delineate non-ischaemic tissue by the presence of blue staining12 as detailed in Supplementary material online, Methods.

2.4 Myocardial function and haemodynamics

LV function indices were derived from the pressure–volume (PV) measurements using a miniaturized PV conductance catheter, as detailed in Supplementary material online, Methods. Briefly, CD36+/+ mice were divided into four groups of eight mice each: two groups of sham-operated and MI/R mice (vehicle and EP 80317). After 6 or 48 h of reperfusion, a left thoracotomy was performed again, a microtip 1.4 Fr PV catheter (SPR 839, Millar Instruments) was inserted into the LV apex, connected to a transducer system (Millar) to generate PV loops according to Chua et al.13

2.5 Imaging experiments

High resolution imaging experiments were performed with the avalanche photodiode-based small animal μPET scanner.14 Freshly prepared tracer analogues, including [18F]-FTHA (∼37 MBq) and [18F]-FDG (∼20 MBq), as well as the [11C]-acetate (∼20 MBq) tracer, were injected via the caudal vein in vehicle- and EP 80317-treated CD36+/+ and CD36−/− mice at 5.5 h following reperfusion and serial imaging was begun. List-mode dynamic acquisitions were initiated for 20 and 30 min after the injection of [11C]-acetate and [18F]-FTHA (n = 8 mice per group), respectively, according to Ménard et al.15 Similar experiments were performed with [18F]-FDG (n = 14 mice per group). Representative blood input and myocardium [18F]-FTHA and [18F]-FDG uptake curves (Supplementary material online, Figure S1) and detailed data analysis are provided in Supplementary material online, Methods.

2.6 Non-esterified fatty acid kinetics at steady state

The albumin–[14C]-palmitate complex was prepared according to Oakes et al.16 and infused until steady state at a dose of 7.4 KBq/kg/min in vehicle- and EP 80317-treated CD36+/+ and CD36−/− mice (n = 14 mice per group), from 5 h after reperfusion. Four blood samples (50 µL) were obtained at 10 min interval over the last 30 min and plasma palmitate kinetics was determined as detailed in Supplementary material online, Methods.

2.7 Biochemical assays

Blood from non-fasted mice was collected on 4 mM EDTA and plasma lipids and hormones were assayed using commercial kits as described in Supplementary material online, Methods.

2.8 Real-time PCR analysis

cDNA was prepared from total RNA extracted from epididymal fat using the RNeasy Lipid Tissue Mini Kit (Qiagen). Real-time PCR was performed as in Supplementary material online, Methods using β-actin as an internal control.

2.9 Western blotting

Immunoblots of LV Akt, phosphoSer473-Akt (p-Akt), AMPKα1/α2, phosphoThr172-AMPK (p-AMPK) (New England Biolabs), and mouse α-tubulin (Abcam) were performed as described in Supplementary material online, Methods. The signal was quantified by the ImageQuant 5.2 software (Molecular Dynamics).

2.10 Statistical analysis

Data are expressed as mean ± SEM. Comparisons between groups were performed on normally distributed data using the unpaired t-test or one-way ANOVA followed by pairwise multiple comparisons using the Bonferroni post hoc test with GraphPad Prism4 Software. Differences were considered significant at P < 0.05.

3. Results

3.1 Effect of EP 80317 treatment on MI/R injury and post-ischaemic LV function in CD36+/+ mice

The immediate post-operative mortality rate of the surgical procedure was 13% (24/226). MI/R was associated with a consistently large mean area-at-risk (AAR) of 65 ± 2.4% in vehicle-treated CD36+/+ mice, which did not differ between groups, as shown on representative photomicrographs of LV cross-sections at the mid-ventricular level (Figure 1A) and bar graph (Figure 1B). EP 80317 reduced the infarct area (IA) by 34 and 31% (P < 0.05), as quantified by the IA to LV (Figure 1C) and IA to AAR weight ratios (Figure 1D), respectively. Treatment with EP 80317 did not modulate myocardial mRNA levels of insulin growth factor-1 (Igf-1) and insulin receptor substrate-1 (Irs-1) (data not shown). In additional experiments, a 14-day pretreatment with an equimolar dose of EP 51389, a synthetic GHRP tripeptide analogue that is devoid of binding affinity to CD36,17 did not modulate AAR/LV mass (Figure 1E) nor the infarcted area (Figure 1F) when compared with vehicle-treated mice, in contrast to EP 80317 which reduced IA/AAR by 39% (P < 0.05) (Figure 1F).

