Hypoxia-inducible factor as a therapeutic target for cardioprotection

Pharmacology & Therapeutics 136 (2012) 69–81

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Hypoxia-inducible factor as a therapeutic target for cardioprotection Sang-Ging Ong, Derek J. Hausenloy ⁎ The Hatter Cardiovascular Institute, University College London Hospital, 67 Chenies Mews, London WC1E 6HX, United Kingdom

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Keywords: Heart Hypoxia-inducible factor Cardioprotection Prolyl hydroxylase domain-containing enzyme von Hippel–Lindau protein

a b s t r a c t Hypoxia inducible factor (HIF) is an oxygen-sensitive transcription factor that enables aerobic organisms to adapt to hypoxia. This is achieved through the transcriptional activation of up to 200 genes, many of which are critical to cell survival. Under conditions of normoxia, the hydroxylation of HIF by prolyl hydroxylase domain-containing (PHD) enzymes targets it for polyubiquitination and proteosomal degradation by the von Hippel–Lindau protein (VHL). However, under hypoxic conditions, PHD activity is inhibited, thereby allowing HIF to accumulate and translocate to the nucleus, where it binds to the hypoxia-responsive element sequences of target gene promoters. Experimental studies suggest that HIF may act as a mediator of ischemic preconditioning, and that the genetic or pharmacological stabilization of HIF under normoxic conditions, may protect the heart against the detrimental effects of acute ischemia–reperfusion injury. The mechanisms underlying the cardioprotective effect of HIF are unclear, but it may be attributed to the transcriptional activation of genes associated with cardioprotection such as erythropoietin, heme oxygenase-1, and inducible nitric oxide synthase or it may be due to reprogramming of cell metabolism. In this review article, we highlight the role of HIF in mediating both adaptive and pathological processes in the heart, as well as focusing on the therapeutic potential of the HIF-signaling pathway as a target for cardioprotection. © 2012 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The discovery of hypoxia-inducible factor . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Regulation of hypoxia‐inducible factor 1α activity . . . . . . . . . . . . . . . . . . . . . . . 4. Hypoxia‐inducible factor 1α activation and the heart . . . . . . . . . . . . . . . . . . . . . 5. Hypoxia‐inducible factor 1α as a mediator of ischemic preconditioning . . . . . . . . . . . . . 6. Genetic strategies for stabilizing hypoxia‐inducible factor 1α to induce cardioprotection . . . . . 7. Pharmacological strategies for stabilizing hypoxia‐inducible factor 1α to induce cardioprotection . 8. Mechanisms of hypoxia‐inducible factor 1α-mediated cardioprotection . . . . . . . . . . . . . . . 9. Stabilization of hypoxia‐inducible factor 1α in heart failure: beneficial or detrimental? . . . . . 10. Hypoxia‐inducible factor 2α and cardioprotection . . . . . . . . . . . . . . . . . . . . . . . 11. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AP-1, Activator protein 1; A2BR, Adenosine A2B receptor; CH-1, Constant-Domain 1; CoCl2, Cobalt chloride; DMOG, Dimethyloxaloylglycine; ETC, Electron transport chain; EDHB, Ethyl-3,4-dihydroxybenzoate; EPO, Erythropoietin; FIH, Factor Inhibiting HIF; HO-1, Heme oxygenase-1; HIF, Hypoxia-inducible factor; HPC, Hypoxic preconditioning; iNOS, Inducible nitric oxide synthase; IPC, Ischemic preconditioning; IRI, Ischemia–reperfusion injury; LAD, Left anterior descending; LIMD1, LIM domain-containing protein 1; LV, Left ventricle; MAPK, Mitogen-activated protein kinase; mRNA, Messenger RNA; miRNA, MicroRNA; mPTP, Mitochondrial permeability transition pore; NAD+, Nicotinamide adenine dinucleotide; NO, Nitric oxide; p300/CRB, p300/cAMP response element-binding protein; PI3K, Phosphoinositide 3-kinase; PHD, Prolyl hydroxylase domain-containing enzyme; PTEN, Phosphatase and tensin homolog; ROS, Reactive oxygen species; siRNA, Small interfering RNA; SWOP, Second window of preconditioning; VEGF, Vascular endothelial growth factor; VHL, von Hippel–Lindau protein. ⁎ Corresponding author. Tel.: +44 203 447 9894; fax: +44 203 447 5095. E-mail address: [email protected] (D.J. Hausenloy). 0163-7258/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2012.07.005

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Novel therapeutic strategies are required to protect the heart against acute ischemia–reperfusion injury (IRI), in order to preserve left ventricular function, and reduce the onset of heart failure in patients with coronary heart disease. The hypoxia-inducible factor (HIF) has emerged as a critical oxygen-sensitive transcription factor, which orchestrates the body's protective response to hypoxia. This is achieved through the transcriptional activation of up to 200 genes, many of which are critical to cell survival, and may therefore be important in protecting the heart against acute IRI (cardioprotection). Experimental studies have demonstrated that HIF may act as a mediator of ischemic preconditioning (IPC) and that the genetic or pharmacological stabilization of HIF, under normoxic conditions, can protect the heart against acute IRI (Loor & Schumacker, 2008; Tekin et al., 2010; Semenza, 2011). In this review article, we discuss the regulatory mechanisms of HIF including some recently discovered hypoxic regulators, highlighting the role of HIF as a mediator of both adaptive and pathological processes in the heart, and focus on the therapeutic potential of the HIF signaling pathway as a target for cardioprotection.

expression of proteins in various biological pathways including but not limited to glucose metabolism, cell proliferation, neovascularization, inflammation and cellular differentiation. For a more in-depth understanding of the various pathways regulated by HIF, the reader is advised to refer to several excellent reviews which have been published over the last few years (Semenza, 2000; Loor & Schumacker, 2008; Gale & Maxwell, 2010; Heather & Clarke, 2011). HIF-1 is a heterodimeric complex containing an oxygen-labile α-subunit (120 kDa) as well as a constitutively expressed β-subunit (also known as aryl hydrocarbon receptor nuclear transporter) (91–94 kDa), both of which are members of the basic helix–loop– helix Per-Arnt-Sim family of transcription factors (Wang et al., 1995). Mammals possess three isoforms of HIF-α: HIF-1α and HIF-2α which are structurally similar and have been most extensively characterized, as well as a third isoform HIF-3α, which exists as multiple splice variants, some of which can actually inhibit HIF-1α and HIF-2α activities (Makino et al., 2001). For the purpose of this review, the focus will be mainly on HIF-1α, as this particular isoform has been most extensively investigated in the heart, and only a brief mention will be made of HIF-2α, the role of which has only recently been explored in the heart.

2 . The discovery of hypoxia-inducible factor

3 . Regulation of hypoxia‐inducible factor 1α activity

In 1992, Semenza and Wang (1992) first discovered in Hep3B human hepatoma nuclear extracts, a 50 nucleotide nuclear factor induced under hypoxic conditions, which bound to the oxygen-responsive enhancer element of the erythropoietin (EPO) gene resulting in the transcription of the latter. The seminal discovery of the ‘hypoxia-inducible enhancer’ as it was originally termed, which was subsequently termed ‘hypoxia-inducible factor 1’ (HIF-1) in a subsequent publication by the same authors, first implicated HIF-1 as a general transcriptional regulator of gene expression in response to hypoxia (Wang & Semenza, 1993). Its discovery provided the identity of the critical oxygen-sensitive transcription factor, responsible for orchestrating the body's protective response to hypoxia. To date, HIF transcription factors have been shown to induce

The activity of HIF-1α within the cell is oxygen-sensitive, decreasing in response to normoxia and increasing under hypoxic conditions. Jiang et al. (1996) demonstrated in human HeLa cells that HIF-1α levels increase exponentially with a fall in oxygen concentration, with a half-maximal response occurring at 1.5–2.0% O2, and maximal HIF-1α levels induced at b0.5% O2. Under normoxic conditions, the hydroxylation of HIF-1α at two specific proline residues in the oxygen-dependent degradation domain by the prolyl-4-hydroxylase domain-containing enzymes (PHD) targets HIF-1α for polyubiquitination and proteosomal degradation by the von Hippel–Lindau tumor suppressor protein (VHL) (see Fig. 1) (Maxwell et al., 1999; Ivan et al., 2001). Under hypoxic conditions, PHD activity is inhibited, sparing HIF-1α from polyubiquitination

1 . Introduction

Fig. 1. HIF activity can be regulated by several different mechanisms including oxygen-dependent pathways such as PHD and VHL, and oxygen-independent factors such as Sirtuins, protein kinases, mitochondrial ROS and microRNAs.

