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Cardiovascular Research Advance Access first published online on October 11, 2007
This version [Corrected Proof] published online on November 2, 2007
Cardiovascular Research, doi:10.1093/cvr/cvm035
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Complete loss of ischaemic preconditioning-induced cardioprotection in mice with partialdeficiency of HIF-1{alpha}

Zheqing Cai1,2,5, Hua Zhong1,2, Marta Bosch-Marce1,2, Karen Fox-Talbot3, Lei Wang4, Chiming Wei4, Michael A. Trush7 and Gregg L. Semenza1,2,5,6,*

1 Vascular Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
2 McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Suite 671,733 North Broadway, Baltimore, MD 21205, USA
3 Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
4 Department of Surgery, The Johns Hopkins University School of Medicine, Baltimore,MD 21205, USA
5 Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
6 Departments of Pediatrics, Oncology, and Radiation Oncology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
7 Department of Environmental Health Sciences, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD 21205, USA

* Corresponding author. Tel: +1 410 955 1619; fax: +1 443 287 5618. E-mail address: gsemenza{at}jhmi.edu

Time for primary review: 33 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: We investigated whether hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) plays a role in the acute phase of ischaemic preconditioning (IPC).

Methods and results: Hearts from wild-type (WT) mice and mice heterozygous for a null allele at the locus encoding HIF-1{alpha} (HET) were subjected to IPC (10-min ischaemia/5 min reperfusion, or two cycles of 5 min ischaemia/5 min reperfusion), followed by 30 min ischaemia and reperfusion. Left ventricular-developed pressure, heart rate, and coronary flow rate were measured continuously. Apoptosis and infarct size were assessed by TUNEL assay, cleaved caspase 3 immunohistochemistry, and triphenyltetrazolium chloride staining. Production of reactive oxygen species (ROS) in isolated cardiac mitochondria was measured by a chemiluminescence assay. The phosphatase and tensin homologue (PTEN) and AKT (protein kinase B) were analysed by immunoblot assay. IPC improved functional recovery and limited infarct size and apoptosis after prolonged ischaemia–reperfusion in WT hearts, but not in HET hearts. Mitochondrial ROS production, PTEN oxidation, and AKT phosphorylation were impaired in HET hearts. WT and HET hearts were protected by adenosine, which acts via an ROS-independent mechanism.

Conclusion: HIF-1{alpha} is required for IPC-induced mitochondrial ROS production and myocardial protection against ischaemia–reperfusion injury.

KEYWORDS Hypoxia; Myocardial infarction; Reactive oxygen species

Received September 18, 2007; accepted October 18, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Myocardial infarction can cause sudden death or the subsequent development of heart failure. Current approaches to limit infarct size involve restoration of blood flow to ischaemic tissue by percutaneous coronary intervention or thrombolytic therapy. Despite the importance of restoring blood flow to ischaemic tissue, it is recognized that reperfusion itself may cause irreversible myocardial damage.1–3 Ischemic preconditioning (IPC), consisting of one or several episodes of brief ischaemia followed by reperfusion, generates profound protection against a subsequent prolonged episode of ischaemia–reperfusion.4 The protection occurs in two phases: one of which is acute, occurring immediately following the IPC stimulus, and the other is delayed, occurring 12–24 h after the IPC stimulus.5 Many different signal transduction pathways have been implicated in IPC, including (but not limited to) protein kinase C-{varepsilon}, nitric oxide, cGMP-dependent protein kinase, AKT (protein kinase B), ERK and p38 MAP kinases, AMP-dependent protein kinase, and the mitochondrial ATP-dependent potassium channel.5

Several studies reported that reactive oxygen species (ROS) are involved in triggering IPC.5 The ROS scavenger N-2-mercaptopropionyl glycine completely blocked the cardioprotection induced by an IPC stimulus consisting of 5 min ischaemia followed by 10 min reperfusion in rabbit.6,7 Moreover, modest concentrations of free radicals are sufficient to induce protection in the heart.6–14 The source of IPC-induced ROS appears to be the mitochondrial electron transport chain.12 Multiple episodes of IPC may induce the protection through an ROS-independent pathway.7,13 ROS are required to trigger both the acute and delayed phases of IPC-induced cardioprotection.5

