Cardiovascular Pharmacology
Cardioprotection from ischaemia–reperfusion injury by a novel flavonol that reduces activation of p38 MAPK

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Abstract

Oxidative stress, activation of intracellular protein kinases and cardiomyocyte apoptosis are known mediators of cardiac ischaemia/reperfusion injury. The sites at which NP202, a novel water soluble pro-drug of 3′,4′-dihydroxyflavonol (DiOHF), acts in this cascade to cause cardioprotection are unknown. In this study we examined the ability of NP202 to reduce infarct size after a prolonged period of ischaemia and reperfusion. In addition, we tested whether NP202 inhibits pro-apoptotic signalling, apoptosis and inflammation following myocardial ischaemia and reperfusion. Sheep were anaesthetised, the heart exposed and the 2nd branch of the left anterior descending coronary artery isolated. The artery was occluded for 3 h and, five minutes before 3 h of reperfusion was commenced, sheep were treated with intravenous vehicle or NP202. At the end of reperfusion infarct size was measured and normal left ventricle, non-infarcted area-at-risk and infarcted myocardium were collected to identify polymorphonuclear leukocytes (PMN) or apoptotic cells (TUNEL-positive), or assessed for activation of mitogen-activated protein kinase (MAPK) pathways by Western blot analysis. Compared with vehicle treatment, NP202 reduced infarct size (− 20 ± 4%, P < 0.05) and decreased the number of PMNs and TUNEL-positive cells in the area-at-risk (− 35 ± 16% and − 52 ± 19%, respectively) and infarcted tissue (− 57 ± 9 and − 81 ± 5%, respectively, P < 0.05). Furthermore, NP202 significantly reduced I/R-induced elevated p38 MAPK phosphorylation (by 67 ± 4%, P < 0.05) in the area-at-risk zone. In conclusion, the novel aqueous flavonol NP202 provided significant cardioprotection from clinically relevant prolonged myocardial ischaemia when administered just before reperfusion. Efficacy of NP202 was also associated with reduced p38 MAPK activation, inflammation and apoptotic cell death.

Introduction

An emerging strategy to prevent ischaemia and reperfusion (I/R) injury depends on altering the interplay between pro-death and pro-survival cell signalling pathways in the myocardium at the time of reperfusion (Hausenloy and Yellon, 2007, Lecour, 2009). Investigation has focussed on the mitogen-activated protein kinase (MAPK) signal transduction cascades that are activated after myocardial I/R (Omura et al., 1999, Ravingerova et al., 2003, Hamacher-Brady et al., 2006, Hausenloy and Yellon, 2006). Myocyte apoptosis is induced during reperfusion by activating pro-injurious kinases, including p38-mitogen-activated protein kinase (MAPK) and c-jun NH2 terminal kinase (JNK) (Yue, et al. 2000). In contrast, cardioprotection is afforded by activating the endogenous reperfusion injury survival kinases (RISK), including extracellular regulated kinase (ERK1/2) and AKT (Hausenloy and Yellon 2007).

There are currently no drugs for clinical use to reduce reperfusion injury. Despite the success of several compounds in reducing infarct size following I/R in animal studies, these have not been effective in human trials (Downey and Cohen, 2009). A possible reason for this lack of translation is that many animal studies are sub-optimally designed with relatively brief periods of ischaemia (30–90 min), whereas patients generally experience longer periods of ischaemia (3–5 h) before restoration of coronary perfusion (Schomig et al., 2006). This is concerning, since the duration of ischaemia is a key determinant of vulnerability to arrhythmias, myocardial stunning and lethal myocyte injury after reperfusion (Grech et al., 1995).

In previous studies we reported that the synthetic flavonol 3′,4′-dihydroxyflavonol (DiOHF) substantially reduced cardiac I/R injury (Wang et al., 2004, Wang et al., 2009a, Wang et al., 2009b). We proposed that the cardioprotective effect of DiOHF resulted from its antioxidant, vasorelaxant and anti-inflammatory actions (Wang et al., 2004, Woodman and Chan, 2004, Jiang et al., 2009) and its ability to reduce calcium overload (Woodman and Chan 2004). Notably, in the setting of prolonged 4 weeks reperfusion following ischaemia, DiOHF reduced post-infarction left ventricular remodelling by preventing myocyte apoptosis (Wang et al., 2009a), a key pathological process (Abbate et al., 2006). These multifaceted activities of DiOHF indicate its potential as a possible therapeutic intervention to ameliorate I/R injury and ventricular remodelling, but it remains unclear whether DiOHF has effects on activation of cardioprotective kinases or pro-injurious kinases. Furthermore, these studies used relatively short periods of ischaemia (1 h), and it is unknown whether DiOHF maintains its effectiveness after prolonged ischaemia. The flavonol DiOHF is highly lipophilic and able to rapidly enter cardiomyocytes, but it cannot be administered in aqueous solution. We have, therefore, developed a water-soluble pro-drug of DiOHF (NP202), which bears an ionisable carboxylic acid carrier group that is cleaved by endogenous esterases in vivo to release DiOHF.

The objectives of the present study were to characterise the consequences of prolonged 3 h myocardial ischaemia and 3 h reperfusion in anaesthetised sheep and assess the cardioprotective effects of NP202 in this I/R injury model. In addition, to address the potential mechanisms involved, we analysed the phosphorylation status of important MAPK family members and the prevalence of inflammation and apoptosis in sheep treated with NP202 (Williams et al., 2011).

Section snippets

Materials and methods

An expanded methods section is available in the on-line Data Supplement.

Preliminary NP202 dose–response study

NP202 treatment at doses 2.7, 6.6, 13.2, 19.8 or 39.6 mg/kg just before reperfusion, resulted in infarct sizes of 62 ± 2%, 46 ± 1%, 60 ± 3%, 58 ± 4% and 56% (IS/AR), respectively, compared with vehicle (80 ± 3%, IS/AR). Since treatment with 6.6 mg/kg NP202 caused the greatest reduction in infarct size, this dose was used in the main study to determine efficacy of the drug following prolonged ischaemia and reperfusion.

Effect of NP202 on haemodynamic parameters following prolonged I/R injury

During the 30 min control period after surgical instrumentation, and prior to myocardial

Discussion

The present study demonstrated that a single dose of the novel water soluble flavonol NP202, a pro-drug of DiOHF, significantly reduced infarct size induced by prolonged 3 h ischaemia and 3 h reperfusion in anaesthetised sheep. In addition, NP202 reduced inflammation, apoptosis and the activation of pro-apoptotic p38 MAPK in the ischaemic myocardium. The cardioprotective features of NP202 reported in the present study closely resemble the beneficial effects of its parent flavonol, DiOHF.

Sources of Funding

This work was supported by a grant-in-aid from the National Heart Foundation of Australia (G 08M 3761). CNM was supported by a Research Fellowship from NHMRC, Australia (566819). DN is supported by a NHFA/NHMRC Fellowship (404126).

Disclosures

O.L. Woodman and C.N. May are shareholders in NeuProtect, a company which holds a patent for use of flavonols as a treatment for myocardial ischaemia–reperfusion injury.

Acknowledgements

We thank Alan McDonald and Tony Dornom for their expert technical assistance with surgical preparation of the animals. Neuprotect Pty. Ltd. generously provided NP202.

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