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and proteosomal degradation, thereby allowing it to accumulate and translocate to the nucleus where it dimerizes with HIF-1β, and binds to the hypoxia-responsive element sequences of target gene promoters (see Fig. 1). In the nucleus the transcriptional activity of the HIF-1 heterodimer is regulated by the hydroxylase, factor inhibiting HIF-1 (FIH-1) (Mahon et al., 2001). 3.1 . The prolyl hydroxylase domain-containing enzymes (PHD) The hydroxylation of HIF-1α by PHD is an oxygen-regulated enzymatic reaction which also requires iron and α-ketoglutarate. The oxygen-sensitivity of this hydroxylation process therefore confers the redox sensitivity of HIF-1α activity in the cell. Three functionally active isoforms of PHD have been identified with evidence demonstrating selective regulation of PHD isoforms by specific HIF-α isoforms. For example, PHD-2, the most ubiquitously expressed of the PHDs is specifically induced by HIF-1α whereas PHD-3 is responsive to both HIF-1α and HIF-2α (Appelhoff et al., 2004). All 3 PHD isoforms are present in the heart, but the most abundant PHD isoforms under basal conditions are PHD-2 and PHD-3 (Willam et al., 2006). In response to either myocardial ischemia or infarction, mRNA expression of both PHD-2 and PHD-3 is increased (Willam et al., 2006). 3.2 . von Hippel–Lindau protein (VHL) The hydroxylation of HIF-1α by PHD under normoxic conditions facilitates the binding of the von Hippel–Lindau protein (VHL), which together with elongin B, elongin C, Rbx1 and Cul2 then forms the E3 ubiquitin ligase complex, which subsequently targets HIF-1α for proteolytic degradation by the ubiquitin-proteasome pathway (Maxwell et al., 1999; Ivan et al., 2001). 3.3 . Factor inhibiting hypoxia‐inducible factor 1 (factor inhibiting HIF‐1) While PHD and VHL serve to regulate HIF-1α stability, there is another control switch in the C-terminal of HIF-1α regulating its transcriptional activity. In the presence of oxygen, hydroxylation by factor inhibiting HIF-1 (FIH-1), of an asparagine residue (Asn803 and Asn851 in human HIF-1α and HIF-2α, respectively) takes place which reduces the interaction between HIF-1α and the CH-1 domain of the transcriptional co-activator, p300/CBP, that is essential for activation of target genes (Lando et al., 2002). Importantly, a specific subset of genes transcribed by HIF-1α appears to be particularly sensitive to this O2-dependent regulation by FIH-1 (Mahon et al., 2001; Dayan et al., 2006). 3.4 . LIM domain-containing protein 1 (LIMD1) LIMD1 is a tumor suppressor and member of the Ajuba family of LIM domain-containing proteins, possessing three conserved carboxyterminal LIM domains as well as a proline/serine-rich amino-terminal (pre-LIM) region. Foxler et al. (2012) have recently demonstrated through co-immunoprecipitation studies that PHD bound to the pre-LIM region whereas VHL bound to the LIM-domain region of LIMD1, forming a PHD–LIMD1–VHL axis. As VHL does not associate directly with PHDs, the presence of this complex enables VHL and PHD to associate together, thereby enhancing the local concentration, enabling more efficient sequential modification and degradation of HIF-1α (Foxler et al., 2012). The authors demonstrated that the overexpression of LIMD1 affected HIF-1 transcriptional activity in a negative manner whereas depletion of LIMD1 in either normoxia or hypoxia led to increased HIF activity (Foxler et al., 2012). Interestingly, this PHD2-LIMD1-VHL complex may be functional in hypoxia, potentially as part of the adaptive response to chronic hypoxia where a significant proportion of cellular HIF-1α constantly undergoes LIMD1-dependent

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degradation, which is dependent on hydroxylase and proteasomal activities (Foxler et al., 2012). 3.5 . Regulation of hypoxia‐inducible factor 1α by protein kinase B and extracellularly‐regulated kinase‐1 and ‐2 HIF-1α activity has also been demonstrated to be under the regulation of protein kinase phosphorylation. In 1999, Richard et al. (1999) first demonstrated in non-cardiac cells that HIF-1α could be phosphorylated by the mitogen-activated protein kinase (MAPK), Erk1/2, but not p38 or Jun MAPK. Importantly, Erk1/2-induced phosphorylation of HIF-1α was linked to HIF-1α mediated transcription activity (Richard et al., 1999). Subsequent experimental studies have reported similar findings with Akt, showing that the activation of the PI3K-Akt pathway using either, insulin, epidermal growth factor or PTEN suppression resulted in HIF-1α activation and downstream transcription of vascular endothelial growth factor (VEGF) in cancer cell lines (Zundel et al., 2000; Jiang et al., 2001). Interestingly, the protein kinases, Akt and Erk1/2, have both been implicated as mediators of cardioprotection elicited by ischemic preconditioning (see Section 5.3.2). Despite various studies demonstrating the importance of protein kinase cascade in HIF-1 regulation, there have been studies which have shown that protein kinases are dispensable for induction and activation of HIF-1. For example, p38 mitogen-activated protein kinase deficient cells were found to be able to activate HIF-1 in response to anoxia or iron chelators during normoxia, although the same cells failed to activate HIF-1 under hypoxic conditions, suggesting that p38 mitogenactivated protein kinase is required for hypoxia signaling but not during anoxia (Emerling et al., 2005). In a separate study, Sutton et al. (2007) found that ERK1/2 signaling is required for HIF-1 induction in response to insulin growth-factor 1 yet dispensable for the induction and activation of HIF-1 in response to hypoxia. These discrepancies highlight the need for more in-depth studies for ascertaining the role of protein kinases in regulating HIF-1. 3.6 . The role of mitochondria in the regulation of hypoxia‐inducible factor 1α There is a growing body of evidence implicating mitochondria as potential regulators of HIF-1α stabilization under conditions of hypoxia. This involvement may be through two potential mechanisms. The first one is that mitochondrial reactive oxygen species (ROS) generated under conditions of hypoxia stabilize HIF-1α (Chandel et al., 1998, 2000). In this regard, Chandel and colleagues (Chandel et al., 1998, 2000; Bell et al., 2007) have reported in human Hep3B cells, that under hypoxic conditions (1.5% O2), complex III of the mitochondrial electron transport chain (ETC) generated ROS which then stabilized HIF-1α. These authors found that rho-zero cells, which are deficient in mitochondrial DNA, 1 were unable to stabilize HIF-1α under hypoxic conditions, whereas exogenous hydrogen peroxide under normoxic conditions did stabilize HIF-1α in these cells (Chandel et al., 1998, 2000). Furthermore, the mitochondrial ROS generated under hypoxic conditions were demonstrated to stabilize HIF-1α by preventing hydroxylation of HIF-1α by PHD. In contrast to these findings, Vaux et al. (2001) found that rho-zero cells and other mutant cell-lines deficient in components of the mitochondrial ETC were able to stabilize HIF-1α under conditions of hypoxia (0.1% O2), suggesting that the ETC was not required for HIF-1α stabilization. The difference in findings may have been due to the degree of hypoxia used to stimulate HIF-1α stabilization with moderate hypoxia (1.5% O2) used by Chandel et al. (1998, 2000), and severe hypoxia (0.1% O2) utilized by Vaux et al. (2001). In this regard, Schroedl et al. (2002) went on to demonstrate that rho-zero cells were able to stabilize HIF-1α under anoxic but not hypoxic conditions, suggesting that mitochondrial ROS can stabilize HIF-1α under hypoxic conditions, but not anoxic conditions. The regulation of HIF-1α by mitochondrial ROS has been recently implicated in the tumor-suppressive effects of the

