Skip Navigation


Cardiovascular Research Advance Access first published online on January 25, 2008
This version [Corrected Proof] published online on February 21, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn017
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
78/1/116    most recent
cvn017v2
cvn017v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Gonon, A. T.
Right arrow Articles by Pernow, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gonon, A. T.
Right arrow Articles by Pernow, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Adiponectin protects against myocardial ischaemia-reperfusion injury via AMP-activated protein kinase, Akt, and nitric oxide

Adrian T. Gonon1,*, Ulrika Widegren2, Aliaksandr Bulhak1, Firoozeh Salehzadeh2, Jonas Persson3, Per-Ove Sjöquist1 and John Pernow1

1 Department of Medicine, Unit of Cardiology, Karolinska Institutet, 171 77 Stockholm, Sweden
2 Department of Molecular Medicine and Surgery, Section of Integrative Physiology, Karolinska Institutet, 171 77 Stockholm, Sweden
3 Danderyd University Hospital, Karolinska Institutet, 171 77 Stockholm, Sweden

* Corresponding author: Department of Cardiology, Karolinska University Hospital, Solna, 171 76 Stockholm, Sweden. Tel: +46 8 517 70 000; fax: +46 8 31 10 40. E-mail address: adrgon{at}ki.se

Received 29 June 2007; revised 14 January 2008; accepted 15 January 2008

Time for primary review: 29 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Cardiovascular disease and type 2 diabetes mellitus are associated with low plasma concentration of adiponectin. The aim of this study was to investigate whether adiponectin exerts cardioprotective effects during myocardial ischaemia-reperfusion and whether this effect is related to the production of nitric oxide (NO).

Methods and results: Isolated rat hearts were subjected to 30 min of either global or local ischaemia followed by 60 min of reperfusion. The hearts received vehicle, adiponectin (3 µg/mL), the NO-synthase inhibitor nitro-L-arginine (L-NNA) (0.1 mM), or a combination of adiponectin and L-NNA at the onset of ischaemia. Haemodynamics, infarct size, and expression of endothelial NO-synthase (eNOS), AMP-activated protein kinase (AMPK), and Akt were determined. Adiponectin significantly increased left ventricular function and coronary flow during reperfusion in comparison with the vehicle group. Co-administration of L-NNA abrogated the improvement in myocardial function induced by adiponectin. Infarct size following local ischaemia-reperfusion was 40 ± 6% of the area at risk in the vehicle group. Adiponectin reduced infarct size to 19 ± 2% (P < 0.01). L-NNA did not affect infarct size per se but abolished the protective effect of adiponectin (infarct size 40 ± 5%). Phosphorylation of eNOS Ser1177, AMPK Thr172, and Akt Ser 473 was increased in the adiponectin group (P < 0.05).

Conclusion: Adiponectin protects from myocardial contractile dysfunction and limits infarct size following ischaemia and reperfusion by a mechanism involving activation of AMPK and production of NO.

KEYWORDS Adiponectin; Nitric oxide; Ischaemia; Reperfusion; Heart


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Obesity and type 2 diabetes mellitus are increasing worldwide and are associated with increased risk for cardiovascular disease. Adiponectin is an adipocyte-specific protein playing a key role in the regulation of glucose and lipid metabolism. Decreased plasma concentration of adiponectin is related to obesity, insulin resistance, type 2 diabetes mellitus, and coronary artery disease.1–8 Adiponectin circulates in plasma as a trimer (low molecular weight), a hexamer (medium molecular weight), and a multimeric form (high molecular weight) that is supposed to be the most biologically active form.9 Adiponectin exerts beneficial vascular and anti-inflammatory actions. Thus, adiponectin reduces expression of endothelial cell adhesion molecules, suppresses endothelial cell apoptosis, enhances production of nitric oxide (NO) in endothelial cells and reduces cytokine production from macrophages.9,10 Accordingly, endothelium-dependent vasodilation is reduced in adiponectin-knockout mice and in patients with hypoadiponectinaemia.11,12

