Detailed Materials and Methods

Murine model of myocardial ischemia and IP. All animal protocols were in accordance with the German guidelines for use of living animals and were approved by the Institutional Animal

Care and Use Committee of the University Hospital Tübingen and the Regierungspräsidium

Tübingen. C57BL/6 mice were obtained from Charles River (Sulzfeld, Germany). Mice deficient of the A2BAR or CD73 on the C57BL/6 strain were generated, validated and characterized as described previously 1. In pharmacological studies, age, gender and weight matched C57BL/6 mice were used. In studies of genetically targeted mice, homozygous mice for a specific gene deletion were compared with littermate controls, matched in age, gender and weight. Anesthesia was induced (70 mg/kg body weight i.p.) and maintained (10 mg/kg/h) with sodium pentobarbital. Mice were placed on a temperature-controlled heated table (RT, Effenberg,

Munich, Germany) with a rectal thermometer probe attached to a thermal feedback controller to maintain body temperature at 37°C. The tracheal tube was connected to a mechanical ventilator

(Servo 900C, Siemens, Germany) with pediatric tubing and the animals were ventilated with a pressure controlled ventilation mode (peak inspiratory pressure of 10 mbar, frequency 110 breaths/min, positive end-expiratory pressure of 3 mbar, FiO2 = 0,3). Blood gas analysis revealed normal paO2 (115±15 mmHg) and paCO2 (38±6 mmHg) levels with our ventilator regime. After induction of anesthesia, animals were monitored with a surface electrocardiogram (ECG,

Hewlett Packard, Böblingen, Germany). Fluid replacement was performed with normal saline,

0.1ml/h i.p. The carotid artery was catheterized for continuous recording of blood pressure with a statham element (WK 280, WKK, Kaltbrunn, Switzerland). Operations were performed under an upright dissecting microscope (Olympus SZX12). Following left anterior thoracotomy, exposure of the heart and dissection of the pericardium, the left coronary artery (LCA) was visually identified and an 8.0 nylon suture (Prolene, Ethicon, Norderstedt, Germany) was placed around the vessel. Atraumatic LCA occlusion for ischemia and IP studies was performed using a hanging weight system 2, 3. Successful LCA occlusion was confirmed by an immediate color change of the vessel from light red to dark violet, and of the myocardium supplied by the vessel from bright red to white, as well as the immediate occurrence of ST-elevations in the ECG.

During reperfusion, the changes of color immediately disappeared when the hanging weights were lifted and the LCA was perfused again. Infarct sizes were determined by calculating the percentage of myocardial infarction compared to the area at risk (AAR) using a previously described double staining technique with Evan’s blue and triphenyltetrazolium chloride (TTC) 3.

Evan’s blue is excluded from the area of the heart perfused by the LCA and thus allows one to identify the AAR. TTC stains all cells red except those that are depleted in NADPH and therefore allows one to visualize the white infarcted tissue. AAR and the infarct size were determined via planimetry using the NIH software Image 1.0 and the degree of myocardial damage was calculated as percent of infarcted myocardium from the AAR 4. Hemodynamic parameters are summarized in supplemental Table 1 and Table 2.

In vivo siRNA repression. To achieve in vivo repression of HIF-1 within cardiac tissues, we developed a system to apply siRNA/transfection agent via continuous infusion into the left ventricle, in order to achieve sufficient intracardial siRNA concentrations for successful siRNA repression. For this purpose, we advanced a carotid artery catheter into the left ventricle and confirmed its correct position by pressure monitoring. Once the appearance of a diastolic left ventricular pressure reading confirmed the position of the catheter in the left ventricle (diastolic pressure values of 5-10 mmHg), the infusion of siRNA together with transfection agent (siPORT

Amine, Ambion, Austin, TX, USA) was started (1,5µg siRNA/g body weight over 2 hrs in all experiments). SMARTpool siRNA targeting HIF-1 was synthesized by Dharmacon (Lafayette,

CO, USA). SMARTpool reagents combine four SMARTselection-designed siRNAs into a single pool, resulting in even greater probability that the SMARTpool reagent will reduce target mRNA to low levels. As control a siRNA (Dharmacon Lafayette, CO, USA) with at least four mismatches to any human, mouse, or rat gene that was used. In subsets of experiments, a similar approach was used for PHD1, PHD2 or PHD3 (Dharmacon, Lafayette, CO, USA). This approach was associated with successful cardiac gene repression following 2h of siRNA infusion.

Sequences of HIF1, PHDs and control siRNA are summarized in supplemental Table 1.

Transcriptional analysis. To assess the influence of IP on transcript level, IP was performed, the area at risk (AAR) was delineated by Evan’s blue staining and excised at indicated time periods, followed by isolation of RNA and quantification of transcript levels by real-time RT-

PCR (iCycler; Bio-Rad Laboratories, Munich, Germany), as previously described determined 1.

Primer sets and PCR conditions for ARs, and PHDs are summarized in supplemental Table 2.