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mitochondrial deacetylase, Sirtuin 3 (see Section 3.7) (Bell et al., 2011; Finley et al., 2011). The second model implicating mitochondria as regulators of HIF-1α activity has proposed that mitochondrial oxygen consumption results in less oxygen being available for activating PHD in the cytosol, leading to reduced PHD activity and HIF-1α stabilization (Hagen et al., 2003; Doege et al., 2005). A recent study by Yang et al. (2012) has suggested that ROS and other mitochondrial mechanisms work in a concerted manner to contribute to HIF-1α stabilization in hypoxia. In this elegant study, the authors described the role of CHCHD4, a protein localized to the mitochondria in regulating HIF-1α in hypoxia (Yang et al., 2012). Knockdown of CHCHD4 was found to decrease basal oxygen consumption rate and cellular ATP levels as well as inhibiting HIF-1α protein induced in response to hypoxia, whereas overexpression of CHCHD4 led to increased HIF-1 protein stability during hypoxia (Yang et al., 2012). Interestingly, CHCHD4 knockdown did not significantly affect HIF-1α induced in response to deferoxamine mesylate (DFX). To investigate the role of ROS in HIF-1α stabilization, the use of antioxidant, N-acetyl cysteine, was found to partially block HIF-1α protein levels induced in hypoxia demonstrating that ROS is important to a certain extent in stabilizing HIF-1α (Yang et al., 2012). However, the authors found that the enhanced stabilization of HIF-1α protein induced by CHCHD4 overexpression in hypoxia was not sensitive to N-acetyl cysteine treatment (Yang et al., 2012). On the contrary, inhibition of complex IV activity using sodium azide blocked CHCHD4-mediated enhancement of HIF-1α protein levels in hypoxia even when CHCHD4 was overexpressed suggesting that CHCHD4 is linked to cytochrome c and COX implicating the importance of an intact mitochondria in regulating HIF (Yang et al., 2012). Clearly, it is evident that there remains much to be done to fully elucidate the involvement of both ROS and mitochondria on HIF regulation. What is especially important is to define the oxygen levels used to study the role of mitochondria as clear as possible since the oxygen concentration is a critical determinant as to whether mitochondria is required for modulation of HIF-1 stability and activity. Advancement of genetic techniques will also allow a more in-depth analysis in ascertaining the role of mitochondria on HIF regulation where specific components of the mitochondria in various cell types can be targeted for knockdown or overexpression under differing oxygen levels. 3.7 . MicroRNAs as regulators of hypoxia‐inducible factor 1α MicroRNAs (miRNAs) are a class of single-stranded non-coding RNAs, which negatively regulate gene expression. A number of experimental studies have investigated the role of miRNAs in regulating HIF-1α stabilization, and the downstream activation of its target genes. Conversely, other studies have implicated HIF-1α to also act as a regulator of miRNA function. Rane et al. (2009) have demonstrated using neonatal cardiomyocytes that the stabilization of HIF-1α and the subsequent activation of pro-apoptotic proteins such as p53, during sustained hypoxia, required the down-regulation of miRNA-199a. This finding makes the suppression of miRNA-199a to stabilize HIF-1α in the heart, a potential therapeutic strategy for cardioprotection in which both hypoxic preconditioning (HPC) or knockdown of miR-199a was able to activate Sirtuin-1 (a class III histone deacetylase), inhibit PHd-2 and stabilize HIF-1α in neonatal cardiomyocytes under normoxia. Interestingly, both HPC and knockdown of miR-199a induced the translocation of HIF-1α to mitochondria, where mitochondrial potential was preserved following simulated IRI (Rane et al., 2009). In a recent experimental study using human endothelial cells, hypoxia was demonstrated to increase miRNA-424 expression, which targeted cullin 2, part of the E3 ubiquitin ligase responsible for degrading HIF-1α, thereby stabilizing HIF-1α isoforms (Ghosh et al., 2010). Interestingly, apart from stabilizing HIF-1α, several miRNAs have been discovered to negatively regulate HIF-1α expression. For example, miRNA-20b and miRNA-519c have

both been found to target the 3′ untranslated region of HIF-1α, and the overexpression of either was found to be associated with decreased protein levels of HIF-1α (Lei et al., 2009; Cha et al., 2010). 3.8 . Sirtuins as regulators of hypoxia‐inducible factor 1α The Sirtuins are a group of nicotinamide adenine dinucleotide (NAD +)-dependent redox-sensitive enzymes which deacetylate proteins as part of the cellular response to stress. Recent evidence suggests that the Sirtuins (Sirt) can deacetylate and regulate HIF-1α and HIF-2α activities, providing cross-talk between the redox-sensitive and oxygen-sensitive responses of the cell. In 2009, Rane et al. (2009) provided the first evidence linking Sirt1 to HIF-1α, by demonstrating that the down-regulation of miRNA-199a stabilized HIF-1α in neonatal cardiomyocytes through the activation of Sirt1 and the inhibition of PHD-2. However, a subsequent study has suggested that Sirt1 is able to interact with and directly deacetylate HIF-1α at Lys674, thereby blocking p300 recruitment, and suppressing the transcription of HIF-1α target genes (Lim et al., 2010). However, under conditions of hypoxia, NAD+ levels fall, resulting in down-regulation of Sirt1 and the subsequent acetylation and activation of HIF-1α (Lim et al., 2010). However, the story is complicated by the fact that during hypoxia, Sirt1 activity may in itself be regulated by HIF-1α, providing a two way communication between Sirt1 and HIF-1α (Chen et al., 2011). Two very recently published studies (Bell et al., 2011; Finley et al., 2011) have provided data suggesting that the mitochondrial Sirtuin, Sirt3, may suppress HIF-1α stability by attenuating the mitochondrial production of ROS, an effect which was demonstrated in these studies to attenuate tumor growth. Sirt6 has also been reported to act as a suppressor of HIF-1α with Sirt6-deficient cells showing HIF-1α stabilization and increased glucose uptake, up-regulation of glycolysis, and reduced mitochondrial respiration (Zhong et al., 2010). In terms of HIF-2α regulation, Dioum et al. (2009) have demonstrated that in response to hypoxia, Sirt1 interacted with and deacetylated HIF-2α, allowing it to be stabilized and transcribe its downstream gene, EPO. 4 . Hypoxia‐inducible factor 1α activation and the heart There are a number of situations in which the heart is subjected to conditions of hypoxia and in which myocardial HIF-1α would be expected to be stabilized. These include during embryonic development, in response to acute ischemia–reperfusion injury (IRI), and adaptation to heart failure. In this review article the focus will be on HIF-1α stabilization in the setting of acute IRI and its role as a target for cardioprotection. One of the first experimental studies to investigate HIF-1α stabilization in cardiac cells was in 1999 by Nguyen and Claycomb (1999). Using the HL-1 cardiac cell line, these authors demonstrated that hypoxia (1% O2) induced both the mRNA expression of HIF-1α and the protein expression of HIF-1α after 4 h. Upregulated mRNA expression of HIF-1α was confirmed in both neonatal hearts and cardiomyocytes subjected to chronic hypoxia by Jung et al. (2000). Later that same year, Lee et al. (2000) were the first to demonstrate increased myocardial HIF-1α mRNA expression in response to acute ischemia or infarction in human myocardial biopsies harvested from patients undergoing CABG surgery, providing the first evidence of the relevance of the HIF-1α signaling pathway to man. In the adult rat heart, Kim et al. (2002) reported increased mRNA expression of myocardial HIF-1α in both the ischemic and non-ischemic zones, 12 h following permanent left anterior descending (LAD) coronary artery ligation. In that study mechanical stretch was also shown to stabilize myocardial HIF-1α (Kim et al., 2002). The first experimental study to implicate HIF-1α as a mediator of cardioprotection was by Shyu et al. (2002), who demonstrated that following permanent LAD ligation in the rat heart, the injection of plasmid DNA encoding for either VEGF or HIF-1α into the infarcted zones, limited MI size, increased capillary