Based on its anti-inflammatory actions and beneficial vascular effects, adiponectin has been implicated in the regulation of myocardial ischaemia-reperfusion injury. Exogenous adiponectin protects against apoptosis/necrosis during ischaemia and reperfusion in adiponectin-knockout mice.13 The mechanism behind these effects is suggested to be related to AMP-activated protein kinase (AMPK), cyclooxygenase (COX)-2, and tumour necrosis factor (TNF)-alpha,13 as well as inhibition of the inducible isoform of NO synthase (iNOS).14 Interestingly, AMPK is of importance for phosphorylation of the endothelial isoform of NOS (eNOS) on Ser1177.15 This may imply that adiponectin following activation of AMPK downstream may phosphorylate eNOS during myocardial ischaemia-reperfusion.8 NO produced from eNOS exerts important biological effects such as vasodilatation, inhibition of superoxide accumulation, attenuation of platelet adhesion and aggregation, regulation of interaction between leukocytes and endothelium, and modulation of cardiac myocyte function.16 NO also reduces myocardial oxygen consumption.17 Accordingly, NO produced from eNOS is an important mediator of protection against myocardial ischaemia and reperfusion. Administration of L-arginine, the substrate for NO production, or NO donors protect from ischaemia-reperfusion injury.17 Furthermore, protection against ischaemia-reperfusion injury induced by other pharmacological agents is dependent on intact production and release of NO.18–22 Thus, based on the finding that adiponectin stimulates AMPK and further downstream activates eNOS, phosphorylation of eNOS may be an important target for adiponectin during ischaemia-reperfusion. Previous studies have not addressed the issue whether adiponectin protects against ischaemia-reperfusion injury via a mechanism involving NO in animals with intact adiponectin production. Therefore, the aim of this study was to investigate whether supplementation with adiponectin protects from myocardial ischaemia and reperfusion injury via a mechanism that is dependent on production of NO using pharmacological inhibition of NOS and determining eNOS protein phosphorylation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
All parts of the investigations were approved by the regional ethics committee for animal research and conform with the Guide for the care and use of laboratory animals published by the US National Institute of Health (NIH publication No 85-23, revised 1985).

2.1 Animal preparation
Male Sprague-Dawley rats (weight 250–350 g, B&K Universal AB, Sollentuna, Sweden) were heparinized and anaesthetized with a mixture of fluanisonum, fentaylum, and midazolam (2.5, 0.08, and 1.25 mg/kg, respectively, i.m.; Janssen, Beerse, Belgium and Hoffmann-La Roche, Basel, Switzerland). The hearts were excised, the ascending aorta was cannulated, and immediately retrogradely perfused with non-circulating modified Krebs-Henseleit solution containing (in millimolar) NaCl 118, KCl 4.7, CaCl2 1.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25.2, and glucose 11.1. The perfusion pressure was kept constant at 90 cm H2O. The perfusate was bubbled with 95% O2 and 5% CO2 and kept at 37°C. A Transonic flow probe (Transonic, Ithaca, NY, USA) connected to a Transonic flow meter (T208) was placed in the circuit proximal to the aortic cannula for continuous measurement of coronary flow (CF). To assess contractile function in protocol 1 (see below), a latex balloon connected to a pressure transducer was inserted into the left ventricular (LV) cavity via the left atrium. Left ventricular end-diastolic pressure (LVEDP) was set at 5 mmHg by inflating the balloon with physiological saline. LV pressure and its electronically differentiated derivative dP/dt were continuously recorded on a personal computer equipped with PharmLab V3.0 (AstraZeneca R&D, Mölndal, Sweden). Heart rate was determined from the pressure registration every 5 min. A side arm connected to a mixing chamber in connection to the aortic cannula was used for the administration of drugs at the onset of ischaemia.

2.2 Experimental protocol
All hearts were allowed to stabilize for 30 min. They were included in either of the two different experimental protocols: (i) global ischaemia and or (ii) regional ischaemia. In protocol 1, warm global ischaemia was induced by clamping the buffer perfusion circuit for 30 min followed by 60 min of reperfusion. All combinations of drugs were infused in volumes of 3 mL for 1 min into the aortic cannula starting at the onset of ischaemia. After reperfusion, the heart was immediately frozen at –80°C for subsequent protein analysis. In protocol 2, the perfusion was stopped and the different combinations of drugs were given as 3 mL infusions as in protocol 1. At the end of the infusion, regional ischaemia was induced by ligation of the left main artery with a 5.0 silk while perfusion was restored to the rest of the coronary circulation. This procedure lasted for 1.5 min and the subsequent regional ischaemia was therefore maintained for 28.5 min followed by 60 min reperfusion.