The most well-characterized targets of ROS are protein tyrosine phosphatases. They share with phosphatase and tensin homologue deleted on chromosome 10 (PTEN) an active centre containing a CX5R motif.15 H2O2 oxidizes cysteine residue 124 at the catalytic site of PTEN to form a disulphide bond with cysteine residue 71, leading to PTEN inactivation.16 PTEN is a dual lipid and protein tyrosine phosphatase. Phosphatidylinositol-3,4,5-triphosphate, which is produced by phosphatidylinositol-3-kinase (PI3K) and required for the activity of AKT, is dephosphorylated to phosphatidylinositol-4,5-biphosphate by PTEN. The activities of PI3K and phosphoinositide-dependent kinase 1 (PDK1) are required for IPC.2,5,17 We recently demonstrated that ROS-mediated oxidation and inactivation of PTEN leads to increased AKT phosphorylation in preconditioned hearts.18 Numerous studies have shown that AKT promotes cardioprotection2,19 and also inhibits cell apoptosis by regulating multiple substrates. AKT phosphorylates and inactivates pro-apototic proteins including glycogen synthase kinase-3ß and caspase 9, the upstream activator of caspase 3.20,21 Phosphorylation of the pro-apoptotic Bcl-2 family member Bad prevents it from binding to the mitochondrial permeability transition pore (MPTP), thereby inhibiting the initiation of apoptosis.22 AKT activates the anti-apoptotic Bcl-2 protein, increasing MPTP stability.23 AKT phosphorylates nitric oxide synthase and increases the production of nitric oxide, leading to PKC-{varepsilon} activation.24 Activated PKC-{varepsilon} associates with the MPTP and inhibits its opening.25

Hypoxia-inducible factor 1 (HIF-1) consists of HIF-1{alpha} and -1ß subunits.26 The HIF-1ß subunit is constitutively expressed and the HIF-1{alpha} subunit is upregulated in response to hypoxia or ischaemia in the heart.27,28 Both the stability and transcriptional activity of the HIF-1{alpha} subunit are inhibited under normoxic conditions by O2-dependent hydroxylation reactions.29 Homozygous inactivation of the Hif1a gene encoding HIF-1{alpha} causes embryonic lethality because of failed cardiac and vascular development,30,31 whereas heterozygous-null Hif1a+/– (HET) mice with partial deficiency of HIF-1{alpha} develop normally but manifest impaired responses to hypoxia compared with their wild-type (WT) littermates.32,33 In this study, we report that IPC induces mitochondrial ROS production, PTEN oxidation, AKT phosphorylation, and cardioprotection in WT, but not in HET mice.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Animals
All experiments were performed with 5–7-month-old male littermate mice that were either WT or heterozygous (HET) for the Hif1atm1jhu mutant allele in which exon 2 has been replaced with a neoR gene.30 All procedures were approved by the Johns Hopkins University Institutional Animal Care and Use Committee and conform to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1996).

2.2 Mouse Langendorff perfusion system
Mice were anesthetized by intraperitoneal injection of pentobarbital (70 µg/g). After the chest was opened, the heart was excised and the ascending aorta was cannulated with a blunt needle. The heart was perfused at a constant pressure of 100 cm H2O with Krebs–Henseleit buffer (in mmol/L, glucose 17, NaCl 120, NaHCO3 25, CaCl2 2.5, KCl 5.9, MgSO4 1.2, and EDTA 0.5), which was maintained at 37°C and bubbled continuously with a mixture of 95% O2 and 5% CO2. Coronary flow rate (CFR) was continuously monitored via a flow meter (T106, Transonic systems, Ithaca, NY, USA). A water-filled plastic balloon was inserted into the left ventricle and connected to a transducer, amplifier, and data processor (MLT844/D, ML110, PowerLab/4sp, respectively, ADInstruments, Colorado Spring, CO, USA) to continuously monitor heart rate (HR) and left ventricular (LV) pressure. The LV-developed pressure (LVDP) was calculated as the difference between the systolic and end-diastolic LV pressures. Global ischaemia was induced by cessation of perfusion for 5–30 min, followed by reperfusion for 5–120 min. Two different IPC protocols were used: a single cycle of 10 min ischaemia and 5 min reperfusion; or two cycles of 5 min ischaemia and 5 min reperfusion. Adenosine was administered for 15 min at a final concentration in the perfusate of 100 µmol/L.

