Electronic Supplementary Material

Materials and methods

Animals

The experiments were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals. Both the University Animal Care Committee and the Federal Authorities for Animal Research of the Regierungsprasidium Darmstadt (Hessen, Germany) approved the study protocol. Male Sprague Dawley rats (250-350g) were obtained from Charles River Laboratories (Sulzfeld, Germany). Rats were randomized to receive either solvent (ethanol, 0,5 vol% final concentration), Levosimendan (Orion Pharma, Espoo, Finland; calculated dosage 3mg/kg body weight/d), combined Levosimendan and glibenclamide (Levo; calculated dosage 3mg/kg bw/d; glibenclamide [Sigma, Deishofen, Germany] calculated dosage: 5mg/kg bw/d) or Nicorandil (Merck, Darmstadt, Germany; calculated dosage: 7,5mg/kg bw/d) with their drinking water. Pharmacological treatment started 3 days prior monocrotaline (MCT) injection. Control animals received water ad libitum.

Monocrotaline treatment

Monocrotaline (MCT, Sigma) was dissolved in 1 M HCl, adjusted to pH 7.4 with 1 M NaOH and administered as a single subcutaneous injection in a dose of 60 mg/kg body weight, as described [1]. Control rats received an equal volume of isotonic saline.

Histological analysis

7 and 21 days after MCT injection, rats (n=4-5 for each group and time point) were sacrificed by final isoflurane anesthesia. Abdominal vessels and thoracic cavity were opened, a canula was inserted into the right ventricle and the animals were perfused with phosphate-buffered saline (PBS). The left lungs were dissected and shock frozen in liquid nitrogen. Afterwards, right lungs were perfused and fixed with 4% paraformaldehyde/PBS solution, paraffin-embedded and sectioned in slices (~4µm). Hearts were removed and right ventricle walls were dissected. Afterwards, the ratio of the right ventricle free wall weight to left ventricle and septum weight (RV/LV+S) was calculated for each animal sacrificed.

Morphometrical analysis of pulmonary arteries

For morphometrical analysis of pulmonary vessels, a computerised morphometric analysing system was used (Leica Q Win Standard Analyzing Software; Leica Microsystems GmbH). Briefly, at 400x magnification 80-100 small pulmonary vessels of each animal (n=4-5 for each group) ranging 10–100 µm in external diameter. Percentage of medial wall thickness was calculated by: (2xmedia thickness/external diameter) x 100. Media thickness was defined as the distance between the lamina elastica interna and the lamina elastica externa. Slides were analyzed by light microscopy by one observer in a blinded manner.

Evaluation of in situ pulmonary arterial SMC proliferation

Six days after monocrotaline challenge, animals received an intraperitoneal injection of 100mg/kg bw 5-Bromo-2-deoxy-Uridine [BrdU; Roche Applied Science, Mannheim, Germany) dissolved in PBS. 24hours later, respectively 7days after MCT injection, animals were sacrificed (n=5 for each group) and BrdU was visualized by a commercially available kit (Roche Applied Science) in deparaffinized pulmonary histological sections with the modification that the antigens were recovered by heating in citrate buffer (pH6.1). BrdU was detected with an anti-BrdU primary antibody (Roche Applied Science) and visualized by an Alexa 546 coupled (1:300; Invitrogen, Karlsruhe,Germany) secondary antibody. α-smooth muscle actin was detected by a directly labeled fluorescein isothiocyanate (FITC)-coupled antibody (1:200; Sigma) and nuclei were counterstained using ToPro-3 iodide (1:1000). Images of each right lung lobe (image size 900x900µm) were acquired by laser scanning microscopy (LSM 510meta; Carl Zeiss, Microimaging, Jena, Germany), and BrdU positive vascular smooth muscle cells were counted for each animal by observers blinded to the study protocol.

Western blot analysis

Lungs were mortared in liquid nitrogen, dissolved in triton-X-100 (1%) lysis buffers containing protease inhibitors and subjected to SDS-PAGE as described previously [2]. After Western blotting the following proteins were detected by antibodies: endothelial NO synthase (eNOS), AKT and β-Actin.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide pulmonary arterial smooth muscle cells proliferation assay

Rat pulmonary artery smooth muscle cells (PASMCs) were seeded in multiwell plates after isolating them from pulmonary arteries of untreated rats. When a subconfluent growth state was reached, cells were subjected to 1 day serum starving with 0.1% bovine serum albumin (BSA) in standard cell culture medium. Afterwards, the culture medium was replaced by a 0.5% fetal calf serum (FCS) containing medium, Levosimendan (100nmol/l; 1µmol/l, 10µmol/l, 100µmol/l final concentrations) or solvent (DMSO) was added and proliferation was subsequently stimulated by platelet-derived growth factor-BB (PDGF-BB; 30 ng/ml). After 2 days plates were washed and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added. Following a further incubation period (2h, 37°C, 5% CO2), acidified isopropanol was added to dissolve the precipitated formazan. Absorbance was analyzed by a spectrophotometer (550nm wavelength; Wallac Victor; EG&G Wallac, Freiburg, Germany).