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density and augmented regional myocardial blood flow. Since then a large number of studies have implicated HIF-1α as a critical mediator of cardioprotection. 5 . Hypoxia‐inducible factor 1α as a mediator of ischemic preconditioning The investigation of HIF-1α mediated cardioprotection has been closely linked to ischemic preconditioning (IPC), an endogenous phenomenon, in which one or more brief episodes of non-lethal myocardial ischemia and reperfusion, protects the heart against a subsequent myocardial infarction (Murry et al., 1986; Yellon & Downey, 2003). The IPC stimulus induces two distinct windows of protection, the “first window of protection” or “classical” IPC manifests immediately following the IPC stimulus and lasts up to 3–4 h, following which the protective effect wanes and re-appears 12–24 h later and lasting up to 72 h (termed the “Second Window Of Protection” [SWOP] or delayed IPC) (Kuzuya et al., 1993; Marber et al., 1993; Hausenloy & Yellon, 2010). The SWOP or delayed IPC requires the transcriptional activation of specific cardioprotective genes, raising the possibility that HIF-1α may act as a mediator of delayed IPC to transcribe cardioprotective genes in this setting. As a result, many of the studies implicating HIF-1α as a mediator of cardioprotection have used experimental models of SWOP or delayed IPC. However, more recent evidence suggests that HIF-1α may also mediate the acute phase of IPC. Finally, ischemic postconditioning, an endogenous intervention in which reperfusion is interrupted by short-lived episodes of myocardial ischemia, has been reported to enhance HIF-1α activity in adult rat hearts subjected to acute IRI, although the significance of this finding was not explored in this study (Zhao et al., 2009). 5.1 . Hypoxia‐inducible factor 1α as a mediator of delayed ischemic preconditioning One of the first experimental studies to implicate HIF-1α as a potential mediator of delayed IPC was by Cai et al. (2003). These authors demonstrated that mice partially deficient in HIF-1α (HIF-1α +/−) were resistant to the infarct-limiting effects of intermittent hypoxia and reoxygenation (the delayed IPC stimulus) administered 24 h earlier, when compared to wild-type mice, suggesting that HIF-1α was required as a mediator of delayed IPC. In neonatal rat ventricular cardiomyocytes, Date et al. (2005) demonstrated that hypoxic preconditioning (HPC) increased protein expression of HIF-1α immediately following the preconditioning stimulus, confirming the activation of HIF-1α in response to the preconditioning stimulus. However, the mechanism through which the IPC stimulus actually stabilizes HIF-1α is unclear, although a recent experimental study suggests that the IPC-mediated production of mitochondrial ROS may be absent in mice partially deficient in HIF-1α (Cai et al., 2008) (see Section 5.3.1.). 5.2 . Hypoxia‐inducible factor 1α as a mediator of acute cardioprotection There is emerging evidence that HIF-1α activation may contribute to the acute cardioprotective effect elicited by the ‘first window of protection’ or classical IPC. In 2008, Cai et al. (2008) subjected wild type mice and mice partially deficient in HIF-1α (HIF-1α+/−) to IPC (10 min ischemia/5 min reperfusion or two-5 min cycles of ischemia/ reperfusion) immediately prior to 30 min ischemia followed by 30 min reperfusion on the Langendorrf-apparatus. As expected in the wildtype hearts IPC resulted in a reduction in MI size, preservation of LV systolic function, and less apoptotic cell death (Cai et al., 2008). However, hearts deficient in HIF-1α were found to be resistant to the beneficial effects of IPC, suggesting that HIF-1α may act as a mediator of cardioprotection in the acute phase of IPC (Cai et al., 2008). Because the mice were partially deficient in HIF-1α from birth, the mechanism

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underlying the resistance to IPC must be due to a chronic defect in the signaling pathway. However, a subsequent study has investigated the effects of acute HIF-1α ablation on IPC protection. The mediatory role of HIF-1α in classical IPC was described in an elegant study by Eckle et al. (2008), who demonstrated stabilization of HIF-1α in the cytosol and nucleus of murine hearts within 3 h of the IPC stimulus. These authors then demonstrated that the intravenous infusion of HIF-1α siRNA into the left ventricle of IPC-treated hearts maximally suppressed HIF-1α by 2 h, and this finding was associated with the abrogation of IPC cardioprotection (Eckle et al., 2008). These findings suggest that the acute phase of IPC is mediated by the stabilization of HIF-1α which was reported to occur about 30 min following the IPC stimulus (Eckle et al., 2008). Whether HIF-1α stabilization during the acute phase of IPC has sufficient time to protect the myocardium against IRI through the transcription of new proteins is not known. It may be well that HIF-1α exerts direct effects on mitochondrial function which are independent on transcription and therefore compatible with the acute phase of IPC. Despite the growing body of evidence implicating the role of HIF-1 in preconditioning, Czibik et al. (2008) reported in a study published in 2008 that ischemic preconditioning improved cardiac function in patients undergoing stable coronary artery bypass grafting with stable or unstable angina which involved nuclear factor kappa β-regulated genes but not HIF-regulated genes. Clearly, further work is required to elucidate the mechanism through which HIF-1α stabilization mediates the acute phase of IPC. 5.3 . Hypoxia‐inducible factor 1α and the signaling pathways underlying ischemic preconditioning The actual mechanism through which IPC protects the heart against acute IRI remains unclear. The current paradigm suggests that the IPC stimulus generates autocoids within the heart (such as adenosine, bradykinin, opioids) which activate their respective G-protein coupled receptors on the cardiomyocyte plasma membrane, resulting in the recruitment of a number of complex signaling pathways (including PKC, PTEN, PI3K-Akt, MEK1/2-Erk1/2, eNOS, NO, PKG, signaling ROS) many of which converge on the mitochondria and prevent mitochondrial dysfunction during acute IRI (Yellon & Downey, 2003). The data from above suggests that HIF-1α may act as a mediator of IPC cardioprotection through its interaction with different components of the signaling pathways underlying IPC. 5.3.1 . Hypoxia‐inducible factor 1α and reactive oxygen species signaling in ischemic preconditioning Murry et al. (1988) first demonstrated that antioxidants could abolish the protective effect elicited by IPC, implicating for the first time a role for ROS as a critical mediator of IPC, a finding which was later supported by several experimental studies (Tanaka et al., 1994; Chen et al., 1995; Baines et al., 1997). It was subsequently demonstrated that the administration of exogenous ROS could mimic IPC protection (Tritto et al., 1997), and that the source of the ROS was complex III (Vanden Hoek et al., 1998) and/or complex I (Andrukhiv et al., 2006) of the mitochondrial ETC. The mitochondrial ROS is believed to activate pro-survival protein kinases such as PKC (Baines et al., 1997), Erk1/2 (Samavati et al., 2002), and p38 MAPK (Yue et al., 2001) which then act as mediators of the IPC signal. The actual mechanism through which IPC generates mitochondrial ROS remains unclear, although the opening of the ATP-sensitive mitochondrial potassium (MitoKATP) channel (Andrukhiv et al., 2006) and the transient opening of the mitochondrial permeability transition pore (mPTP) (Hausenloy et al., 2004, 2010) have both been implicated. Cai et al. (2008) investigated the role of HIF-1α and mitochondrial ROS signaling in the acute phase of IPC, in which cardioprotection is elicited immediately and lasts for 2–3 h. These authors demonstrated that mitochondrial ROS production, PTEN oxidation and Akt phosphorylation (all critical events in IPC signaling) (Tong et al., 2000; Cai &

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Semenza, 2005) were impaired in hearts partially deficient in HIF-1α. These findings suggest that HIF-1α is required to generate the mitochondrial ROS, which mediates IPC cardioprotection, although the reason why HIF-1α is needed for this process is not known. How these findings tally with the emerging data implicating mitochondrial ROS as being a stabilizer of HIF-1α is unclear (Chandel et al., 1998, 2000; Schroedl et al., 2002; Bell et al., 2007). Whether the mitochondrial ROS, generated in response to IPC, can also stabilize HIF-1α, which then mediates cardioprotection, is also unknown. Further experimental work is required to elucidate the exact interplay of HIF-1α and mitochondrial signaling ROS, especially in the context of IPC signaling. 5.3.2 . Hypoxia‐inducible factor 1α and pro-survival protein kinase signaling in ischemic preconditioning The activation of pro-survival protein kinases such as PKC, Erk1/2 and Akt, in response to the IPC stimulus, but prior to the index ischemia, is critical in the signaling pathway underlying IPC (Hausenloy & Yellon, 2006). Furthermore, the kinases, Akt and Erk1/2, have been implicated as upstream regulators of HIF-1α stabilization in non-cardiac cells (see Section 3.4) (Richard et al., 1999). The interaction between HIF-1α and this signal transduction pathway in the setting of both classical and delayed IPC has been the subject of investigation. Using neonatal rat cardiomyocytes subjected to simulated IRI, Liu et al. (2003), implicated Erk1/2 mediated phosphorylation of HIF-1α in the SWOP elicited by hypoxic preconditioning (HPC). These authors demonstrated that treatment with the MEK1/2 inhibitor, PD98059, during the HPC stimulus, abolished both the phosphorylation of HIF-1α and the cardioprotective effects of HPC, 24 h later. In terms of the acute phase of IPC, Eckle et al. (2008) have found that the stabilization of HIF-1α in the adult murine heart induced by IPC at 2 h was abrogated in the presence of a PKC inhibitor, suggesting that PKC was also upstream of HIF-1α, although the mechanism linking the two was not explored.