2.3 Measurement of mitochondrial reactive oxygen species production
Mitochondrial ROS production was assessed by using lucigenin- and luminol-derived chemiluminescence.34 Hearts were homogenized in isolation buffer (in mmol/L, sucrose 320, EDTA 1, Tris–HCl 10, pH 7.4.) and then centrifuged at 1500 g for 15 min. The supernatant was again centrifuged at 13 500 g for 15 min. Mitochondrial protein (100 µg) was added to 1 mL of air-saturated respiration buffer (in mmol/L, mannitol 220, sucrose 70, HEPES 20, KH2PO4 2.5, EDTA 0.5, 0.1% bovine serum albumin, pH 7.4). To determine superoxide production, 5 µmol/L lucigenin (Sigma) was added to the buffer, and chemiluminescence was monitored using bioluminometer (Berthold LB9505) at 37°C for 60 min after supplementation with 6 M succinate. The production of hydrogen peroxide was determined with a similar procedure, except that 5 µmol/L luminol and 10 µmol/L horseradish peroxidase are added instead of lucigenin. Paired control and IPC hearts were assayed on the same day.

2.4 Measurement of myocardial infarct size
Hearts were frozen at –80°C for 10 min, sliced into five sections, incubated in 1% triphenyltetrazolium chloride for 30 min, and scanned.35 Viable myocardium stained brick red and infarct tissue appeared pale white. Infarct and LV areas were measured by Image J1.30 software (NIH). Infarct size was calculated as infarct area divided by LV area.

2.5 Assessment of myocardial apoptosis
Apoptosis was quantified by the terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick-end labelling (TUNEL) technique (ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit, Chemicon International). Hearts were fixed with formalin, embedded in paraffin, and 4-µm-thick sections were incubated with proteinase K (20 µg/ml) and then with TdT. After washing, anti-digoxigenin conjugate and peroxidase substrate were applied to the sections. Finally, they were counterstained with hematoxylin. For each section, 8 random fields were examined under an Olympus light microscope (x20 objective). Apoptosis was also analysed by immunohistochemistry, using an antibody that recognizes cleaved caspase 3 (Cell Signaling Technology).

2.6 Immunoblot assay
Hearts were homogenized in cell lysate buffer [in mmol/L, Tris 20 (pH 7.5), NaCl 150, EDTA 1, EGTA 1, Na3VO4 1, and 1% Triton X-100), and centrifuged at 10 000 g for 15 min. Supernatants were fractionated by 10% SDS–PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in 1x TBS and 0.1% Tween-20 (TBS/T) for 1 h, then incubated in primary antibody diluted in TBS/T for 1.5 h. After washing three times for 5 min each with TBS/T, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. After washing three times, the membrane was incubated with ECL reagent (Amersham) for 1 min. Signal intensity was determined by Image J1.30 software (NIH). Rabbit polyclonal anti-phospho-AKT antibody was purchased from Biosource. Mouse monoclonal antibody against AKT was from Santa Cruz Biotechnology.

2.7 Analysis of oxidized and reduced PTEN
Oxidized PTEN is protected from alkylation and can be distinguished from alkylated (reduced) PTEN because of its increased mobility in non-reducing gels.16 Briefly, isolated mouse hearts were homogenized in cell lysate buffer containing the alkylating agent iodoacetamide (50 mmol/L), which attacks thiols in reduced PTEN. The products of the alkylation reactions were fractionated by electrophoresis on 10% non-reducing SDS-polyacrylamide gels, followed by immunoblot assay of PTEN using a rabbit polyclonal antibody (Cell Signaling Technology).