The hearts of both protocols were randomized to four treatments groups: vehicle (n = 7 and 11 in protocol 1 and 2, respectively), human globular adiponectin (AstraZeneca; 3 µg/mL; n = 7 and 9), NO-synthase inhibitor N{omega}-nitro-L-arginine (L-NNA, Sigma, Stockholm, Sweden, 100 µM; n = 7 and 9) or combination of L-NNA and adiponectin (adiponectin + L-NNA; n = 7 and 9).

In protocol 1, haemodynamic parameters were determined at pre-ischaemia and every 5 min during reperfusion. In protocol 2, CF and infarct size were determined.

2.3 Determination of area at risk and infarct size
At the end of reperfusion, the hearts of protocol 2 were perfused with 2% Evans blue to demarcate the area at risk. After dismounting the heart, the LV was cut perpendicular to the base-apex axis into six slices that were scanned from both sides for planimetry of the area at risk. They were then transferred to 0.8% triphenyl tetrazolium chloride at 37°C for 12 min and scanned for the determination of viable myocardium. The area at risk and viable myocardium were calculated by counting pixels with Adobe Photoshop 7.0.

2.4 Immunoblotting of endothelial nitric oxide-synthase, AMP-activated protein kinase, and Akt
2.4.1 Tissue processing
The myocardium from hearts included in protocol 1 were used for protein analysis of phosphorylated and total eNOS, AMPK, and Akt. Additional groups of hearts given vehicle (n = 7) or adiponectin (n = 7) as in protocol 1 were collected immediately following 30 min of ischaemia for analysis of AMPK and Akt. Frozen hearts were homogenized by microdismembrator (Polytron PT1200, Kinematica AG, Switzerland) in 400 µL ice-cold lysis buffer containing 20 mM Tris (pH 7.8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% Triton X-100, 10% (w/v) glycerol, 10 mM NaF, 1 mM ethylenediaminetetraacetic acid, 5 mM Na-pyrophosphate, 0.5 mM Na3VO4, 1 µg/mL leupeptin, 0.2 mM phenylmethyl sulfonyl fluoride, 1 µg/mL aprotinin, 1 mM dithiothreitol, 1 mM benzamidine. Homogenates were solubilized by rotation for 30 min at 4°C and subjected to centrifugation (12 000 g for 15 min at 4°C). Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA).

2.4.2 Western blot analysis
Expression of total and phosphorylated eNOS, AMPK, and Akt were determined. An aliquot of tissue lysate (50 µg protein) was mixed in Laemmli sample buffer containing β-mercaptoethanol. Proteins were separated by 10% (eNOS) or 7.5% (AMPK and Akt) SDS–PAGE, transferred to nitrocellulose membrane (eNOS), and polyvinylivenediflouride membrane (AMPK and Akt; Millipore, Bedford, MA, USA), respectively, and blocked in 7.5% non-fat dried milk in Tris-buffered saline with 0.02% Tween (TBST) for 2 h at room temperature. Membranes were incubated overnight at 4°C with phospho-specific antibodies against Ser1177 (1:1000, BD Biosciences Pharmingen, USA), AMPK Thr172, or Akt Ser473 (1:1000, Cell Signaling Technology, Beverly, MA, USA). After washing in TBST, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse for eNOS Ser1177 (1:10 000, BD Biosciences Pharmingen), and anti-rabbit IgG for AMPK Thr172 and Akt Ser473 (1:25 000, Bio-Rad, Hercules, CA, USA) for 1 h at room temperature, followed by additional washing. Proteins were visualized by enhanced chemiluminescence (ECL plus; Amersham, Arlington Heights, IL, USA) and quantified using densitometry and Molecular Analyst Software (Bio-Rad, Richmond, CA, USA). After immunoblotting with phospho-specific antibodies, membranes were stripped as described and immunoblotted for either total eNOS (1:1000, Affinity BioReagents, USA), AMPK (1:1000, Update/Chemicon, Sweden), or Akt proteins (1:1000, Cell Signaling Technology).

