SUPPLEMENTAL MATERIAL. Supplementary Methods

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1 SUPPLEMENTAL MATERIAL Supplementary Methods Culture of cardiomyocytes, fibroblasts and cardiac microvascular endothelial cells The isolation and culturing of neonatal rat ventricular cardiomyocytes was performed as previously described with some modifications. After digesting the ventricular tissue, the cells were pelleted and the cells were re-suspended in Dulbecco s modified Eagle s medium (DMEM) containing 10% fetal bull serum (FBS) and 1% penicillin-streptomycin. The resulting cell mixture was pre-plated for 1 h to plate out cardiac fibroblasts. The isolated cardiomyocytes were plated into 6-well plates and cultured for 4-5 days. Pre-plated cardiac fibroblasts were cultured for 2 days and then 2-4 cells were used to passage. Primary neonatal rat cardiac microvascular endothelial cells (ECs) were obtained and cultured as described elsewhere with minor modifications. In brief, left ventricles from one week old Sprague-Dawley rats were isolated, and immersed in 70% ethanol for 10 s to devitalize epicardial mesothelial and endocardial ECs and then washed with calcium-free Hanks balanced salt solution (HBSS). The tissue was finely minced and digested in 15 ml HBSS containing 30 mg collagenase at 37 with gentle shaking for 20 min. After a second digestion under the same conditions with the addition of 3 mg trypsin, dissociated cells were filtered through a 100 μm mesh filter, suspended in 10ml HBSS, and centrifuged at 800 rpm for 5 min. Cells were resuspended in M199 medium supplemented with 20% (v/v) FBS and 1% penicillin-streptomycin. After 4 h, plating and removal of non-adherent cells by washing, cells usually reached confluence after 7-9 days, and displayed uniform cobblestone morphology. All experiments were carried out using primary culture cells. The confirmation of cell types was performed using immunochemistry assays (antimyoactin antibody for cardiomyocytes and anti- viementin antibody for fibroblasts (both antibodies were obtained from Wuhan Boster Biological Technology., LTD, China), anti-cd31 and factor VIII antibodies (RB, USA) for microvascular ECs). Anoxia/Reoxygenation protocol Before anoxia exposure, the cardiomyocytes medium was replaced with simulated ischemia buffer without glucose (in mm: NaCl 98.5, KCl 10, MgSO4 1.2, CaCl2 1.0, HEPES 20, sodium lactate 40, ph 6.8, 37 C). Then, the cardiomyocytes were transferred to and kept in an anaerobic incubator (Thermo, USA) at Nitrogen gas was then flushed into the incubator to bring the oxygen content down to 0.5% as monitored by an oxygen probe (Japan Storage Battery, Japan). After 3 h incubation, the cardiomyocytes were then removed to reoxygenate for another 2 h with reoxygenation buffer (in mm: NaH2PO4 0.9, NaHCO3 20.0, CaCl2 1.0, MgSO4 1.2, HEPES 20.0, NaCl 129.5, KCl 5.0, glucose 5.5, ph 7.4, 37 C) in normal air 1

2 incubator (CO2 incubator). As a control, cardiomyocytes were exposed to normoxia (room air). Global ischemia protocol C57BL/6 male mouse aged weeks were anesthetized with pentobarbital, the hearts were quickly excised, and perfused in Langendorff modeling using Krebs-Henseleit buffer (in mmol/l; NaCl, 118.5; NaHCO3,25.0; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; glucose, 11.1; CaCl2, 2.4). The buffer was bubbled with 95% O2 / 5% CO2 at 37 C and perfusion was performed under a hydrostatic pressure of 55 mm Hg. For global ischemia,the perfusion was suspended. Hearts were exposed to 20 min stabilization, 40 min global ischemia, and then 60 min reperfused with K-H buffer. Echocardiographic Measurements Both cardiac function and remodeling were evaluated by echocardiography using a Sequoia 512 system with a 15L-8 probe (Siemens, Germany) at 7, 14, 30, and 42 days after surgery. Mice were fixed in waking state to avoid the influence of anesthesia on the parameter measurements. Two-dimensional echocardiographic views of the mid-ventricular short axis were obtained at the level of the papillary muscle tips below the mitral valve. From M-mode tracings, the LV end-diastolic diameter (LVEDd) and LV end-systolic diameter (LVESd) were measured, while LVFS was obtained by the following equation: (LVEDd - LVESd) / LVEDd 100. Polymerase Chain Reaction Total RNA was extracted from culture cells and mouse heart tissues (total RNA isolation system, Omega, USA). Reverse transcription was carried out in 20 μl of reaction mixture containing 1 μg of total RNA. Digital images of ANP, ICAM-1, and β-actin bands separated on ethidium bromide-stained agarose gels were obtained. We further quantitatively checked the expression of ANP, ICAM-1, MMP-9, transforming growth factor beta (TGF-β), procollagen I, procollagen III and β-actin in cultured cardiac cells of neonatal rats and whole hearts of mice using Quantitect SYBR Green real-time PCR method as described elsewhere. Primer sequences were listed in Table S1. Western Blotting The following antibodies were used for the western blotting analysis: anti-fractalkine (R&D), anti-lc3 (microtubule-associated protein 1 light chain 3, P38, P-P38, ERK, P-ERK, anti-atg5 conjugate (all from Cell Signaling Technology) or anti-β-actin antibody (Boster) Proteins were obtained from whole-heart homogenates. Samples containing equal amounts of protein were separated by 12-15% SDS-PAGE and transferred onto Polyvingyl difluoride membranes. The membranes were blocked with 5% skim milk at room temperature for 2 h, and then incubated overnight at 4 2

3 with the primary antibodies. The blots were detected using a Supersignal ECL kit (Invitrogen, USA) in a Western blotting detection system (Kodak Digital Science, Rochester, NY) and quantified by densitometry using the Image J Analysis Software. Table S1. Sequences of primers for Real-time-PCR 3

4 Supplementary Results 4

5 Figure S3. Monodancylcadaverine (MDC, the highlighted green fluorescent) in neonatal rat cardiomyocytes. MDC stands for autophagosome. (A) Representative images of MDC. (B) Quantitative analysis of MDC in four groups. A/R: anoxia/reoxygenation, RES: resveratrol, FKN: fractalkine. 100 cells in each group were used to perform statistical analysis. 5

6 Figure S4. RES treatment inhibited phosphorylation of p38 and ERK in cultured cardiomyocytes. FKN was incubated for 30 minutes after pretreatment with RES for 1 hour. (A) Representative images of western blotting. (B) Quantitative analysis of MAPKs activities. N = 5 in each group. FKN: fractalkine, RES: resveratrol, MAPKs: Mitogen activated protein kinases, ERK: extracellular signal-regulated kinase. 6