2.8 Statistical analysis
Differences between the groups were analysed for statistical significance (P ≤ 0.05) by one-way ANOVA or two-way t-test.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Ischaemic preconditioning does not promote functional recovery of HET hearts following ischaemia–reperfusion
To determine whether hearts from HET mice have an impaired response to IPC, isolated Langendorff-perfused hearts from WT and HET littermate mice were subjected to an IPC stimulus (10 min ischaemia and 5 min reperfusion) or control perfusion (CON), followed by prolonged ischaemia (30 min) and reperfusion (45 min) (Figure 1A). Twenty-three hearts were analysed under one of four conditions (WTIPC+I/R, n = 7; HETIPC+I/R, n = 7; WTI/R, n = 5; and HETI/R, n = 4). The mean body weights of the 12 WT and 11 HET mice in the study were not significantly different (29.4 ± 1.1 vs. 29.8 ± 1.1 g), but the mean heart weight was significantly less in HET than in WT mice (0.189 ± 0.012 vs. 0.223 ± 0.012 g, P < 0.05). There was no difference in pre-ischaemic LVDP, HR, or CFR between WT and HET hearts (Table 1). However, IPC significantly increased the recovery of LVDP after 30 min ischaemia and 45 min reperfusion in WT hearts (WTIPC+I/R vs. WTI/R = 31.7 ± 4.5 vs. 13.6 ± 3.5 mmHg, P < 0.001; Figure 1B) but not in HET hearts (HETIPC+I/R vs. HETI/R = 9.9 ± 1.9 vs. 15.5 ± 3.3; Figure 1B). IPC did not significantly affect HR or CFR in WT or HET hearts (Figure 1C and D).


Figure 1
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  		Figure 1 Effect of ischaemic preconditioning (IPC) on the functional recovery of wild type (WT) and Hif1a heterozygous-null (HET) hearts after prolonged ischaemia. (A) WT and HET hearts were perfused in oxygenated buffer for 15 min (red bars). Perfusion was continued for an additional 15 min or the hearts were subjected to an IPC stimulus consisting of 10 min global no-flow ischaemia (blue bars) and 5 min reperfusion. All hearts were then subjected to 30 min global (no-flow) ischaemia followed by 45 min reperfusion. (B–D) The recovery of left ventricular developed pressure [LVDP, (B)], heart rate [HR, (C)], and coronary flow rate [CFR, (D)] during the 45 min reperfusion period are shown (mean ± SEM). The zero time point corresponds to the end of the 30 min ischaemic period. *P < 0.001 compared with WT (n = 3–7 for each condition).

 


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  		Table 1 Haemodynamic analysis of isolated WT and HET mouse hearts before ischaemia

 
3.2 Apoptosis is increased in preconditioned HET hearts after ischaemia–reperfusion
Following IPC and 30 min ischaemia/45 min reperfusion (Figure 2A), WT and HET hearts were fixed, embedded, and sectioned for TUNEL assay (Figure 2B). The number of apoptotic cells was significantly greater in sections from HET compared with WT hearts (HETIPC+I/R vs. WTIPC+I/R = 23 ± 1 vs. 16 ± 2 cells/field, P < 0.05, n = 3; Figure 2C). Thus, both functional and immunohistochemical assays demonstrate that the IPC stimulus induced significantly greater protection against ischaemia–reperfusion injury in WT compared with HET hearts.


Figure 2
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  		Figure 2 Analysis of apoptosis in hearts subjected to IPC and prolonged ischaemia–reperfusion. (A) WT and HET hearts were subjected to an IPC stimulus (10 min ischaemia/5 min reperfusion) followed by 30 min ischaemia and 45 min reperfusion. (B) TUNEL assay of sections from WT and HET hearts (x200). Arrows point to apoptotic cells with TUNEL-stained nuclei. (C) Mean (±SEM) number of apoptotic cells in eight random fields in tissue sections from three hearts of each genotype. *P < 0.05 compared with WT.