Figure 1

EP 80317 reduces infarcted areas following MI/R. Representative photomicrographs of (A) the mid-ventricular myocardium showing the infarct area (IA) on the anterior section and bar graphs of (B) area-at-risk (AAR)/LV, (C) IA/LV and (D) IA/AAR. EP 51389, a GHRP analogue devoid of affinity to CD36 did not modulate (E) AAR/LV and (F) IA/AAR, in contrast to EP 80317. Data are mean ± SEM of n= 5–6 mice. *P< 0.05 vs. vehicle. Scale bar, 1 mm.

LV function was measured invasively in sham-operated and MI/R CD36+/+ mice pretreated with vehicle or EP 80317. A representative PV loop at steady state of vehicle-treated, sham-operated, and MI/R mice is shown in Supplementary material online, Figure S2. The body weight and the tibial length were similar in both sham-operated and MI/R groups (Table 1). Overall, early cardiac function declined in post-MI/R mice compared with sham-operated mice, as shown by a significant decline in maximal rate pressure rise (dP/dtmax) (38%) (P < 0.05), dP/dtmin (40%) (P < 0.05), ejection fraction (EF) (36%) (P < 0.001), stroke work (SW) (65%) (P < 0.001), and preload recruitable stroke work (PRSW) (39%) (P < 0.05), in vehicle-treated mice at 6 h post-MI/R. The heart rate (HR) fluctuated, but did not reach statistical differences among the groups. The load-dependent parameters of systolic function including stroke volume (SV), cardiac output (CO), EF, and SW were improved after treatment with EP 80317. In particular, SW and EF were increased by 95 (P < 0.05) and 49% (P < 0.01) in EP 80317-treated mice, respectively, while the load-independent parameter, PRSW, was not significantly different between EP 80317- and sham-operated mice. The relaxation time constant (τ) was prolonged by MI/R suggesting impaired LV relaxation, yet it was not significantly modulated by EP 80317. Arterial elastance (Ea) was elevated in post-M/IR in mice, but declined to the level observed in sham-operated mice after treatment with EP 80317, suggesting an initial vascular response. In a similar manner, total peripheral resistance (TPR) was elevated post-MI/R in mice, returning back to sham-operated levels in EP 80317-treated mice. Although the decline in cardiac function appeared to be moderate at 48 h post-MI/R, the effect of the treatment showed similar trends as those observed at 6 h (Table 1). Together, these results show that decreased cardiac contractility and increased vascular resistance following MI/R are largely improved by EP 80317 pretreatment.

View this table:
Table 1

Left ventricular function of sham-operated and MI/R mice 6 or 48 h post-ischaemia