2.5 Calculation and statistical analysis
Left ventricular-developed pressure (LVDP) is the difference between LV systolic and end-diastolic pressures. Rate pressure product (RPP) was calculated as heart rate multiplied by LVDP. The recovery of myocardial performance is expressed in percent of the pre-ischaemic value. All values are presented as mean ± SEM. Comparison between four groups were made by analysis of variance (ANOVA, area at risk, and infarct size) or two-way analysis of variance (haemodynamics) followed by Fisher's paired least significant difference (PLSD) test. Comparison of data from western blots were analysed by Mann–Whitney U test. The significance level was set at 0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Global ischaemia-reperfusion (Protocol 1)
CF, LVDP, dP/dt, heart rate, or RPP did not differ between the groups before ischaemia (Table 1). The post-ischaemic recoveries of LVDP, dP/dt, and RPP were significantly better in the adiponectin group than in the vehicle group (P < 0.01; Figure 1A–C). Administration of L-NNA completely reversed the beneficial effect of adiponectin on LVDP, dP/dt, and RPP (Figure 1A–C). Heart rate increased in all groups during the initial phase of reperfusion but returned to pre-ischaemic levels at the end of reperfusion. There were no significant differences in heart rate between the groups. L-NNA did not affect the recovery of myocardial function per se. LVEDP during reperfusion was significantly lower in the adiponectin group (48 ± 6 mmHg at 60 min reperfusion) than in the groups given vehicle or adiponectin in combination with L-NNA (62 ± 4 mmHg and 62 ± 5 mmHg, respectively, at 60 min reperfusion; Figure 2). L-NNA slightly but significantly reduced LVEDP during reperfusion in comparison with the vehicle group (Figure 2). Recovery of CF was higher in the adiponectin group than in the vehicle group throughout the reperfusion period (Figure 1D). CF in the L-NNA group and in the group given the combination of L-NNA and adiponectin did not differ significantly from the vehicle group.


Figure 1
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1 The percent recoveries of (A) left ventricular (LV)-developed pressure, (B) first derivative of LV pressure (dP/dt), (C) rate-pressure product, and (D) coronary flow during reperfusion in rat hearts given vehicle, adiponectin, the NOS inhibitor L-NNA, or the combination of adiponectin and L-NNA. Data are presented as mean ± SEM. Significant differences from the vehicle group during the entire reperfusion period are shown (two-way analysis of variance); ***P < 0.001.

 


Figure 2
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2 Left ventricular end-diastolic pressure during ischaemia and reperfusion of rat hearts given vehicle, adiponectin, the NOS inhibitor L-NNA, or the combination of adiponectin and L-NNA. Data are presented as mean ± SEM. Significant differences from the vehicle group during the entire reperfusion period are shown (two-way analysis of variance); ***P < 0.001.

 


View this table:
[in this window]
[in a new window]

  		Table 1 Pre-ischaemic values of the hearts in protocol 1 (global ischaemia)

 
3.2. Regional ischaemia-reperfusion (Protocol 2)
The sizes of the area at risk are depicted in Figure 3A. The areas at risk in the groups receiving L-NNA and the combination of L-NNA and adiponectin were slightly but statistically larger when compared with the vehicle group. The area at risk of the adiponectin group did not differ significantly from any of the groups.


Figure 3
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3 Infarct size expressed in percent of the area at risk after ischaemia and reperfusion in the groups receiving vehicle, adiponectin, the NOS inhibitor L-NNA, or the combination of adiponectin and L-NNA. Data are presented as mean ± SEM. Significant difference from the vehicle group is shown; *P < 0.05 and **P < 0.01.

 
The infarct size was significantly smaller in the group receiving adiponectin (19 ± 2% of area at risk) than that of the vehicle group (41 ± 6%; Figure 3). There was no significant difference in infarct size between the vehicle group and the groups receiving L-NNA or the combination of L-NNA and adiponectin. On the other hand, the infarct size was significantly larger in the group receiving the combination of L-NNA and adiponectin than in the group receiving adiponectin only (P < 0.01, Figure 3).

CF during reperfusion was similar in the vehicle group and the adiponectin group (Figure 4). Both groups receiving L-NNA had lower CF than the vehicle and the adiponectin groups.


Figure 4
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4 The percent recovery of coronary flow during reperfusion in rat hearts subjected to regional ischaemia and reperfusion given vehicle, adiponectin, the NOS inhibitor L-NNA, or the combination of adiponectin and L-NNA. Data are presented as mean ± SEM. Significant differences from the vehicle group during the entire reperfusion period are shown (two-way analysis of variance); ***P < 0.001.

