Supplementary material

Supplemental Methods

Animal model construction

Male C57BL/6 mice were anesthetized with 2 % isoflurane, and an incision was made between the fourth and fifth left ribs to expose the heart. Notch3 siRNA (20μg) or scrambled siRNA was diluted in 30μl of vivo-jetPEITM (Invitrogen, USA) and 10% glucose mixture. Scrambled siRNA (30μl) or Notch3 siRNA solution was delivered via six separate intramyocardial injections (5 μl per injection) into the left ventricle apex and anterolateral wall using a 30-gauge needle [1]. Notch3 overexpression lentivirus was injected into the free anterior wall of the left ventricle (LV) at six different sites. Three days after infection of siRNA or Notch3 cDNA lentivirus, the incision was opened under anesthetic condition once again. Cardiac I/R injury model was constructed as previously described [2]. A 6-0 silk suture slipknot was placed at the proximal one-third of the left anterior descending artery. After 30 min of ischemia, the slipknot was released, and the myocardium was reperfused for 3 h. Sham group underwent the same surgical protocols except that the suture placed under the left coronary artery was not tied.

Determination of Myocardial Apoptosis

Caspase-3 activity was measured with the ApoAlert Caspase-3 Assay Plate (Clontech, Mountain View, Calif) according to the manufacturer’s instructions [2].

Notch3 siRNA or lentivirus carrying Notch3 cDNA construction

Notch3 siRNA oligonucleotides were purchased from GenePharma Company (Shanghai, China). The RNAi sequence targeting Notch3 against murine is sense 50-GGCCAGUUUACUUGCAU

CUTT-30 and antisense 50-AGAUGCAAGUAAACGGCCTT-30，rat is sense 50-GGCA

CACAUUGCCAAUAUATT-30 and antisense 50-UAUAUUGGCAAUGUGUGCCTT-30. The siRNA was transfected into cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Lentivirus carrying Notch3 cDNA was purchased from GeneChem Company (Shanghai, China).

Isolation mitochondria from hearts

Mitochondria was isolated from hearts as previously described [5].We used the ice-cold medium A (120 mM NaCl, 2 mM MgCl2, 20 mM HEPES, 1 mM EGTA, and 5 g/l bovine serum albumin; pH 7.4) to rinse the hearts in order to remove any residual blood. Cardiac tissue was minced in ice-cold medium A and homogenized. The homogenate was centrifuged (600×g, 10 min, 4 ℃). The supernatant was subsequently centrifuged (17,000×g, 10 min, 4 ℃). The pellet containing the mitochondria was re-suspended in medium A, and then centrifuged (7,000×g, 10 min , 4 ℃). After the last centrifugation, the pellet was re-suspended in medium B (2 mM HEPES, 300 mM sucrose, 0.1 mM EGTA; pH 7.4) and re-centrifuged (3,500×g, 10 min, 4 ℃). The resulting pellet containing the heart mitochondria was suspended in a small volume of medium B.

Western Blot Evaluation

Total proteins from cardiomyocytes were separated by SDS-PAGE, blotted and probed with anti-β-actin antibody (Santa Cruz, CA, USA), anti-OSM, anti-Oβ (R&D systems, USA), anti-Notch3 intracellular domain (Notch3 ICD) (Abcam, Cambridge, MA,UK), anti-p-STAT3, anti-STAT3, anti-p-p70S6k (Thr389), anti-p70S6k, anti-p-Akt (ser473), anti-Akt, anti-p-mTOR (Ser2448), anti-mTOR (Cell Signaling, Danvers, MA,USA), p-AMPK (Thr172), adenine mononucleotide protein kinase (AMPK), acetylated- Lysine, peroxisome proliferator-activated 5 receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (Nrf-1), mitochondrial6 transcription factor A (Tfam) (Cell Signaling Technology, Beverly, MA,USA), anti-Bcl-2, anti-Bax (Sigma, St Louis, MO, USA). The Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) was used to quantify protein concentrations. The blots were visualized with a chemiluminescence system (Amersham Bioscience, Buchinghamshire, UK). The signals were quantified by densitometry and normalized to β-actin.

Assessment of cardiomyocyte injury

Myocardial cellular damage was evaluated by measuring lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) activity in plasma. LDH and CK-MB released from ischemic tissue were determined from arterial blood drawn from the carotid catheter 3 h after reperfusion. LDH and CK-MB activity were measured spectrophotometrically with a commercially available assay.

Immunostaining assay

For immunofluorescence staining, H9C2 cardiomyocytes slides were fixed with 4% paraformaldehyde for 10 min and later incubated with 10 % normal goat serum containing0.3 % bovine serum albumin (BSA; Sigma) for 1 h, after which slides were incubated with rabbit anti-Notch3 (diluted 1:150; Santa Cruz, CA, USA) overnight at 4℃. The slides were incubated with a Cy3-conjugated anti-rabbit (Sigma) secondary antibodies and 4,6-diamidino-2-phenylindole (DAPI) (Sigma) stained all cell nuclei.