 6 h48 h
ShamMI/RShamMI/R
0.9% NaClEP 803170.9% NaClEP 803170.9% NaClEP 803170.9% NaClEP 80317
n77876667
Body weight, g30 ± 129 ± 129 ± 129 ± 129 ± 128 ± 128 ± 130 ± 1
Tibial length, mm18 ± 018 ± 018 ± 018 ± 018 ± 018 ± 017 ± 018 ± 0
HR, b.p.m.406 ± 15420 ± 25369 ± 19374 ± 19462 ± 13426 ± 17405 ± 18435 ± 12
SV, μL16 ± 118 ± 18 ± 1***,###13 ± 1##,λλ15 ± 115 ± 110 ± 1**,##16 ± 1λλλ
CO, mL/min7 ± 07 ± 13 ± 0***,###5 ± 0*,##,λ7 ± 06 ± 04 ± 1***,#7 ± 0λλ
EF, %71 ± 566 ± 346 ± 2***,##68 ± 5λλ80 ± 476 ± 273 ± 674 ± 4
Ea, mmHg/μL5.3 ± 0.64.7 ± 0.49.4 ± 1.2**,###5.9 ± 0.3λ5.4 ± 0.55.9 ± 0.38.4 ± 1.3*5.7 ± 0.4
TPR, mmHg min/mL13 ± 111 ± 126 ± 4**,##16 ± 112 ± 114 ± 121 ± 3**14 ± 1λ
Systolic
 Pes, mmHg89 ± 1685 ± 575 ± 478 ± 489 ± 490 ± 585 ± 395 ± 3
 Ves, μL7 ± 19 ± 19 ± 17 ± 24 ± 15 ± 04 ± 16 ± 1
 dP/dtmax, mmHg/s6745 ± 6136607 ± 8174212 ± 334*,#4889 ± 4636833 ± 2536797 ± 6866030 ± 4597782 ± 275λλ
 SW, mmHg/μL1180 ± 1091158 ± 111409 ± 45***,###799 ± 84*1070 ± 851066 ± 91732 ± 1011147 ± 81λ
 PRSW, mmHg90 ± 1278 ± 1155 ± 7*75 ± 1295 ± 975 ± 1176 ± 687 ± 6
Diastolic
 Ped, mmHg2 ± 02 ± 13 ± 13 ± 12 ± 12 ± 02 ± 03 ± 1
 Ved, μL24 ± 227 ± 216 ± 2#20 ± 320 ± 220 ± 014 ± 122 ± 2
 dP/dtmin, mmHg/s−5501 ± 571−5649 ± 746−3276 ± 405*,#−3852 ± 449−6069 ± 396−6245 ± 587−5385 ± 384−6646 ± 367
 τ, ms12.4 ± 0.812.1 ± 1.524.1 ± 3.8**,##21.4 ± 3.2*,#10.7 ± 1.010.9 ± 0.812.6 ± 1.010.8 ± 0.6
  • In vivo cardiac function was measured by the Millar conductance catheter system; HR, heart rate; SV, stroke volume; CO, cardiac output; EF, ejection fraction; Ea, arterial elastance; TPR, total peripheral resistance (Pes/CO); Pes, end-systolic pressure; Ves, end-systolic volume; dP/dtmax (or min), maximal rate of pressure increase (or decline); SW, stroke work; PRSW, preload recruitable stroke work; Ped, end diastolic pressure; Ved, end diastolic volume; τ, relaxation time constant. Data are mean ± SEM. *P< 0.05, **P< 0.01, and ***P< 0.001 vs. vehicle-treated sham mice; #P< 0.05, ##P< 0.01, and ###P< 0.001 vs. EP 80317-treated sham mice; λP< 0.05, λλP< 0.01, and λλλP< 0.001 vs. vehicle-treated MI/R mice.

3.2 Effect of EP 80317 treatment on myocardial energy substrate uptake, NEFA clearance and mobilization, and hormone levels following MI/R in CD36+/+ mice

Serial myocardial imaging of [18F]-FTHA, a marker of non-esterified fatty acids (NEFA) sarcolemmal and mitochondrial uptake in the myocardium,18 was used to estimate plasma NEFA fractional extraction and myocardial uptake rates following reperfusion of ischaemic hearts, according to the protocol outlined in Figure 2A. Whereas plasma NEFA fractional extraction was unaffected by EP 80317 treatment (Figure 2B), myocardial [18F]-FTHA uptake was reduced by 43% (P < 0.01) (Figure 2C). Imaging of [18F]-FDG revealed unaltered plasma fractional extraction (Figure 2D) and myocardial metabolic rate of glucose (MMRG) (Figure 2E) in EP 80317-treated CD36+/+ mice. In addition, CD36+/+ mice, treated or not with EP 80317, had a similar myocardial blood flow index (Figure 2F) and oxidative metabolism (O2 uptake) (Figure 2G) as assessed using [11C]-acetate compartmental kinetic analysis.19 Plasma NEFA were reduced by 33% (P < 0.001 vs. vehicle) in CD36+/+ mice treated with EP 80317, returning to baseline levels by 48 h (Figure 2H). No significant effect of EP 80317 was observed on glycaemia (Figure 2I), insulin plasma levels (Figure 2J), catecholamines (Figure 2K), and triglycerides (results not shown). LV function was also assessed by μPET ventriculography at 6 h post-MI/R. SV was increased by 33% (P< 0.01) in EP 80317-treated mice vs. vehicle (Figure 2L). In addition, neither HR (Figure 2M) nor mean arterial blood pressure (Figure 2N) were changed by the treatment.

Figure 2

EP 80317 reduces circulating NEFA and myocardial NEFA uptake and the decline in stroke volume (SV) as assessed by μPET and ventriculography. (A) Schematic representation of the experimental protocol involving sequential administration of radiotracers in mice pretreated with EP 80317 or vehicle. Bar graphs of (B) NEFA fractional extraction, (C) myocardial NEFA uptake, (D) glucose fractional extraction, (E) myocardial metabolic rate of glucose (MMRG), (F) myocardial blood flow, (G) myocardial oxidative metabolism, (H) plasma NEFA, (I) glycaemia, (J) plasma insulin, (K) plasma catecholamines, (L) SV, (M) heart rate (HR), (N) mean arterial blood pressure (MABP). Data are mean ± SEM of n= 6–8 mice for [18F]-FTHA and [11C]-acetate µPET, [18F]-FDG ventriculography and plasma hormones and n = 12–14 mice for [18F]-FDG µPET. *P< 0.05, **P< 0.01 and ***P< 0.001 vs. vehicle and ###P< 0.001 vs. EP 80317. E, epinephrine; NE, norepinephrine.