 
3.3 Immunoblotting of endothelial NO-synthase, AMP-activated protein kinase, Akt
There was no difference in the expression of total eNOS or AMPK between the vehicle and adiponectin group. On the other hand, at the end of reperfusion, phosphorylation of eNOS Ser1177 and AMPK Thr172 increased significantly following the administration of adiponectin (P < 0.05, Figures 5 and 6). There was no significant difference in the expression of phosphorylated Akt Ser473 between the two groups at the end of reperfusion. In the groups collected immediately after ischaemia, there was a significant increase in phosphorylation of Akt Ser473 (Figure 7), but no change in AMPK Thr172.


Figure 5
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5 Representative immunoblots and quantification of myocardial protein extracts for analysis of total endothelial nitric oxide-synthase (eNOS) protein and phosphorylated eNOS Ser1177 obtained from hearts given vehicle or adiponectin and subjected to global ischaemia and reperfusion. Values are depicted as mean ± SEM (n = 7). Significant differences between groups are shown; *P < 0.05.

 


Figure 6
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6 Representative immunoblots and quantification of myocardial protein extracts for analysis of AMP-activated protein kinase (AMPK) and phosphorylated AMPK Thr172 obtained from hearts given vehicle or adiponectin and subjected to 30 min of global ischaemia and 60 min reperfusion. Values are depicted as mean ± SEM (n = 7). Significant differences between groups are shown; *P < 0.05.

 


Figure 7
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 7 Immunoblots and quantification of Akt and phosphorylated Akt Ser473 in myocardial extracts from hearts given vehicle or adiponectin and subjected to 30 min global ischaemia, but without reperfusion. Values are depicted as mean ± SEM (n = 7). Significant differences between groups are shown; *P < 0.05.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
The main findings of this study is that the protective effect of adiponectin against myocardial contractile dysfunction and infarction following ischaemia and reperfusion is inhibited by pharmacological blockade of NO production, and is associated with phosphorylation of AMPK Thr172, Akt Ser473, and eNOS Ser1177. These observations suggest that the protection against myocardial ischaemia and reperfusion injury mediated by adiponectin is related to activation of eNOS and production of NO.

The observation that exogenous adiponectin exerts cardioprotective effects during myocardial ischaemia and reperfusion is in agreement with previous studies performed in adiponectin-deficient mice in vivo.13 Shibata et al.13 further demonstrated that adiponectin inhibits apoptosis by activating AMPK and suppresses the production of TNF-alpha via a COX 2-dependent mechanism. In addition, there are observations suggesting that adiponectin stimulates the production of NO in endothelial cells.23 Production and release of NO during ischaemia and reperfusion are of importance for mediating cardioprotection during ischaemia and reperfusion.18–22 The hypothesis that NO is involved in the cardioprotective effect mediated by adiponectin was tested by investigating both functional recovery following global ischaemia-reperfusion and infarct size following regional ischaemia-reperfusion. Administration of the NOS inhibitor L-NNA completely blocked the cardioprotective effect of adiponectin in both experimental protocols, which supports our hypothesis. Importantly, L-NNA did not affect the recovery of myocardial contractile function or infarct size per se when compared with the vehicle group, suggesting that it interfered with the protective mechanisms induced by adiponectin.

In the global ischaemia experiments, adiponectin increased CF during reperfusion in comparison with the vehicle group. This may suggest that the improvement in myocardial function is due to increased perfusion. However, improved CF may be owing to a reduction in the area of no-reflow and thereby secondary to inhibition of the reperfusion injury. Previous observations suggest that reperfusion injury leads to decrease in myocardial perfusion,24 suggesting that inhibition of reperfusion injury will lead to increased flow. In the regional ischaemia experiments, adiponectin did not affect post-ischaemic CF in comparison with the vehicle group suggesting that changes in CF did not contribute to the difference in infarct size.

To further explore the effect of adiponectin on NO production, the phosphorylation and the expression of eNOS protein was investigated. Administration of adiponectin did not increase total eNOS expression, but it increased the expression of phosphorylated eNOS Ser1177. It was recently demonstrated that adiponectin increases eNOS phosphorylation in the mouse heart but the phosphorylation site was not specified.14 This is of importance since phosphorylation at different sites may result in opposite effects on enzyme activity. Phosphorylation of eNOS at residue Ser1177 is associated with increased enzyme activity and increased production of NO.25 We have previously demonstrated that L-NNA does not influence the phosphorylation of eNOS per se.21 Collectively, these observations suggest that adiponectin stimulates phosphorylation of eNOS at Ser1177 resulting in increased production of NO which mediates the cardioprotection during ischaemia and reperfusion.