 
3.3 Multiple-cycle ischaemic preconditioningdoes not reduce infarct size in HET hearts
We considered the possibility that the defective response of HET hearts might be limited to the particular IPC stimulus that we utilized, which involved only a single cycle of ischaemia–reperfusion. To investigate this possibility, we analysed cardioprotection induced by another commonly used IPC protocol that involves repeated short cycles of ischaemia–reperfusion. We subjected hearts from WT and HET littermates to 30 min ischaemia/120 min reperfusion directly or after an IPC stimulus consisting of two consecutive cycles of 5 min ischaemia/5 min reperfusion (Figure 3A) and determined the percentage of ventricular mass that was infarcted by triphenyltetrazolium chloride staining (Figure 3B). This IPC protocol significantly decreased infarct size in WT mice (WTI/R vs. WTIPC+I/R = 79.6 ± 16.7 vs. 25.5 ± 14.2%; P < 0.05, n = 3; Figure 3C). In contrast, IPC had no protective effect on infarct size in HET hearts (HETI/R vs. HETIPC+I/R = 68.6 ± 23.5 vs. 73.4 ± 21.5%; Figure 3C). Thus, despite using a different protocol with regard to both the IPC stimulus and the measure of heart injury, we again demonstrated a complete loss of cardioprotection in HET mice.


Figure 3
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  		Figure 3 Effect of IPC on infarct size. (A) WT and HET hearts were subjected to IPC (consisting of two cycles of 5 min ischaemia/5 min reperfusion) or control perfusion followed by 30 min ischaemia and 120 min reperfusion. (B) Hearts were sectioned and stained with triphenyltetrazolium chloride. (C) Area of infarcted heart tissue expressed as a percentage of the total ventricular area (mean ± SD). *P < 0.05 compared with no IPC.

 
3.4 Ischaemic preconditioning does not increase mitochondrial ROS production in HET hearts
Ischaemic preconditioning induces the production of ROS, and anti-oxidant treatment blocks the protective effect of IPC against ischaemia–reperfusion injury. To determine whether the loss of IPC-induced protection in HET hearts was associated with deficient ROS production, isolated WT hearts were subjected to IPC (10 min ischaemia/5 min reperfusion) or control perfusion (Figure 4A), followed by mitochondrial isolation and measurement of ROS production. The IPC was associated with 1.6- and 2.6-fold increased mitochondrial production of superoxide and hydrogen peroxide, respectively (Figure 4B). In contrast, in HET hearts, IPC did not induce increased mitochondrial production of superoxide and led to a 50% decrease in hydrogen peroxide production (Figure 4C). Thus, the loss of IPC-induced cardiac protection in HET mice was associated with a loss of IPC-induced ROS production.


Figure 4
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  		Figure 4 Effect of IPC on ROS production in cardiac mitochondria. (A) WT and HET hearts were subjected to IPC (10 min ischaemia/5 min reperfusion) or control perfusion (CON). The mitochondria were isolated, and the production of H2O2 and superoxide were continuously monitored >60 min by lucigenin- and luminol-derived chemiluminescence, respectively. (B) Compared with CON, IPC increased H2O2 (left) and superoxide (right) production in WT hearts. (C) Compared with the WT, H2O2 levels were relatively high in HET in CON condition and decreased in response to IPC (left). Superoxide levels were not affected by IPC in HET hearts (right).

 
3.5 Ischaemic preconditioning does not induce PTEN oxidation in HET hearts
A growing body of evidence indicates that ROS act as signalling molecules in mediating cell growth, differentiation, and survival.15 Protein phosphatases, including PTEN, are inherently sensitive to ROS oxidation.16 To determine whether deficient ROS production leads to decreased PTEN oxidation in HET hearts, we exposed WT and HET mouse hearts to IPC (10 min ischaemia/5 min reperfusion) or control perfusion (Figure 5A). IPC induced a significant increase in PTEN oxidation in WT hearts compared with HET hearts (62 ± 6 vs. 10 ± 8, P < 0.01, n = 3; Figure 5B). Therefore, defective ROS production leads to impaired PTEN oxidation in preconditioned HET hearts.