The systemic clearance of [14C]-palmitate was assessed at steady state following infusion of the tracer. EP 80317 did not change [14C]-palmitate plasma clearance (Figure 3A), yet the appearance rate of NEFA in circulation was reduced by 19% (P < 0.05) (Figure 3B). The potential role of adipose tissue in regulating circulating NEFA levels was investigated by determining the level of expression of selected adipogenic and anti-lipolytic genes. Epididymal fat from EP 80317-treated CD36+/+ mice showed a 1.5-, 1.8-, 1.5-, and 1.5-fold increase (P < 0.05) of CCAAT-enhancer-binding protein alpha (Cebpα) (Figure 3C), cytosolic phosphoenolpyruvate carboxykinase encoding gene 1 (Pck1) (Figure 3D), perilipin 1 (Plin1) (Figure 3E) and diacylglycerol acyltransferase 2 (Dgat2) (Figure 3F) mRNA levels, respectively, but no change in uncoupling protein 1 (Ucp1) was observed (data not shown). Changes in epididymal mRNA levels of the selected genes were transient inasmuch as no change was observed at 48 h following reperfusion of treated animals (Figure 3C–F).

Figure 3

EP 80317 reduces the NEFA apparition rate following MI/R. Bar graphs of (A) [14C]-palmitate plasma clearance, (B) NEFA apparition rate and mRNA levels of adipocyte genes (C) Cebpα, (D) Pck1, (E) Plin1, and (F) Dgat2. Statistical analysis of Cebpα and Plin1 were performed on log-transformed data. Data are mean ± SEM of n= 6–8 mice (mRNA) and 12–13 mice ([14C]-palmitate). *P< 0.05 vs. vehicle.

3.3 Effect of EP 80317 treatment on myocardial AMPK and Akt phosphorylation after MI/R in CD36+/+ mice

Western blots analysis of myocardial protein homogenates showed that the relative ratio of pThr172-AMPK to total AMPK band density was increased, by 121% (P < 0.01) (Figure 4A and B) at 6 h after reperfusion of ischaemic hearts in EP 80317-treated CD36+/+ mice. In a similar manner, the ratio of pSer473-Akt to total Akt was increased by 57% (P < 0.05) (Figure 4A and C). Although the AMPK phosphorylation ratio was unchanged compared with that of vehicle-treated mice at 48 h post-reperfusion, the Akt phosphorylation ratio was still elevated by 89% (P < 0.01 vs. vehicle) (Figure 4A–C).

Figure 4

EP 80317 increases AMPK and Akt phosphorylation following MI/R. (A) Western blots of band densities of pThr172-AMPK and AMPK or pSer473-Akt and Akt at 6 or 48 h after MI/R. Bar graphs represent the relative band intensity ratios that have been normalized to α-tubulin band intensity. Data are mean ± SEM of 5 mice per group. *P< 0.05 and **P< 0.01 vs. vehicle.

3.4 Effect of EP 80317 treatment after transient myocardial ischaemia in CD36−/− mice

In CD36−/− mice, AAR to the total LV area did not differ between groups, as shown on representative photomicrographs of LV cross-sections at the mid-ventricular level (Figure 5A) and bar graph (Figure 5B). EP 80317 had no cardioprotective effect on myocardial injury (Figure 5C and D). In a similar manner, µPET data analysis did not reveal change in NEFA or glucose myocardial uptake (Figure 5E–H), or myocardial perfusion and oxidative metabolism after 6 h of reperfusion (Figure 5I and J). In addition, neither [14C]-palmitate plasma clearance (Figure 5K) nor NEFA appearance rate (Figure 5L), as well as NEFA, glycaemia, insulin, or catecholamine plasma levels (Figure 5M–P), was modulated by a pretreatment with EP 80317 in CD36−/− mice.