To study the possible link between adiponectin and phosphorylation of eNOS, we determined the expression and phosphorylation of AMPK and Akt at different time points. AMPK is an important regulatory mechanism involved in the generation of adenosine triphosphate, fatty acid oxidation, glycolysis, increasing muscle glucose uptake, and inhibiting energy-consuming anabolic pathways.26 Adiponectin stimulates AMPK signalling in isolated cardiomyocytes27 and in the ischaemic hindlimb.28 Furthermore, adiponectin protects the murine heart against ischaemia and reperfusion injury via an AMPK-dependent pathway.13 In this study, we show that phosphorylation of the Thr172 residue of AMPK is increased by adiponectin in the myocardium 60 min following ischaemia. Since it has been demonstrated that AMPK phoshorylates eNOS Ser1177 in rat heart extracts and endothelial cells,15,23,29 these observations suggest that adiponectin stimulates phosphorylation of AMPK, which results in downstream phosphorylation of eNOS in the ischaemic/reperfused myocardium. The effect of adiponectin has also been suggested to involve the PI3-kinase/Akt signalling pathway.29 In the present study we did not detected any change in Akt at the end of reperfusion, but at the onset of reperfusion there was a significant increase in phosphorylation of Akt in the group receiving adiponectin. Taken together these observations indicate that adiponectin activates both AMPK and Akt, but at different time points during myocardial ischaemia-reperfusion.

There are limitations with the present study. The studies were performed on isolated hearts and the results need to be confirmed in vivo. On the other hand, it has been demonstrated that adiponectin reduces infarct size in adiponectin knockout mice in vivo.13 The present study extends earlier knowledge by demonstrating that adiponectin improves post-ischaemic myocardial function and reduces infarct size via an effect that is dependent on the availability of NO. In an earlier study it has been shown that adiponectin may be locally produced in cardiomyocytes.30 However, the present study indicates that local production of adiponectin does not give optimal efficacy and supplemental administration of adiponectin during ischaemia could exert additional protective effects. A further limitation is that the authors, in this presently used model, cannot determine which molecular form of adiponectin that is protective. Previous data suggest the high molecular weight fraction to be of biological importance,9 but this issue remains to be elucidated in ischaemia-reperfusion studies. Further, the degree of tissue penetration and thereby the target cell type for the effect of adiponectin under these experimental conditions is unknown. The molecular size of adiponectin may indicate that its effect is confined to the vasculature and the endothelial cells. An effect on the endothelial cells may be sufficient in order to release NO, which in turn may affect cells on the abluminal side of the vasculature including cardiomyocytes.

In conclusion, administration of adiponectin at the onset of ischaemia followed by reperfusion induces phosphorylation of AMPK Thr172, Akt Ser473, and eNOS Ser1177 and protects from myocardial contractile dysfunction and infarction. Pharmacological blockade of NOS abrogated the cardioprotective effect of adiponectin. These observations suggest that the cardioprotective effect of adiponectin is mediated via NO by phosphorylation of eNOS.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work was supported by the Swedish Research Council Medicine (10857); the Swedish Heart and Lung Foundation; the Stockholm County Council (ALF); Novo Nordisk Foundation; the Commission of the European Communities (Contract No. LSHM-CT-2004-512013 EUGENEHEART); Swedish Medical Association; and Åke Wiberg Foundation.


   Acknowledgements
 
We thank Andreas Lundqvist, AstraZeneca, Mölndal, Sweden for producing and supplying adiponectin. We are grateful to Mrs. Marita Wallin for technical assistance.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