Determination of Myocardial Apoptosis

Myocardial apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining as previously described [2]. TUNEL staining was performed with fluorescein-dUTP (In Situ Cell Death Detection Kit; Roche Diagnostics) for apoptotic cell nuclei and 4’,6-diamidino-2-phenylindole (DAPI) (Sigma) stained all cell nuclei. Additional staining was performed using a monoclonal antibody against Troponin I (cTnI, Santa Cruz) for the identification of myocardium. Apoptotic index (AI) was determined. AI= number of TUNEL-positive myocytes / total number of myocytes stained with DAPI from a total of 40 fields per heart (n=5). All of these assays were performed in a blinded manner.

Flow cytometry analysis

Flow cytometric analysis of cellular apoptosis was performed as previously described [3]. In brief, H9C2 cardiomyocyteswere harvested and stained with annexin V (Invitrogen) and propidium iodide (PI). Data acquisition and analysis were performed using a flow cytometer (FACSort-B0008; Becton–Dickinson, Franklin Lakes, NJ, USA) and Cell Quest Pro software, respectively.

Measurement of mitochondrial membrane potential (ΔΨm)

The fluorescent, lipophilic and cationic probe, JC-1 (Beyotime, China), was employed to measure the mitochondrial membrane potential (DYm) of H9C2 cells according to the manufacturer’s directions. H9C2 cells in 35 mm2 dishes were suspended in HEPES saline buffer and the mitochondrial membrane potential (ΔΨm) was detected as previously described [4]. Then the cells were incubated with JC-1 (5μM) in growth medium for 30 min at 37 °C. The cultures were washed three times using fresh growth medium. Fluorescence was examined with a confocal laser scanning microscope (Spectra MaxGeminiXS, spectra Max, Atlanta, GA, USA) at an excitation wavelength of 490 nm. Green fluorescence represented the monomeric form of JC-1, appearing in the cytosol after mitochondrial membrane depolarization. Red emission represents a potential-dependent aggregation in the mitochondria, reflecting mitochondrial membrane potential measurement. Colocalization appeared as an orange red color because of the mixing of the red and yellow signals. Fluorescence of each sample was read at an excitation wavelength of 490 nm and an emission wavelength of 530 (green) and 590 (red) nm. Results in fluorescence intensity were expressed as 590-to-530-nm emission ratio. The DYm of cardiomyocytes in each treatment group was calculated as the uorescence ratio of red to green and was expressed as a multiple of the level in the control groups. All experiments were repeated at least three times.

Transmission electron microscopy (TEM)

Mice were anesthetized with 3% sodium pentobarbital, then they were perfused from the left ventricle with 30 ml of wash-out solution, and cardiectomy was performed. Ventricles were cut perpendicular to the long axis into 1-2 mm wide rings. After fixing overnight in 4 % glutaraldehyde, the sections were postfixed in 1 % osmium tetroxide for 1 h, dehydrated using agraded ethanol immersion series, and embedded in resin. Semi-thin sections were mounted on glass slides and stained with 1 % azure II in 1 % sodium borate. Ultrathin sections were cut at 80 nm thick on a LKB-NOVA, stained with uranium acetate and lead citrate, and observed with an electron microscope (JEM-2000EX TEM, Japan).Random sections were taken and analyzed by two technicians blinded to the treatments [5].

References

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Figure S1 OSM inhibited cardiomyocyte apoptosis induced by cardiac I/R injury in mice.

Caspase-3 activity analysis demonstrated OSM decreased caspase-3 expression in the mice that underwent cardiac I/R injury. *p＜0.05 vs Sham, # p＜0.05 vs Sham+OSM, §p＜0.05 vs I/R.

Figure S2 Oβ knockout enhanced cardiomyocyte apoptosis in mice that underwent

cardiac I/R injury

Caspase-3 activity analysis demonstrated Oβ knockout increased caspase-3 expression in the mice that underwent cardiac I/R injury. *p＜0.05 vs Sham+ Oβ+/+, # p＜0.05 vs Sham + Oβ-/-, §p＜0.05 vs Sham +OSM+ Oβ-/-, $p＜0.05 vs I/R+ Oβ+/+.

Figure S3 Notch3 overexpression inhibited cardiomyocyte apoptosis induced by cardiac I/R injury in mice

Caspase-3 activity analysis demonstrated Notch3 overexpression decreased caspase-3 expression in the mice that underwent cardiac I/R injury. *p＜0.05 vs Sham, # p＜0.05 vs I/R, §p＜0.05 vs I/R+siControl, $p＜0.05 vs I/R+siNotch3, p＜0.05 vs I/R+Control vector, □p＜0.05 vs I/R+Notch3.