Western blots for HIF-1, PHD1-3 and A2BAR. C57BL/6 mice were anesthetized and the

AAR was excised and immediately frozen at -80°C (remaining blood was removed before)

Western blotting was done as described previously using the following antibodies:

HIF1- (rabbit polyclonal antibody, synthetic peptide conjugated to KLH derived from within residues 500 - 600 of Human HYPB / HIF-1, Abcam, Cambridge, UK), PHD1 (goat polyclonal antibody, epitope mapping near the N-terminus of PHD1 of human origin, Santa Cruz Biotechnology Inc., Santa Cruz, California, USA), PHD2 (goat polyclonal antibody, epitope mapping near the C-terminus of PHD2 of human origin, Santa Cruz Biotechnology Inc., Santa

Cruz, California, USA), PHD3 (rabbit polyclonal antibody, synthetic peptide made to an internal fragment of the human protein sequence of PHD3, Abcam, Cambridge, UK) and A2BAR (rabbit polyclonal antibody, epitope corresponding to amino acids 293-332 mapping at the C-terminus,

Santa Cruz Biotechnology Inc., Santa Cruz, California, USA).

Immunohistochemistry. Hearts were fixed in Tissue-Tek (Sakura) for 24 h. The whole heart was cut coronary in 5 µm slices starting from the lower third of the left ventricle and mounted on glass slides, air dried and postfixed in acetone/methanol (1:1) for 10 min at room temperature and stored at – 20°C. For treated animals as well as for control mice, histological and immunohistochemical evaluation was performed for one heart tissue sample each time. For general assessment of the tissues, routine hematoxylin-eosin staining was performed.

Immunohistochemical stainings were performed with an A2BAR rabbit polyclonal antibody

(epitope corresponding to amino acids 293-332 mapping at the C-terminus, Santa Cruz

Biotechnology Inc., Santa Cruz, California, USA) or an HIF1- rabbit polyclonal antibody

(Synthetic peptide conjugated to KLH derived from within residues 500 - 600 of Human HYPB /

HIF-1, Abcam, Cambridge, UK) . For controls, both negative controls with omission of the first antibody and isotype controls with a corresponding IgG concentration (unspecific goat anti- mouse IgG) were performed. All histological and immunohistochemical stainings were performed on serial sections. Evaluation of the histological and immunohistochemical stainings of whole coronary sections and photographic documentation was performed using an Olympus

Vanox AH-3 light microscope (Hamburg, Germany). Heart Enzyme Measurement. Blood was collected by central venous puncture for troponin I

(cTnI) measurements using a quantitative rapid cTnI assay (Life Diagnostics, Inc., West Chester,

PA, USA).

Adenosine measurements. Cardiac adenosine measurements were performed after siRNA repression of HIF-1, control siRNA treatment, i.p. DMOG treatment (1mg) or vehicle control.

Ischemic preconditioning (4 cycles, 5 min ischemia, 5 min reperfusion) or shame operations were performed in cd73-/- mice or littermate controls. Preconditioned cardiac tissue was snap- frozen with clamps pre-cooled to the temperature of liquid nitrogen. The cardiac tissues were pulverized under liquid nitrogen and tissue protein was precipitated with 0.6 N ice-cold perchloric acid. Tissue adenosine levels were determined via HPLC as described previously.5

Data analysis. For comparison of two groups, the nonparametric Mann Whitney test was performed. When comparing more than two groups, the Kruskal-Wallis test with a Dunn´s post test was performed. Statistical significance was accepted at a level of P<0.05. All values are expressed as mean ± SEM from 6 animals per condition. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written. References

1. Eckle T, Krahn T, Grenz A, Kohler D, Mittelbronn M, Ledent C, Jacobson MA, Osswald H, Thompson LF, Unertl K, Eltzschig HK. Cardioprotection by ecto-5'-nucleotidase (CD73) and A2B adenosine receptors. Circulation. 2007;115(12):1581-1590.

2. Dewald O, Frangogiannis NG, Zoerlein MP, Duerr GD, Taffet G, Michael LH, Welz A, Entman ML. A murine model of ischemic cardiomyopathy induced by repetitive ischemia and reperfusion. Thorac Cardiovasc Surg. 2004;52(5):305-311.

3. Eckle T, Grenz A, Kohler D, Redel A, Falk M, Rolauffs B, Osswald H, Kehl F, Eltzschig HK. Systematic evaluation of a novel model for cardiac ischemic preconditioning in mice. Am J Physiol Heart Circ Physiol. 2006.

4. Eckle T, Grenz A, Kohler D, Redel A, Falk M, Rolauffs B, Osswald H, Kehl F, Eltzschig HK. Systematic evaluation of a novel model for cardiac ischemic preconditioning in mice. Am J Physiol Heart Circ Physiol. 2006;291(5):H2533-2540.

5. Delabar U, Kloor D, Luippold G, Muhlbauer B. Simultaneous determination of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in biological samples using solid-phase extraction and high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl. 1999;724(2):231-238.