  1. Bacci S, Menzaghi C, Ercolino T, Ma X, Rauseo A, Salvemini L, et al. The +276 G/T single nucleotide polymorphism of the adiponectin gene is associated with coronary artery disease in type 2 diabetic patients. Diabetes Care (2004) 27:2015–2020.[Abstract/Free Full Text]
  2. Hotta K, Funahashi T, Arita Y, Takahashi M, Matsuda M, Okamoto Y, et al. Plasma concentrations of a novel, adipose-specific protein, adiponectin, in type 2 diabetic patients. Arterioscler Thromb Vasc Biol (2000) 20:1595–1599.[Abstract/Free Full Text]
  3. Kumada M, Kihara S, Sumitsuji S, Kawamoto T, Matsumoto S, Ouchi N, et al. Association of hypoadiponectinemia with coronary artery disease in men. Arterioscler Thromb Vasc Biol (2003) 23:85–89.[Abstract/Free Full Text]
  4. Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun (1999) 257:79–83.[CrossRef][Web of Science][Medline]
  5. Aso Y, Yamamoto R, Wakabayashi S, Uchida T, Takayanagi K, Takebayashi K, et al. Comparison of serum high-molecular weight (HMW) adiponectin with total adiponectin concentrations in type 2 diabetic patients with coronary artery disease using a novel enzyme-linked immunosorbent assay to detect HMW adiponectin. Diabetes (2006) 55:1954–1960.[Abstract/Free Full Text]
  6. Lacquemant C, Froguel P, Lobbens S, Izzo P, Dina C, Ruiz J. The adiponectin gene SNP+45 is associated with coronary artery disease in Type 2 (non-insulin-dependent) diabetes mellitus. Diabet Med (2004) 21:776–781.[CrossRef][Web of Science][Medline]
  7. Ohashi K, Ouchi N, Kihara S, Funahashi T, Nakamura T, Sumitsuji S, et al. Adiponectin I164T mutation is associated with the metabolic syndrome and coronary artery disease. J Am Coll Cardiol (2004) 43:1195–1200.[Abstract/Free Full Text]
  8. Hopkins TA, Ouchi N, Shibata R, Walsh K. Adiponectin actions in the cardiovascular system. Cardiovasc Res (2007) 74:11–18.[Abstract/Free Full Text]
  9. Kobayashi H, Ouchi N, Kihara S, Walsh K, Kumada M, Abe Y, et al. Selective suppression of endothelial cell apoptosis by the high molecular weight form of adiponectin. Circ Res (2004) 94:e27–e31.[Abstract/Free Full Text]
  10. Han SH, Quon MJ, Kim JA, Koh KK. Adiponectin and cardiovascular disease: response to therapeutic interventions. J Am Coll Cardiol (2007) 49:531–538.[Abstract/Free Full Text]
  11. Ouchi N, Ohishi M, Kihara S, Funahashi T, Nakamura T, Nagaretani H, et al. Association of hypoadiponectinemia with impaired vasoreactivity. Hypertension (2003) 42:231–234.[Abstract/Free Full Text]
  12. Nystrom T, Nygren A, Sjoholm A. Increased levels of tumour necrosis factor-alpha (TNF-alpha) in patients with Type II diabetes mellitus after myocardial infarction are related to endothelial dysfunction. Clin Sci (Lond) (2006) 110:673–681.[Medline]
  13. Shibata R, Sato K, Pimentel DR, Takemura Y, Kihara S, Ohashi K, et al. Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2-dependent mechanisms. Nat Med (2005) 11:1096–1103.[CrossRef][Web of Science][Medline]
  14. Tao L, Gao E, Jiao X, Yuan Y, Li S, Christopher TA, et al. Adiponectin cardioprotection after myocardial ischemia/reperfusion involves the reduction of oxidative/nitrative stress. Circulation (2007) 115:1408–1416.[Abstract/Free Full Text]
  15. Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, et al. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett (1999) 443:285–289.[CrossRef][Web of Science][Medline]
  16. Cotton JM, Kearney MT, Shah AM. Nitric oxide and myocardial function in heart failure: friend or foe? Heart (2002) 88:564–566.[Abstract/Free Full Text]
  17. Heusch G, Post H, Michel MC, Kelm M, Schulz R. Endogenous nitric oxide and myocardial adaptation to ischemia. Circ Res (2000) 87:146–152.[Abstract/Free Full Text]
  18. Bulhak AA, Gourine AV, Gonon AT, Sjoquist PO, Valen G, Pernow J. Oral pre-treatment with rosuvastatin protects porcine myocardium from ischaemia/reperfusion injury via a mechanism related to nitric oxide but not to serum cholesterol level. Acta Physiol Scand (2005) 183:151–159.[CrossRef][Web of Science][Medline]