5.3.3 . Hypoxia‐inducible factor 1α and adenosine signaling in ischemic preconditioning Liu et al. (1991) first demonstrated that the cardioprotection elicited by IPC could be abolished by pharmacological inhibition of adenosine, and could be recapitulated using an adenosine agonist to mimic IPC, suggesting for the first time that IPC was a receptor-mediated effect. HIF-1α has been implicated as a mediator of hypoxia-induced activation of the ecto-5′-nucleotidase, CD73 (an enzyme which mediates extracellular adenosine generation) (Thompson et al., 2004) and the adenosine A2B receptor (A2BR) (Kong et al., 2006) in endothelial and epithelial cells. Furthermore, the human A2BR promoter has been reported to have a functional HIF-1α binding site (Kong et al., 2006). On this background, Eckle et al. (2008) have recently investigated the interaction between HIF-1α stabilization and the adenosine signaling pathway in the acute phase of IPC. These authors found that IPC increased the extracellular accumulation of adenosine in adult murine hearts, and this effect was shown to be associated with the HIF-1α dependent mRNA and protein up-regulation of CD73 and the A2BR (Eckle et al., 2008). The stimulatory effect of IPC on CD73 and A2BR was recapitulated using either DMOG or PHD-2 siRNA to stabilize HIF-1α (Eckle et al., 2008). The PHD inhibitor, DMOG, failed to increase cardiac adenosine and limit MI size in mice deficient in CD73 (Eckle et al., 2008). Finally, it was found that mice deficient in A2BR were resistant to the infarct-limiting effects of either DMOG or PHD-2 siRNA (Eckle et al., 2008). Taken altogether, these findings suggest that HIF-1α dependent adenosine signaling through the C73-A2BR pathway is essential for the acute phase of IPC (Eckle et al., 2008). The role of HIF-1α as a mediator of cardioprotection has been investigated using a variety of experimental strategies to stabilize HIF-1α under normoxic conditions. These have included both genetic and pharmacological approaches and are reviewed in the following sections (see Fig. 2).

Fig. 2. Short-term stabilization of HIF-1 in the heart has beneficial effects including protection against acute IRI, the mediation of IPC and possibly IPostC. Therapeutically this can be achieved using either genetic or pharmacological activation of HIF. However, the long-term stabilization of HIF-1 in the heart may be detrimental, and this may be treated by HIF-1 inhibitors.

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6 . Genetic strategies for stabilizing hypoxia‐inducible factor 1α to induce cardioprotection 6.1 . Genetic over-expression of hypoxia‐inducible factor 1α One of the first experimental studies to investigate the effect of HIF-1α over-expression on cardioprotection was by Date et al. (2005), who demonstrated that constitutive over-expression of HIF-1α in neonatal rat cardiomyocytes, 48 h prior to an episode of simulated lethal IRI reduced cell death, and this protective effect was associated with the mRNA expression of known HIF-1α target genes, VEGF, GLUT-1, GLUT4 and iNOS. Later that year, Kido et al. (2005) investigated the effect of genetically over-expressing HIF-1α in the adult murine heart. These authors found that the constitutive over-expression of HIF-1α in the heart protected it against in situ acute IRI as evidenced by a reduction in MI size and preserved cardiac function at 4 weeks, when compared to wild-type mice, although there was no difference in acute MI size at 24 h (Kido et al., 2005). The beneficial effects of HIF-1α over-expression were associated with increased capillary density and myocardial expression of VEGF and iNOS in the infarcted and peri-infarcted zones (Kido et al., 2005). Whether the hearts over-expressing HIF-1α had a specific phenotype, which may have been expected, were not commented upon in this study. Recent data by Czibik et al. (2009, 2011) has investigated in vivo gene delivery of HIF-1α as a therapeutic strategy for cardioprotection. These authors reported that the injection of DNA for HIF-1α into the quadricep muscles of mice, 3 days prior to MI, resulted in significant limitation in MI size and less adverse LV remodeling at 4 weeks (Czibik et al., 2009, 2011). The mechanism underlying cardioprotection was attributed to the local activation of hemoxygenase-1 (HO-1) within the muscle, and the subsequent release of the anti-oxidant bilirubin (a haem breakdown product), into the blood-stream (Czibik et al., 2009, 2011). The findings from this intriguing study suggest that the myocardium may be remotely protected against acute IRI by stabilizing HIF-1α in an organ or tissue away from the heart (Czibik et al., 2009, 2011), drawing an obvious analogy with the endogenous cardioprotective phenomenon of delayed remote ischemic preconditioning, in which the application of brief episodes of ischemia and reperfusion to an organ or tissue away from the heart renders the myocardium tolerant to acute IRI, 24 h later (Hausenloy & Yellon, 2008).

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PHD-2 mRNA (8% in the heart), on susceptibility to acute IRI (Hyvarinen et al., 2010). When subjected to acute IRI on a Langendorrf-apparatus, hearts deficient in PHD-2 displayed better recovery of cardiac function and coronary flow rate, had less myocardial injury (as measured by lactate dehydogenase), and better preserved ATP levels when compared to WT mice. Finally, a recent study has reported that cardiomyocyte-specific deletion of PHD-2 resulted in hearts with smaller MI size, less apoptotic cardiomyocyte death, and preserved LV systolic function after 3 weeks, following permanent LAD ligation (Holscher et al., 2011). Recently, the role of PHD-1 as a target for cardioprotection has been investigated by Adluri et al. (2011), who demonstrated that mice deficient in PHD-1 had increased myocardial protein expression of HIF-1α, a finding which was associated with a smaller MI size and less apoptotic cell death following IRI, when compared to wild littermate controls. In terms of cardioprotection and of direct clinical relevance, the experimental data suggests that the diabetic heart may be resistant to IPC and other cardioprotective maneuvers, a proposal which has been recently investigated with respect to HIF-1α stabilization as a cardioprotective strategy. Using Leprdb diabetic mice, Natarajan et al. (2008) demonstrated that HIF-1α stabilization by PHD-2 siRNA reduced MI size and preserved LV systolic function in isolated perfused hearts, showing that the diabetic heart was amenable to HIF-1α-mediated cardioprotection. These experimental studies implicate PHD, in particular the PHD-2 isoform, as a therapeutic target for protecting the heart against acute IRI. 6.3 . Genetic ablation of von Hippel–Lindau protein We have recently demonstrated that the acute cardiomyocyte-specific ablation of VHL in the adult murine heart enhanced myocardial expression of HIF-1α, and reduced MI size following in vivo acute IRI (Ging et al., unpublished). Isolated cardiomyocytes deficient in VHL when subjected to simulated IRI had attenuated cell death, produced less ROS, and had reduced susceptibility to mPTP opening, effects which were abrogated with the pharmacological inhibition of HIF-1α (Ging et al., unpublished). These findings suggest that the acute VHL ablation and HIF-1α stabilization protect the adult murine heart against acute IRI through its beneficial effects on mitochondrial function, although the actual mechanism remains to be explored (Ging et al., unpublished). Although, the acute ablation of VHL appears to be cardioprotective, chronic VHL deletion and long-term HIF-1α stabilization may actually be detrimental to the heart (Lei et al., 2008) (see Section 10).