Figure 5
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  		Figure 5 Effect of IPC on PTEN and AKT. (A) WT and HET hearts were subjected to IPC [10 min ischaemia (grey bars) and 5 min reperfusion] or control perfusion (CON; white bars) (n = 3 for each condition). (B) Heart lysates were treated with alkylating agent and analysed for oxidized (Ox) and reduced (Re) PTEN by non-reducing SDS–PAGE and immunoblot assay. Representative immunoblot (top) and the results of densitometric analysis of Ox-PTEN in arbitrary units (AU; bottom) are shown. *P < 0.01 compared with WT CON or HET IPC. (C) Heart lysates were analysed for phosphorylated AKT (p-AKT) and total AKT (AKT) by immunoblot assay. Representative immunoblots (top) and the results of densitometric analysis of p-AKT in AU (bottom) are shown. *P < 0.01 compared with WT CON or HET IPC.

 
3.6 Ischaemic preconditioning does not increase protein kinase B phosphorylation in HET hearts
Ischaemic preconditioning is believed to inhibit apoptosis in part by inducing the phosphorylation and activation of AKT, which, in turn, phosphorylates and inactivates pro-apoptotic signalling proteins. Tissue samples from WT and HET hearts that were subjected to IPC or control perfusion (Figure 5A) were analysed for the levels of total AKT protein and AKT phosphorylated at serine residue 473 (phospho-AKT). Phospho-AKT levels were significantly increased in lysates of WTIPC compared with HETIPC hearts (145 ± 5 vs. 72 ± 18, P < 0.01, n = 5; Figure 5C). The failure to induce phosphorylation of AKT after IPC provides a molecular basis for the increased apoptosis in preconditioned HET hearts following ischaemia–reperfusion.

3.7 Adenosine infusion protects HET heartsagainst ischaemia–reperfusion injury
A variety of pharmacological agents induce preconditioning. Among these, adenosine is of particular interest because unlike preconditioning induced by ischaemia or other pharmacological agents, adenosine preconditioning is not blocked by ROS scavengers.36 We reasoned that if HIF-1{alpha} was required for ROS generation in response to IPC, then an agent acting via an ROS-independent mechanism may induce protection in HET hearts. Hearts were subjected to control perfusion, IPC, or adenosine perfusion followed by 30 min ischaemia/45 min reperfusion. Immunohistochemical analysis of cleaved (activated) caspase 3 expression was performed as a measure of apoptosis (Figure 6A). The number of cells that stained positive for cleaved caspase 3 was significantly reduced in WT hearts exposed to IPC (47 ± 31, mean ± SD, n = 3) or adenosine treatment (35 ± 24) compared with control perfusion (94 ± 15). In HET hearts, cleaved caspase 3 staining was similar in hearts subjected to control perfusion (106 ± 48) or IPC (99 ± 21), but was significantly reduced after adenosine perfusion (25 ± 12).


Figure 6
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  		Figure 6 Effect of adenosine infusion. WT and HET hearts (n = 3 for each condition) were subjected to the following conditions: control perfusion(I/R); IPC consisting of two cycles of 5 min ischaemia/5 min reperfusion (IPC + I/R); or perfusion with buffer containing 100 µM adenosine for 15 min (Ado + I/R). All hearts were then subjected to 30 min ischaemia and 45 (A) or 120- (B) min reperfusion. White bar, control perfusion; dark grey bar, ischaemia; light grey bar, adenosine perfusion. (A) The hearts were formalin-fixed, paraffin-embedded, and immunohistochemistry was performed on tissue sections using an antibody specific for cleaved caspase 3. (B) Hearts were sectioned and stained with triphenyltetrazolium chloride as in Figure 3. Data are presented as mean ± SD. *P ≤ 0.05 vs. I/R.