Determination of 3´,5´-cyclic guanosine monophosphate (cGMP) in cultured human umbilical vein endothelial cells

First passage human umbilical vein endothelial cells (HUVEC´s), isolated as previously described [3], were seeded in culture dishes containing MCDB medium and 20% FCS supplemented with penicillin (50U/ml) and streptomycin (50µg/ml). After 1h of serum deprivation, isobutylmethyl xanthine (0.5mmol/l), a non-specific phosphodiesterase inhibitor was added to the medium for 30min followed by the addition of Levosimendan (100nmol/l, 1µmol/l, 10µmol/l) or solvent (dimethyl sulfoxide) for another 10 minutes. Sodium nitroprusside dihydrate (SNP; 30µmol/l, 3 min) was used as a positive control. The reaction was stopped by adding trichloracetic acid (6%), cGMP was extracted and determined by radioimmunoassay as described previously [4].

Determination of blood glucose level

Rats (n=5 for each group) were anesthetized repetitively with isoflurane at the time points indicated, blood samples were obtained from the sublingual vein, blood glucose levels were determined with a conventional dry-chemistry blood glucose meter .

mRNA expression in HUVEC´s

2nd passage HUVECs were grown to confluence in 3.5 cm dishes. FCS was reduced to 0.5% for a period of 4 hours. Then, Levosimendan (10µmol/l, 1µmol/l, 100nmol/l), glibenclamide (10µmol/l; Sigma, Deishofen, Germany) or solvent (DMSO) were added and cells were stimulated with tumor necrosis factor α (TNF-α, 10ng/ml) for additional 4hours. RNA isolation was done with Absolutely RNA Miniprep Kit (Stratagene), cDNA synthesis with SuperScript III Reverse Transcriptase (Invitrogen) and random hexamer primers, semiquantitative real-time PCR with ABsolute QPCR SYBR Green Mix and ROX as reference dye (Thermo Scientific) in Mx4000 (Stratagene) with the following primers: ICAM-1 ICAM-1 (Inter-Cellular Adhesion Molecule 1): 5’-CAGTGGGCAAGAACCTTAC-CCTAC-3’, 5’-GTTCAGTGCGGCACGAGAAATTGG-3’; MCP-1 (monocyte chemotactic protein-1): 5’-CAAGCAGAAGTGGGTTCAGGAT-3’, 5’-TTAGCTGCAGATTCTTGGGT-TGT-3’; COX 2 (cyclooxygenase-2): 5’-CCAGCACTTCACGCATCAGT-3’; 5’-ACGCTGT-CTAGCCAGAGTTTCAC-3’; E-Selectin: 5’-GTCCTGAAGGATGGACGCTCAATG-3’, 5’-GCAGCAGAAAGTCCAGCTACCAAG-3’. Relative expressions of target genes were normalized to eukaryotic translation elongation factor 2 (EEF2), analyzed by delta-delta-Ct method and given as percentage compared to control experiments.

Reporter gene assays

Experiments were performed similar as reported [2]. In brief: HUVECs were seeded on 3.5 cm dishes and transiently transfected with luciferase reporter constructs for AP1, NFκB and HIF-1α contained in the pGL3p vector. Control cells were transfected with pGL3p. Cells were subsequently treated with or without TNF-α and levosimendan in the concentrations indicated over night, subsequently lysed and subjected to protein determination. Luciferase activity was measured in a LB9505 chemiluminescence machine (Berthold) and normalized to the maximal activity.

Data analysis

All values are mean ± SEM. Statistical analysis was performed by analysis of variance (ANOVA) followed by Fisher’s LSD test or, wherever appropriate by an unpaired t-test. Survival analysis was performed by Log-rank (Mantel-Cox) Test. A P value less than 0.05 was considered statistically significant.

Reference List

1. Schermuly RT, Kreisselmeier KP, Ghofrani HA, Samidurai A, Pullamsetti S, Weissmann N, Schudt C, Ermert L, Seeger W, Grimminger F (2004) Antiremodeling effects of iloprost and the dual-selective phosphodiesterase 3/4 inhibitor tolafentrine in chronic experimental pulmonary hypertension. Circ Res 94:1101-1108.

2. Schroder K, Kohnen A, Aicher A, Liehn EA, Buchse T, Stein S, Weber C, Dimmeler S, Brandes RP (2009) NADPH oxidase Nox2 is required for hypoxia-induced mobilization of endothelial progenitor cells. Circ Res 105:537-544.

3. Wiemer G, Scholkens BA, Becker RH, Busse R (1991) Ramiprilat enhances endothelial autacoid formation by inhibiting breakdown of endothelium-derived bradykinin. Hypertension 18:558-563.

4. Fisslthaler B, Loot AE, Mohamed A, Busse R, Fleming I (2008) Inhibition of endothelial nitric oxide synthase activity by proline-rich tyrosine kinase 2 in response to fluid shear stress and insulin. Circ Res 102:1520-1528.

1