6.2 . Genetic ablation of prolyl hydroxylase domain‐containing enzyme The heart contains all 3 isoforms of PHD although the predominant isoforms are PHD-2 and PHD-3. One of the first experimental studies to investigate the effect of genetic PHD ablation on cardioprotection was by Natarajan et al. (2006). The authors demonstrated in mice that the in vivo administration of siRNA to PHD-2 resulted, 24 h later, in a significant reduction in MI size in isolated perfused hearts (31.6% vs 9.7% of the area at risk) (Natarajan et al., 2006). The infarct-limiting effects of PHD-2 ablation were blocked by 1400 W (a pharmacological inhibitor of iNOS), and were associated with a reduction in myocardial mRNA expression of PHD-2, an elevation in cardiac nuclear HIF-1α, and enhanced cardiac expression of iNOS mRNA. Importantly, mice deficient in iNOS were shown to be resistant to the infarct-limiting effects of PHD-2 ablation (Natarajan et al., 2006). In a subsequent study by the same research group, the cardioprotective effect of PHD-2 siRNA was associated with a significant reduction in the release of pro-inflammatory chemokines (Natarajan et al., 2007). Eckle et al. (2008) demonstrated that an intravenous infusion of siRNA to PHD-2 but not PHD-1 or 3 into the left ventricle of an adult murine heart resulted in stabilization of cardiac HIF-1α after 2 h, an effect which was associated with infarct-limitation. A recent experimental study has investigated the effects of whole body deletion of PHD-2, using a transgenic mouse line that expresses decreased amounts of wild-type

7 . Pharmacological strategies for stabilizing hypoxia‐inducible factor 1α to induce cardioprotection 7.1 . Pharmacological inhibition of prolyl hydroxylase domain‐containing enzyme Under normoxic conditions the hydroxylation of specific proline residues on HIF-1α by PHD is critical for the degradation of HIF-1α. Therefore, the pharmacological inhibition of PHD to stabilize HIF-1α under normoxic conditions is a potential therapeutic strategy for cardioprotection. However, many of the agents used to inhibit PHD are not specific, yet alone specific for a particular isoform. 7.1.1 . Cobalt chloride as a pharmacological prolyl hydroxylase domain‐containing enzyme inhibitor Cobalt chloride (CoCl2) has been used to chemically induce hypoxia in experimental models investigating erythropoiesis and angiogenesis in vivo. A number of experimental studies have reported that CoCl2 could protect the heart and other organs from acute IRI (Conrad et al., 1984; Endoh et al., 2000). The first experimental study to implicate HIF-1α as a potential mediator of CoCl2-induced cardioprotection was by Xi et al. (2004). These authors demonstrated using mice that a single dose of CoCl2 reduced MI size 24 h later, and that this cardioprotective

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effect was associated with the up-regulation of HIF-1α and activator protein-1 (AP-1) (Xi et al., 2004). In neonatal piglets, pre-treatment with CoCl2 attenuated myocardial apoptosis following acute IRI, a finding which was associated with enhanced cardiac expression of phosphorylated Akt and the anti-apoptotic protein, Bcl-2, as well as decreased production of the pro-apoptotic protein, Bax (Kerendi et al., 2006). The mechanism through which CoCl2 stabilizes HIF-1α is unclear, although it has been attributed to the antagonism of Fe 2+ (an essential co-factor with oxygen for the prolyl hydroxylation of HIF-1α) or the production of ROS (Xi et al., 2004). 7.1.2 . Desferroxamine as a pharmacological prolyl hydroxylase domain‐containing enzyme inhibitor The iron chelator, desferroxamine (DFO), has been reported to protect the heart against acute IRI by acting as a free radical scavenger (Reddy et al., 1989). However, the chelation of iron by DFO has also been demonstrated to stabilize HIF-1α, by preventing the hydroxylation of HIF-1α by inhibiting PHD. Dendorfer et al. (2005) found that a single intraperitoneal dose of DFO administered 2–3 days prior to IRI reduced MI size, and this cardioprotective effect was associated with the mRNA expression of activation of the HIF-1α target genes, aldose reductase and COX-2. Interestingly, the infarct-limiting effect of DFO was dependent on the generation of signaling ROS, although the relation between this finding and HIF-1α stabilization was not explored (Dendorfer et al., 2005). The cardioprotective effects of DFO were confirmed in a subsequent study by Philipp et al. (2006a) who demonstrated that DFO limited MI size in rabbit hearts when administered on the Langendorff-apparatus as a classical preconditioning agent. This protective effect was abrogated by pharmacological inhibitors of NO, PKG and ROS (Philipp et al., 2006a). Pre-treatment of isolated rabbit cardiomyocytes with DFO increased ROS production (an effect which was blocked by pharmacological inhibition and the mitochondrial KATP channel) (Philipp et al., 2006a). In this latter study of acute cardioprotection, whether HIF-1α or its target genes were actually contributing to cardioprotection was not tested. 7.1.3 . Dimethyloxalylglycine as a pharmacological prolyl hydroxylase domain‐containing enzyme inhibitor One of the first experimental studies to investigate the effect of pharmacological PHD inhibition as a therapeutic strategy for cardioprotection was by Ockaili et al. (2005). To inhibit PHD, these authors used dimethyloxalylglycine (DMOG), which is a non-specific PHD inhibitor. They administered DMOG to rabbits 24 h prior to in vivo IRI and demonstrated MI size reduction, attenuation of plasma IL-8, less polymorphonuclear neutrophil accumulation in the myocardium, and activation of myocardial heme-oxygenase-1 (HO-1) (Ockaili et al., 2005). One of the first experimental studies to investigate whether DMOG could induce acute cardioprotection was by Eckle et al. (2008), who demonstrated that a single intraperitoneal injection of DMOG resulted in maximal HIF-1α stabilization after 2 h, and this was associated with a significant reduced in MI size to a level comparable to classical IPC. Importantly, the reduction in MI size elicited by DMOG was completely abrogated in hearts pre-treated with siRNA to HIF-1α, confirming that the cardioprotective effect of DMOG was actually mediated by HIF-1α stabilization (Eckle et al., 2008). Finally, the cardioprotective effects of intraperitoneally administered DMOG 24 h prior to Langendorrf-perfusion have been confirmed in the rat heart, a finding which was associated with myocardial activation of HIF-1α and VEGF (Poynter et al., 2011). 7.1.4 . Ethyl-3,4-dihydroxybenzoate as a pharmacological prolyl hydroxylase domain‐containing enzyme inhibitor Ethyl-3,4-dihydroxybenzoate (EDHB) has been reported to be a specific cell-permeable PHD inhibitor that competitively binds to both the ascorbate- and α-ketoglutarate-binding sites of the prolyl hydroxylase active domain (Majamaa et al., 1986; Sasaki et al., 1987). Wright et al. (2003) reported that PHD inhibition with EDHB

protected neonatal murine cardiomyocytes against simulated ischemia (using cyanide and de-oxyglucose) and this protective effect was associated with enhanced protein expression of HIF-1α target genes GLUT-1, iNOS, and HO-1. Philipp et al. (2006a) found that pre-treatment of isolated rabbit cardiomyocytes with EDHB generated ROS, and this effect was abolished in the presence of pharmacological inhibitors of NO, PKG, the mitochondrial KATP channel, and the mitochondrial respiratory chain. Finally, pre-treatment of neonatal rat cardiomyocytes with EDHB or DMOG was found to reduce cell death following metabolic inhibition, an effect which was associated with improved mitochondrial function following metabolic inhibition (preserved mitochondrial membrane potential and higher ATP levels) (Sridharan et al., 2007). 7.1.5 . Novel prolyl hydroxylase domain‐containing enzyme inhibitors Novel compounds are starting to be developed which exhibit higher specificity to PHD. Recently, two new PHD inhibitors — TM6008 and TM6089 were introduced which can induce angiogenesis and protect organs against ischemia in vivo (Nangaku et al., 2007). In a more recent study, GSK360A, an orally active reversible inhibitor of all three isoforms of PHD (PHD-1 > PHD-2 = PHD-3) which acts by competing with the α-ketoglutarate site and has little or no activity in inhibiting FIH has been shown to confer beneficial effects on post-MI remodeling in a rat model of post-MI heart failure (Bao et al., 2010). To date, FG-2216 as well as another PHD inhibitor, FG-4592, are currently in phase II clinical trials for the treatment of anemia in chronic disease patients (www.clinicaltrials.gov NCT00456053 and NCT00761657). 8 . Mechanisms of hypoxia‐inducible factor 1α-mediated cardioprotection Up-regulation of HIF-1α during myocardial ischemia is associated with improved myocardial tolerance to acute IRI, and this is inherently due to synergistic cardioprotective effects afforded by various downstream HIF-1α target genes, many of which have been implicated as mediators of delayed IPC. These include HIF-1α a number of target genes including erythropoietin (Cai et al., 2003), heme oxygenase-1 (Ockaili et al., 2005), adiponectin (Natarajan et al., 2008), and inducible nitric oxide synthase (Natarajan et al., 2006). 8.1 . EPO as a mediator of hypoxia‐inducible factor 1α cardioprotection One of the first HIF-1α target genes to be implicated in cardioprotection was erythropoietin (EPO). Cai et al. (2003) demonstrated that an IPC stimulus comprising intermittent hypoxia and reoxygenation reduced MI size 24 h later in wild-type mice, but not in mice partially deficient in HIF-1α. The protective effect was shown to be associated with the production of EPO, which was found to be increased in the plasma of mice treated with IPC (Cai et al., 2003). Whether EPO was responsible for the cardioprotective effect of delayed IPC in this experimental setting was not demonstrated, although the protective effects of EPO have been well-established in the pre-clinical setting (Riksen et al., 2008). 8.2 . Inducible nitric oxide synthase as a mediator of hypoxia‐inducible factor 1α cardioprotection Takano et al. (1998) were the first to implicate iNOS as a mediator of delayed IPC. They found that the pharmacological inhibition of iNOS prior to acute IRI abolished the MI size limitation, elicited by an IPC stimulus administered 24 h earlier. The requirement for iNOS as a mediator of cardioprotection in delayed IPC was subsequently confirmed using mice deficient in iNOS (Guo et al., 1999). Several experimental studies have implicated iNOS as a target gene for HIF-1α mediated cardioprotection. In 2000, Jung et al. (2000) published the first study to investigate the role of HIF-1α on expression of