Figure 5

EP 80317 does not reduce myocardial injury or modulate myocardial substrate uptake in CD36−/− mice. Representative photomicrographs of (A) mid-ventricular myocardium showing the IA on the anterior section and bar graphs of (B) AAR/LV, (C) IA/LV, and (D) IA/AAR. (E) NEFA fractional extraction, (F) myocardial NEFA uptake, (G) glucose fractional extraction, (H) myocardial metabolic rate of glucose, (I) myocardial blood flow index (J), and myocardial oxidative metabolism index. (K) [14C]-palmitate plasma clearance, (L) NEFA apparition rate, (M) plasma NEFA, (N) glycaemia, (O) plasma insulin, and (P) plasma catecholamines. Data are mean ± SEM. n= 5–7 mice (infarct, [18F]-FTHA, [11C]-acetate, [14C]-palmitate and plasma hormones) and 12–13 mice ([18F]-FDG). ***P< 0.001 vs. vehicle and ###P< 0.001 vs. EP 80317. Scale bar, 1 mm.

4. Discussion

The major finding of the present study was that pretreatment with EP 80317, a synthetic hexapeptide ligand of the CD36 receptor, exerts cardioprotective effects that are associated with transient reduction in circulating NEFA levels and myocardial uptake following transient MI/R in mice. Interestingly, daily administration of EP 80317 did not modulate plasma NEFA levels in sham-operated mice (results not shown), which may suggest a selective cardioprotective effect of the peptide in the context of MI/R. Indeed, CD36 is a multiligand receptor involved in multiple functions, including facilitation of a large fraction of cellular long-chain fatty acids entry in the heart, skeletal muscle, and adipocyte, and features scavenging function for specific bacterial and fungal components, apoptotic cells, and oxidatively modified low-density lipoproteins.20

In the present study, myocardial infarction was elicited secondary to the temporary occlusion of the LAD coronary artery in mice, generating reproducible AAR and infarct size at 48 h post-reperfusion in agreement with values previously reported.21 Reduced myocardial damage after pretreatment with EP 80317 was found to be IGF-1 independent (data not shown). In line with a role for CD36 in mediating these cardioprotective effects, neither myocardial injury nor any of the metabolic or hormonal parameters were modulated by treatment with EP 80317 in CD36−/− mice (Figure 5). Furthermore, the administration of an equimolar dose of the GHRP tripeptide analogue, EP 51389, which featured no binding affinity to CD36,17 did not reduce myocardial injury (Figure 1E and F), as previously shown in hypophysectomized rats.6

Cardiovascular function derived from PV loops indicated deterioration of LV function at 6 h after reperfusion of ischaemic hearts in mice treated with vehicle, as shown by a marked decline in SV and CO, increased Ea, elevated vascular resistance, and a prolonged τ, indicating impaired LV relaxation (Table 1). No significant change in HR was observed. This is in agreement with impaired contractile recovery following reperfusion of ischaemic heart associated with high rates of long-chain fatty acid uptake and oxidation.22 Indeed, increased NEFA availability as a consequence of peripheral lipolysis23 may lead to myocardial accumulation of long-chain acyl-CoA esters and increased levels of free oxygen radicals.24 Along this line of events, myocardial NEFA oxidation is associated with increased oxygen consumption and reduced glucose oxidation, accumulation of lactate and intracellular acidification, thereby shifting away ATP from contractile function in order to maintain ionic homoeostasis.25 The decline in the load-dependent contractility indexes, including EF and SW as well as that of the load-independent parameter PRSW, was markedly improved in mice treated with EP 80317 at 6 h post-MI/R (Table 1), in line with results obtained by μPET ventriculography (Figure 2L). In addition, an increase in dP/dtmax was observed at 48 h (Table 1), paralleling with the reduced infarct size. Interestingly, previous studies showed that a 14-day treatment with hexarelin, initiated 1 month after eliciting myocardial infarction in rats, was associated with similar haemodynamic effects including increased SV and reduced TPR.7 Yet, additional studies will be necessary to assess the effect of the treatment on post-ischaemic contractile recovery.

