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SUPPLEMENTAL MATERIAL Supplementary methods Cell culture The neonatal rats were sacrificed by 2% isoflurane inhalation and cervical dislocation. Isolation and culture of ventricular cardiomyocytes, fibroblasts, and cardiac microvascular ECs was performed as described previously 1-3 with 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. 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 10 mL 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. (Figure S1). Infarct size determination To evaluate the extent of cardiac fibrosis, each heart was cut into 4 sections and stained with Masson trichrome. Then the extent of fibrosis (old infarct size) was determined as the ratio of the Masson trichrome-stained area to the total left ventricular area. Briefly, Infarct scar area and the total area of LV myocardium were traced manually in the digital images and measured automatically using Image J. Infarct size, expressed as a percentage, was calculated by dividing the sum of infarct areas from all sections by the sum of LV areas from all sections (including those without infarct scar) and multiplying by 100. (Figure S2)
Caspase-9 activity assay Caspase-9 Fluorometric Assay kit (catalog No. BF7100, R&D) was used for this examination. Cardiomyocytes were collected by centrifugation in a conical tube at 250 x g for 10 minutes. The supernate was gently removed and discarded while the cell pellet was lysed by the addition of 50 µl Lysis Buffer and incubated on ice for 10 minutes. The resulting cell lysate was centrifuged for 1 min at 10,000 × g and the supernatant was collected for enzyme reaction. Each reaction requires 50 μL of cell lysate and 50 μL of 2 x Reaction Buffer containing 0.5 µl DTT. 5 µl of caspase-9 fluorogenic substrate (LEHD-AFC) was added into the respective samples and incubated in the dark at 37 ◦C for 1 h. At the end of the incubation period, the samples were read on a fluorescent microplate reader with excitation at 400 nm and emission at 505 nm. Caspase -9 activity was normalized to the quantity of viable cells. Supplementary references  Korhonen T, Hanninen SL, Tavi P. Model of excitation-contraction coupling of rat neonatal ventricular myocytes. Biophys J 2009;96:1189-1209.  Tiede K, Melchior-Becker A, Fischer JW. Transcriptional and posttranscriptional regulators of biglycan in cardiac fibroblasts. Basic Res Cardiol 2010;105:99-108.  Mountain DJ, Singh M, Menon B, Singh K. Interleukin-1beta increases expression and activity of matrix metalloproteinase-2 in cardiac microvascular endothelial cells: role of PKCalpha/beta1 and MAPKs. Am J Physiol Cell Physiol 2007;292:C867-875.
Table S1. Sequences of primers for routine PCR Table S2. Sequences of primers for real-time PCR
Supplementary Figures C B A D Figure S1. Cell type identification. (A) Cardiomyocytes were confirmed by immunocytochemistry using antibody against myoactin. (B) Cardiac fibroblasts were indicated by positive viementin stain and negative myoactin stain. (C and D) The microvascular endothelial cells displayed a “cobblestone” growth pattern, and were characterized by positive staining with factor VIII and CD31 antibodies. Scale bar = 20 μM. Area: a Area: b Area: c Infarct size = c/(a – b)×100% Figure S2. Example of infarct size measurement. In this section, the external and as well as internal LV cavity area as well as infarct scar area were traced manually and measured automatically using Image J software. The area of LV myocardium was calculated as external LV cavity area (a) - inner LV cavity area (b), infarct size was calculated by dividing the sum of infarct areas from all sections by the sum of LV areas. Scale bar = 1 mm.
A C B endothelia cells fibroblasts ANP cardiomyocytes endothelia cells ANP ICAM-1 MMP-9 D E F MMP-9 cardiomyocytes fibroblasts ICAM-1 β-actin Procollagen I and III TGF-β TNF-α induced FKN G FKN(ng/ml) 0 100 0 100 Figure S3. Amplification plots of targeted genes in cultured neonatal cardiac cells. (A) β-actin /ANP in cardiomyocytes with fractalkine stimulation. (B) β-actin /ICAM-1 in endothelial cells with fractalkine stimulation. (C-E) β-actin /MMP-9, procollagen I and III and TGF-β in firoblast cells with fractalkine stimulation. (F-G) β-actin/FKN in endothelial cells with TNF-αor H2O2 stimulation. H2O2 induced FKN Figure S4. Results of reverse-transcription PCR. Fractalkine (FKN) upregulated ANP in fibroblast cells, MMP-9 in cardiomyocytes and ICAM-1 in both cardiomyocytes and fibroblast cells. Each experiment was repeated for 3 times.