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iNOS in the heart under conditions of hypoxia. They found that subjecting the heart to chronic hypoxia increased iNOS mRNA expression, a response which was abolished if the HIF-1α binding site was mutated or deleted (Jung et al., 2000). Overexpression of constitutive HIF-1α in a transgenic mouse strain resulted in attenuated MI size and improved cardiac function 4 weeks after hearts were subjected to acute IRI, and these observations were linked to an increase in iNOS expression, establishing iNOS as a target gene in HIF-1α-mediated cardioprotection (Kido et al., 2005). Mice deficient in iNOS were found to be resistant to the delayed cardioprotective effect of the PHD inhibitor, CoCl2, suggesting that cardioprotection was dependent on the presence of iNOS (Xi et al., 2004). Finally, Belaidi et al. (2008) provided evidence of a direct interaction between HIF-1α and iNOS in preconditioned hearts, and in addition showed that treatment of rats with cadmium (a metal which induces HIF-1α degradation) abolished the cardioprotective effects of delayed IPC by preventing the stabilization of HIF-1α and activation of iNOS (Belaidi et al., 2008). 8.3 . Heme oxygenase‐1 as a mediator of hypoxia‐inducible factor 1α cardioprotection Another target gene of HIF-1α that is heme oxygenase-1 (HO-1), an enzyme which catalyzes heme oxidation to biliverdin, carbon monoxide, and free ferrous iron, has been reported to have antioxidant and anti-inflammatory effects. Cardiac specific overexpression of HO-1 in the adult murine heart has been reported to be cardioprotective, whereas mice deficient in HO-1 sustain larger MI sizes following acute IRI (Yet et al., 2001). Jancso et al. (2007) found that pharmacological preconditioning using adenosine, epinephrine or opioid in neonatal rat cardiomyocytes conferred delayed cardioprotection and increased the myocardial expression of HO-1, effects which were blocked by siRNA to HO-1. Ockaili et al. (2005) investigated the role of HO-1 as a downstream target gene of HIF-1α mediated cardioprotection. Pre-treatment of rabbits with the PHD inhibitor, DMOG, 24 h prior to acute IRI, resulted in a reduction in MI size, a finding which was associated with enhanced myocardial expression of HO-1, and an anti-inflammatory response (as evidenced by lower plasma IL-8 levels and reduced myocardial myeloperoxidase activity) (Ockaili et al., 2005). 8.4 . Adiponectin as a mediator of hypoxia‐inducible factor 1α cardioprotection Adiponectin that is a circulating adipocytokine, which is secreted by adipose tissue and cardiomyocytes, has been reported to have important cardiovascular effects, including cardioprotection (Smith & Yellon, 2011). It has been found that mice deficient in adiponectin are more susceptible to myocardial IRI, compared to wild type littermate controls and that the exogenous infusion of adiponectin to wild type mice limited MI size (Shibata et al., 2005). Recently, Natarajan et al. (2008) have reported that HIF-1α stabilization in diabetic and non-diabetic mice using PHD-2 siRNA increased the mRNA expression of adiponectin in microvascular endothelial cells and in the heart, a finding which was associated with cardioprotection. 8.5 . Other hypoxia‐inducible factor 1α target genes associated with cardioprotection A number of other HIF-1α target genes have been reported to be associated with the cardioprotection elicited by HIF-1α stabilization. However, for the most part the direct contribution of these target genes to cardioprotection has not been demonstrated. These include aldose reductase, VEGF (Date et al., 2005), GLUT-1 (Date et al., 2005), GLUT-4 (Date et al., 2005; Dendorfer et al., 2005), the β-catenin/eNOS/NFκB and Bcl-2 signaling pathway (Adluri et al., 2011).

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8.6 . Metabolic adaptation to hypoxia The stabilization of HIF-1α allows cells to adapt to conditions of hypoxia by metabolic reprogramming of oxidative phosphorylation to anerobic glycolysis, which may be beneficial in cardiomyocytes subjected to acute IRI. This co-ordinated response to maintain ATP production in a hypoxic environment requires the upregulation of number of specific HIF-1α target genes: GLUT1 and GLUT4 (which increase cellular uptake of glucose); glycolytic enzymes (such as phosphofructokinase 1 and aldolase which breakdown glucose); lactate dehydrogenase A (which increases the conversion of pyruvate to lactate); pyruvate dehydrogenase kinase (which diverts pyruvate away from mitochondrial oxidative phosphorylation); the mitochondrial protease, LON, which mediates a change from COX4-1 to COX4-2 subunits in complex IV (in order to improve the efficiency of this component of the ETC, thereby limiting ROS production); and enhanced mitochondrial autophagy (through the activation of BNIP3) (reviewed in Cadenas et al., 2010). In order to accommodate the additional lactate production arising from enhanced anerobic glycolysis under conditions of hypoxia, HIF-1α up-regulates the plasma membrane monocarboxylate transporter 4, which exports lactate out from the cell (Ullah et al., 2006), and increases the activity of the Na+–H+ exchanger thereby extruding H+ ions (Shimoda et al., 2006). The production of ROS during myocardial IRI is detrimental to the heart, in part through the opening of the mitochondrial permeability transition pore (mPTP), a critical determinant of cell death (Hausenloy & Yellon, 2003). As such the metabolic switch from oxidative phosphorylation to anerobic glycolysis by HIF-1α stabilization would be expected to reduce the generation of mitochondrial ROS during IRI, resulting in reduced susceptibility of mPTP opening at the onset of myocardial reperfusion. In this regard, a recent experimental study has demonstrated that the chronic activation of HIF-1α in mice lacking PHD-1 was found to promote anerobic glycolysis and reduce mitochondrial ROS production in skeletal myofibres, resulting in greater tolerance to ischemia (Aragones et al., 2008). Whether this protective mechanism is operational in IPC-treated hearts is unknown and remains to be explored.