An important observation of the present study is the reduced myocardial NEFA uptake as determined by µPET imaging of [18F]-FTHA (Figure 2C). Yet, neither the fractional plasma extraction rate of NEFA nor myocardial Cd36 mRNA expression levels (results not shown) were altered following treatment with EP 80317, suggesting that reduced circulating levels of NEFA in treated mice largely accounted for the reduced NEFA myocardial uptake. With regard to [18F]-FTHA uptake, it is possible that a larger infarcted area in vehicle-treated mice led to lower fractional uptake rates with an underestimation of total NEFA uptake. This limitation would have reduced our capacity to detect the demonstrated reduction in cardiac NEFA uptake with EP 80317 treatment as the latter displayed reduced infarcted area. Additional µPET imaging studies showed that neither the fractional plasma extraction rate of [18F]-FDG nor the estimated MMRG was modulated in EP 80317-treated mice compared with the vehicle group. Myocardial glucose and NEFA oxidation rates have been assessed through the use of tracer analogues. Yet, the fact that >90% of 18F activity is found in mitochondria after an iv injection of [18F]-FTHA suggests that myocardial [18F]-FTHA uptake is a good marker of myocardial NEFA oxidation.18 Notwithstanding potential limitation in extrapolating FDG data modelling to myocardial glucose metabolism,26 no effect of the treatment on glucose uptake and oxidation could be detected in the present study. Interestingly, neither myocardial perfusion nor myocardial oxidative metabolism, as determined by [11C]-acetate µPET imaging, was significantly modulated by EP 80317 (Figure 2F and G), despite reduced myocardial fatty acid uptake, and no apparent change in MMRG. A possible explanation for this observation may be a compensatory increase in the utilization of alternative sources of energy for oxidative metabolism. In this regard, blood lactate levels were decreased in EP 80317-treated mice in the first minutes following reperfusion of ischaemic hearts, suggesting reduced myocardial lactate accumulation in these mice (data not shown).

Taking into account the lack of apparent change in plasma insulin and catecholamine levels (Figure 2J and K) and the fact that the NEFA appearance rate was reduced (Figure 3B), the role of adipose tissue in regulating circulating fatty acid levels was shown by the relative increase in mRNA levels of Cebpα, Plin1, Pck1, and Dgat2 in epididymal fat tissues of EP 80317-treated CD36+/+ mice at 6 h after reperfusion. No change was observed for Ucp1 (data not shown). These findings support a transient attenuation in adipose tissue fatty acid mobilization and an increase in triacylglycerol storage.27,28

Metabolic stresses such as MI/R are known to rapidly activate AMPK in cardiomyocytes to enhance pathways conducive to ATP generation, targeting key enzymes of the glycolytic and fatty acid oxidation pathways29 in a manner to preserve ATP for the contractile activity of the heart.30 Both GLUT4 and CD36 trafficking to plasma membrane are regulated by AMPK; while AMPK activation appears to be cardioprotective in MI/R,31 caution has been raised regarding its prolonged activation at times of heart exposure to high levels of circulating NEFA, with the potential for fatty acid oxidation burst.32 Our results show a transient 121% increase in phospho-AMPK at 6 h, whereas phospho-Akt was still elevated by 89% at 48 h following reperfusion in EP 80317-treated CD36+/+ mice. The latter has been reported to be rapidly phosphorylated and activated following reperfusion33 and to exert cardioprotective effect through the recruitment of anti-apoptotic pathways,34 activation of glycogen synthesis,35 inhibition of mitochondrial permeability transition pore opening,36 endothelial nitric oxide synthase, and protein kinase C activation.37 In addition, Akt activation may exert metabolic effects, including sarcolemmal translocation of GLUT4 vesicles,38 and enhance glycolysis to glucose oxidation coupling.39 Altogether, these observations support a favourable coupling between glycolytic products and their oxidation in the context of a reduced lipid burden during the early phase of myocardial reperfusion.40,41

In conclusion, our results show a cardioprotective effect of EP 80317, a selective ligand of CD36. The treatment was associated with beneficial myocardial and peripheral transient metabolic changes in the first hours following reperfusion of ischaemic hearts. The cardioprotective effects of the peptide in MI/R, coupled to its potent anti-atherosclerotic activity,9 support the clinical potential of CD36 ligands in the treatment of ischaemic heart disease.

Funding

This work was supported by a grant from Heart and Stoke Foundation of Quebec and the Canadian Institute of Health Research (MOP 97915). V.L.B. and S.M.L. are recipients of the Fonds de la recherche en santé du Québec (FRSQ) and of the Canadian Diabetes Association and V.L.B. and D.N.H. of the Groupe de recherche universitaire sur le médicament studentships, respectively. A.C.C. is a recipient of a FRSQ Senior Scholarship Award.

Acknowledgements

The authors thank Dr Jean-François Tanguay for critical reading of the manuscript and Petra Pohankova and Marc-Antoine Gillis for their skillful technical assistance.

Conflict of interest: none declared.

Footnotes

  • These authors contributed equally to this work.

References

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