  19. Bulhak AA, Sjoquist PO, Xu CB, Edvinsson L, Pernow J. Protection against myocardial ischaemia/reperfusion injury by PPAR-alpha activation is related to production of nitric oxide and endothelin-1. Basic Res Cardiol (2006) 101:244–252.[CrossRef][Web of Science][Medline]
  20. Gonon AT, Bulhak A, Broijersen A, Pernow J. Cardioprotective effect of an endothelin receptor antagonist during ischaemia/reperfusion in the severely atherosclerotic mouse heart. Br J Pharmacol (2005) 144:860–866.[CrossRef][Medline]
  21. Gonon AT, Bulhak A, Labruto F, Sjoquist PO, Pernow J. Cardioprotection mediated by rosiglitazone, a peroxisome proliferator-activated receptor gamma ligand, in relation to nitric oxide. Basic Res Cardiol (2007) 102:80–89.[CrossRef][Web of Science][Medline]
  22. Wright DG, Lefer DJ. Statin mediated protection of the ischemic myocardium. Vascul Pharmacol (2005) 42:265–270.[CrossRef][Web of Science][Medline]
  23. Chen H, Montagnani M, Funahashi T, Shimomura I, Quon MJ. Adiponectin stimulates production of nitric oxide in vascular endothelial cells. J Biol Chem (2003) 278:45021–45026.[Abstract/Free Full Text]
  24. Reffelmann T, Hale SL, Li G, Kloner RA. Relationship between no reflow and infarct size as influenced by the duration of ischemia and reperfusion. Am J Physiol (2002) 282:H766–H772.[Web of Science]
  25. Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol (2003) 284:R1–R12.[Abstract/Free Full Text]
  26. Young LH, Li J, Baron SJ, Russell RR. AMP-activated protein kinase: a key stress signaling pathway in the heart. Trends Cardiovasc Med (2005) 15:110–118.[CrossRef][Web of Science][Medline]
  27. Li L, Wu L, Wang C, Liu L, Zhao Y. Adiponectin modulates carnitine palmitoyltransferase-1 through AMPK signaling cascade in rat cardiomyocytes. Regul Pept (2007) 139:72–79.[CrossRef][Medline]
  28. Shibata R, Ouchi N, Kihara S, Sato K, Funahashi T, Walsh K. Adiponectin stimulates angiogenesis in response to tissue ischemia through stimulation of amp-activated protein kinase signaling. J Biol Chem (2004) 279:28670–28674.[Abstract/Free Full Text]
  29. Ouchi N, Kobayashi H, Kihara S, Kumada M, Sato K, Inoue T, et al. Adiponectin stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in endothelial cells. J Biol Chem (2004) 279:1304–1309.[Abstract/Free Full Text]
  30. Pineiro R, Iglesias MJ, Gallego R, Raghay K, Eiras S, Rubio J, et al. Adiponectin is synthesized and secreted by human and murine cardiomyocytes. FEBS Lett (2005) 579:5163–5169.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Circ Heart FailHome page
K. R. McGaffin, C. S. Moravec, and C. F. McTiernan
Leptin Signaling in the Failing and Mechanically Unloaded Human Heart
Circ Heart Fail, November 1, 2009; 2(6): 676 - 683.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
J. Xu and M.-H. Zou
Molecular Insights and Therapeutic Targets for Diabetic Endothelial Dysfunction
Circulation, September 29, 2009; 120(13): 1266 - 1286.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
Y. Wang, L. Tao, Y. Yuan, W. B. Lau, R. Li, B. L. Lopez, T. A. Christopher, R. Tian, and X.-L. Ma
Cardioprotective effect of adiponectin is partially mediated by its AMPK-independent antinitrative action
Am J Physiol Endocrinol Metab, August 1, 2009; 297(2): E384 - E391.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
Y. Wang, E. Gao, L. Tao, W. B. Lau, Y. Yuan, B. J. Goldstein, B. L. Lopez, T. A. Christopher, R. Tian, W. Koch, et al.
AMP-Activated Protein Kinase Deficiency Enhances Myocardial Ischemia/Reperfusion Injury but Has Minimal Effect on the Antioxidant/Antinitrative Protection of Adiponectin
Circulation, February 17, 2009; 119(6): 835 - 844.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
78/1/116    most recent
cvn017v2
cvn017v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Gonon, A. T.
Right arrow Articles by Pernow, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gonon, A. T.
Right arrow Articles by Pernow, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?