 
Infarct size was also determined by triphenyltetrazolium staining of hearts subjected to 30 min ischaemia/120 min reperfusion after adenosine perfusion (Figure 6B) and compared with the data for control perfusion and IPC that were presented in Figure 3. In contrast to IPC, which provided no protection to HET hearts, infarct size was significantly reduced to 14 ± 4% in adenosine-perfused HET hearts compared with 68 ± 29% in control-perfused HET hearts (mean ± SD, n = 3). These data provide additional evidence in support of the hypothesis that HIF-1{alpha} is specifically required for ROS-dependent cardioprotection induced by IPC.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
In this study, we report three novel, important, and mechanistically related findings. First, acute cardioprotection induced by either single- or multi-cycle IPC was lost in HET mice, as measured by LVDP recovery, TUNEL assay, triphenyltetrazolium chloride staining, and activated caspase 3 immunohistochemistry. These results demonstrate that HIF-1{alpha} is necessary for acute-phase (early window) IPC, as a partial deficiency of HIF-1{alpha} was associated with a complete loss of protection against ischaemia–reperfusion injury. We previously reported that the exposure of mice to intermittent hypoxia induces HIF-1{alpha}-dependent erythropoietin expression in the kidney and increased erythropoietin plasma levels, leading to delayed (late-phase) cardioprotection in WT but not in HET mice.35 The finding that HIF-1{alpha} is also required for the acute phase of preconditioning is surprising.

A priori, there are several potential mechanisms by which HIF-1{alpha} could play an essential role in the acute phase of IPC. The requirement for HIF-1{alpha} may reflect its role in regulating the constitutive expression of a protein that is required for acute IPC. In this model, reduced HIF-1 activity in HET mouse hearts results in altered levels of a dosage-sensitive protein that is required for IPC and induction of HIF-1{alpha} itself is not critical to IPC. Alternatively, the induction of HIF-1{alpha} expression may be required to mediate the response to IPC, either by dimerizing with HIF-1ß and performing its well-established role as a transcriptional activator, or via a mechanism that is independent of HIF-1ß, transcription, or both. Further studies are required to distinguish between these potential mechanisms.

Secondly, increased mitochondrial ROS generation was induced when WT hearts were subjected to IPC, but was lost in HET hearts. A requirement for mitochondrial generation of ROS in triggering cardioprotection has been demonstrated in several models of IPC.2,11,12 Superoxide forms in the mitochondria when electrons prematurely react with O2 at complex I or complex III of the respiratory chain. Mitochondrial superoxide dismutase rapidly converts superoxide to hydrogen peroxide, which functions as a second messenger and is degraded by glutathione peroxidase and catalase. Although the induction of HIF-1{alpha} in hypoxic cells was known to be dependent upon the mitochondrial generation of ROS,37 our data indicate for the first time that HIF-1{alpha} is also required for IPC-induced ROS generation in cardiac mitochondria.

We recently demonstrated increased ROS levels in the brains of WT, but not HET, mice that were exposed to 10 days of chronic intermittent hypoxia.38 In the present study, we have demonstrated a similar lack of ROS generation, but in the isolated hearts of HET mice subjected to an acute IPC stimulus. The impaired ROS generation observed in Hif1a+/– HET mice is in striking contrast to the enhanced ROS production that has been demonstrated in Epas1–/– mice,39,40 which lack expression of HIF-2{alpha}, a protein that dimerizes with HIF-1ß and can regulate gene transcription either in concert with, or in opposition to, HIF-1{alpha}.41 Expression of mRNAs encoding catalase, glutathione peroxidase 1, and superoxide dismutase 1 and 2 were reduced in the livers of HIF-2{alpha}-null mice.39 Using quantitative real-time reverse-transcriptase PCR, we analysed mRNA from fibroblasts derived from WT and Hif1a–/– mice embryos and found no major differences between genotypes in the expression of mRNAs encoding glutathione peroxidase 1 or catalase (data not shown).