9 . Stabilization of hypoxia‐inducible factor 1α in heart failure: beneficial or detrimental? A number of experimental studies have investigated the effect of HIF-1α stabilization, using PHD inhibitors, on post-MI left ventricular (LV) remodeling. These initial studies were conducted with the intention of reducing cardiac interstitial fibrosis in the remote non-infarcted myocardium, through the inhibition of collagen biosynthesis (Nwogu et al., 2001). However, later studies have examined the role of HIF-1α as a therapeutic target for post-MI LV remodeling. Nwogu et al. (2001) found that administering to rats the specific PHD inhibitor, FG041 (starting 48 h after permanent LAD ligation) daily for 4 weeks reduced the extent of myocardial interstitial fibrosis and improved post-MI remodeling (as evidenced by improved LV systolic function and less LV dilatation). In a more recent study the salutatory effects of PHD inhibition in the post-MI remodeled heart were attributed to HIF-1α stabilization, rather than the inhibition of collagen synthesis (Philipp et al., 2006b). These authors found that the administration of the PHD inhibitor, FG-2216, either 48 h prior to MI for 28 days (permanent ligation of LAD) or 48 h after MI for 9 days, improved LV systolic function and prevent LV dilatation with no reduction in MI size, confirming the beneficial effects of PHD inhibition on post-MI LV remodeling. These beneficial effects of PHD inhibition were associated with the stabilization of HIF-1α in the heart but not with any changes in extracellular collagen metabolism (Philipp et al., 2006b). Bao et al. (2010) confirmed the ameliorating effects, on post-MI cardiac remodeling, of chronic HIF-1α stabilization using another novel PHD inhibitor, GSK360A. In this study, the beneficial effects were associated with increased circulating levels of the HIF-1α target gene, EPO, and an elevated hemoglobin. However, the mechanisms through which PHD inhibition

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or HIF-1α stabilization reduces adverse LV remodeling post-MI remain unclear. A recent experimental study by Huang et al. (2011) has investigated the combined effects of PHD-2 and FIH deletion on post-MI LV remodeling and bone marrow cell mobilization in the adult murine heart. They found that the intramyocardial injection of short hairpin RNA sequences to knock-down PHD-2 and FIH, following LAD occlusion in adult murine hearts improved cardiac function, increased recruitment of bone marrow stem cells to the ischemic myocardium, and enhanced myocardial angiogenesis (Huang et al., 2011). Wei et al. (2012) have recently reported that transgenic mice which lack HIF-1α expression in Tie2+ lineage cells (endothelial cells) manifest cardiac decompensation accompanied by cardiac hypertrophy and fibrosis, linked to increased TGF-β signaling when subjected to transverse aortic constriction, demonstrating a critical protective role in the adaptation of the heart to pressure overloading modulated by HIF-1. Despite these studies showing the beneficial modalities afforded by HIF-1 against heart failure, accumulating evidence suggests that the long-term stabilization of myocardial HIF-1α may actually be detrimental (see Fig. 2). Lei et al. (2008) demonstrated that the cardiomyocyte-specific deletion of the gene encoding for VHL protein had detrimental effects in adult mice at 9 months old. The hearts developed lipid accumulation, myofibril rarefaction, altered nuclear morphology, cardiomyocyte loss, and fibrosis, resulting in severe progressive heart failure and premature death (Lei et al., 2008). These findings were associated with the generation of malignant cardiac tumors (Lei et al., 2008). Importantly, these detrimental effects of VHL deletion were absent in mice deficient in both VHL and HIF-1α, confirming the role of HIF-1α in mediating this maladaptive effects in the heart (Lei et al., 2008). Similarly, conditional inactivation of PHD-2 in mice was reported to result in increased red blood cell production, venous congestion and dilated cardiomyopathy (Minamishima et al., 2008). The etiology of the dilated cardiomyopathy was not clear but was attributed to hyperviscosity syndrome and volume overload, although a direct effect of chronic HIF-1α stabilization on the cardiomyocytes could not be excluded (Minamishima et al., 2008). Bekeredjian et al. (2010) found that cardiomyocyte-specific expression of a mutated oxygen-stable HIF-1α induced cardiomyopathy, which was reversed upon inactivation of the transgene. The mechanism for the cardiomyopathy in this study was attributed to reduced mRNA and protein expression of SERCA resulting in attenuated re-uptake of cytoplasmic calcium in cardiomyocytes (Bekeredjian et al., 2010). Finally, a recent study by Moslehi et al. (2010) demonstrated that cardiomyocyte-specific chronic inactivation of PHD-2, resulted in a dilated cardiomyopathy and premature mortality in mice aged 8 weeks old, an effect which was exacerbated when combined with the deletion of PHD-3. These detrimental effects of PHD deletion on cardiac function were associated with enhanced angiogenesis, intracellular accumulation of glycogen and lipids, abnormal mitochondrial biogenesis and morphology, impaired mitochondrial function and enhanced mitophagy (Moslehi et al., 2010). Holscher et al. (2012) have recently demonstrated that chronic stabilization of HIF-1α using a cardiac-specific HIF-1α transgenic mouse resulted in profound cardiac decompensation when subjected to TAC and cardiomyopathy was also noted in aging mice. Furthermore, these authors reported an increased stabilization of HIF-1α in heart samples taken from patients with end-stage heart failure (Holscher et al., 2012). The discrepancies among these studies in trying to elucidate whether HIF-1 is beneficial or detrimental in the context of heart failure can perhaps be explained through the different parameters taken into account in each study. In the studies which demonstrate HIF-1 to be beneficial in halting heart failure, the animals were usually studied for a shorter period of time only or HIF-1 induction was only persistent for several weeks (Bao et al., 2010), whereas in those studies finding detrimental effects with HIF-1, the activation of the latter had been sustained (Holscher et al., 2012; Wei et al., 2012). Therefore,

although the short-term stabilization of HIF-1α may be beneficial for the heart in terms of resisting ischemia and in the early stages of heart failure, the sustained stabilization of HIF-1α may be detrimental, thereby restricting the future therapeutic application of HIF-1α stabilization to the clinical settings of acute IRI such as acute myocardial infarction. Instead, high levels of HIF-1α in heart failure make the inhibition of HIF-1α a potential therapeutic strategy for the treatment of heart failure. In this regard, several HIF-1α inhibitors have been developed as potential treatments for cancer including echinomycin (Cairns et al., 2007), Topotecan (or NSC-609699) (Puppo et al., 2008) and YC-1 (Yeo et al., 2003). Whether these HIF-1α inhibitors are beneficial as a therapy in patients with chronic heart failure remains to be explored.

10 . Hypoxia‐inducible factor 2α and cardioprotection The HIF-2α isoform (previously known as endothelial PAS protein 1) has been much less extensively studied in the heart in comparison to HIF-1α. Experimental studies have suggested that HIF-2α is involved in cardiac and vascular development (Tian et al., 1998; Favier et al., 2001), and more recently, HIF-2α has been reported to mediate protection against acute IRI in the kidney (Kojima et al., 2007) and brain (Ralph et al., 2004). Under basal conditions, HIF-2α is barely detectable in the heart. However, in response to hypoxia (6 h of hypoxic stimulation with 0.1% carbon monoxide), there was an increase in HIF-2α protein expression which began at 1 h and peaked at 3–4 h following the hypoxic stimulus (Warnecke et al., 2003). The role of HIF-2α as a potential mediator of cardioprotection has been recently explored in the heart. Bautista et al. (2009) have provided data suggesting that HIF-2α may mediate delayed cardioprotection through the down-regulation of the β1-subunit of the voltage-dependent K + (maxi-K +) channel. However, it is not clear why the down-regulation of the maxi-K + channel (also known as the mitochondrial calcium-activated K + channel) should be beneficial, given that the opening of this channel has been previously linked to cardioprotection (Xu et al., 2002).

11 . Conclusions The adaptive response of the aerobic organism to survive under conditions of hypoxia is co-ordinated by the oxygen-sensitive transcription factor HIF, and is mediated through the transcription of over 200 of its target genes. Under normoxic conditions, HIF is rapidly degraded by PHDs and VHL, whereas under conditions of hypoxia, HIF is stabilized by PHD inhibition and the mitochondrial production of ROS. Many of the HIF-target genes have been implicated as mediators of IPC, and the IPC stimulus itself has been demonstrated to stabilize HIF prior to index ischemia. Therefore, the short-term stabilization of HIF under normoxic conditions provides a novel therapeutic strategy for protecting the heart and other organs against acute IRI. In experimental studies, this has been achieved by administering either non-specific or specific pharmacological inhibitors of PHD (see Fig. 2). However, the long-term stabilization of HIF under normoxic conditions may be detrimental to the heart and has been reported to induce cardiomyopathy, thereby excluding chronic PHD inhibition as a therapeutic option for ischemia (see Fig. 2). In summary, the HIF signaling pathway provides an important therapeutic target for protecting the heart against acute IRI. In the future, the development of novel and specific inhibitors of PHD (or even VHL) or HIF activators may provide an innovative therapeutic strategy for treating patients with coronary heart disease. Similarly, in patients with ischemic heart failure in which chronic HIF activation is a major problem, the development of novel HIF inhibitors may provide a novel treatment strategy which may be of use also in halting the progress of tumors as the increased presence of HIF is a hallmark of various cancers.

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