We recently demonstrated that HIF-1 plays multiple roles in regulating mitochondrial function. HIF-1 regulates the expression of the gene-encoding pyruvate dehydrogenase kinase 1, which phosphorylates and inactivates the catalytic subunit of pyruvate dehydrogenase, the enzyme that converts pyruvate to acetyl coenzyme A for entry into the tricarboxylic acid cycle.42 HIF-1 also regulates the subunit composition of cytochrome c oxidase (electron transport chain complex IV).43 Finally, HIF-1 mediates the repression of a transcriptional network that promotes mitochondrial biogenesis.44 Whether any of these pathways are relevant to the defect in IPC-induced mitochondrial ROS production in HET mouse hearts will require further investigation. However, in each of these pathways, HIF-1 functions to prevent excess mitochondrial ROS generation under conditions of chronic hypoxia, in contrast to the requirement for HIF-1{alpha} in the generation of mitochondrial ROS in response to acute IPC. Our studies do not address the possible involvement of HIF-1{alpha} in other IPC-associated events occurring outside of the mitochondria, such as alterations in sarcolemmal membrane potential,17 which may contribute to cardioprotection.

The third important result of this study is the additional evidence that ROS function as signalling molecules that regulate cardiac PTEN activity and, by doing so, control AKT activity, which, in turn, is a major determinant of survival during prolonged ischaemia–reperfusion. Previously, we reported that IPC or H2O2 infusion increased PTEN oxidation/inactivation in isolated rat hearts, and that PTEN inactivation was associated with AKT activation and cardioprotection.18 It has been shown that endogenous ROS levels are sufficient to oxidize PTEN15,45,46 and that AKT promotes cell survival in various models.19 Here, we have demonstrated that a genetic defect (heterozygosity for a null allele at the Hif1a locus) that caused loss of IPC-induced ROS production was associated with impaired PTEN oxidation, AKT phosphorylation, and cell survival in the hearts of HET mice (Figure 7).


Figure 7
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  		Figure 7 Involvement of HIF-1{alpha} in IPC-mediated cardioprotection. HIF-1{alpha} is required for IPC-induced ROS production, PTEN oxidation, AKT activation, and protection against ischaemia–reperfusion injury. PTEN inactivation by ROS allows unopposed activation of AKT by phosphatidylinositol 3-kinase (PI3K) and PDK1. Induction of HIF-1{alpha} expression in response to IPC may or may not be required for cardioprotection and HIF-1{alpha} may have additional cardioprotective effects that are independent of its effect on the pathways shown.

 
In addition to the role of HIF-1 in mediating IPC- and hypoxia-induced acute and delayed cardioprotection, HIF-1 promotes vascular remodelling in ischaemic tissue by activating the transcription of genes encoding multiple angiogenic growth factors, including vascular endothelial growth factor, placental growth factor, angiopoietins 1 and 2, platelet-derived growth factor B, and stromal cell-derived factor 1.47–49 Taken together, these data indicate that induction of HIF-1 activity may promote cardioprotection by multiple mechanisms. Recently, a coding sequence polymorphism in the human HIF1A gene was associated with the absence of coronary collaterals in patients with coronary artery disease.50 The fact that partial deficiency of HIF-1{alpha} in mice results in complete loss of IPC-induced cardioprotection suggests that genetic variation at the HIF1A locus may also influence whether patients with acute coronary syndrome present with angina or infarction.

Several studies have indicated that inhibition of the hydroxylases that negatively regulate HIF-1{alpha} results in reduced myocardial ischaemia–reperfusion injury,51,52 although the approaches taken were more relevant to mechanisms of delayed, rather than acute, cardioprotection. The mechanism of action of these agents was unclear because the inhibitors used were not specific for the HIF-1{alpha} hydroxylases and these enzymes regulate other proteins in addition to HIF-1{alpha}.53,54 However, our studies suggest that HIF-1{alpha} activation is likely to play a major role in the cardioprotective effect of hydroxylase inhibitors. Clinical studies are warranted to investigate whether pharmacological approaches to increase HIF-1{alpha} expression may be of therapeutic benefit in the context of acute coronary syndromes.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This work was supported by Public Health Service grant P01-HL65608 from the National Heart, Lung, and Blood Institute.


   Acknowledgements
 
The authors thank William M. Baldwin III for helpful advice.

Conflict of interest: none declared.


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

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