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Supplemental Data Article Detecting early myocardial ischemia in rat heart by MALDI imaging mass spectrometry ALJAKNA KHAN, Aleksandra, et al. Abstract Diagnostics of myocardial infarction in human post-mortem hearts can be achieved only if ischemia persisted for at least 6-12 h when certain morphological changes appear in myocardium. The initial 4 h of ischemia is difficult to diagnose due to lack of a standardized method. Developing a panel of molecular tissue markers is a promising approach and can be accelerated by characterization of molecular changes. This study is the first untargeted metabolomic profiling of ischemic myocardium during the initial 4 h directly from tissue section. Ischemic hearts from an ex-vivo Langendorff model were analysed using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) at 15 min, 30 min, 1 h, 2 h, and 4 h. Region-specific molecular changes were identified even in absence of evident histological lesions and were segregated by unsupervised cluster analysis. Significantly differentially expressed features were detected by multivariate analysis starting at 15 min while their number increased with prolonged ischemia. The biggest significant increase at 15 min was observed for m/z 682.1294 (likely [...] Reference ALJAKNA KHAN, Aleksandra, et al. Detecting early myocardial ischemia in rat heart by MALDI imaging mass spectrometry. Scientific Reports, 2021, vol. 11, no. 1, p. 5135 DOI : 10.1038/s41598-021-84523-z PMID : 33664384 Available at: http://archive-ouverte.unige.ch/unige:150558 Disclaimer: layout of this document may differ from the published version. 1 / 1 Detecting Early Myocardial Ischemia in Rat Heart by MALDI Imaging Mass Spectrometry Online Supplementary Material for Scientific Reports Aleksandra Aljakna Khan1, Nasim Bararpour2,3, Marie Gorka4, Timothée Joye2,3, Sandrine Morel5, Christophe Montessuit5, Silke Grabherr1,2, Tony Fracasso1, Marc Augsburger1,2, Brenda R. Kwak5, Aurélien Thomas1,3, Sara Sabatasso1,2* 1 University Centre of Legal Medicine, Lausanne-Geneva, Rue Michel-Servet 1, 1211 Geneva, Switzerland 2 University Centre of Legal Medicine, Lausanne-Geneva, Rue Vulliette 04, 1000 Lausanne, Switzerland 3 Faculty of Biology and Medicine, Lausanne University Hospital, University of Lausanne, Rue Vulliette 04, 1000 Lausanne, Switzerland 4 Ecole des Sciences Criminelles/School of Criminal Justice, Faculty of Law, Criminal Justice, and Public Administration, University of Lausanne, 1015 Lausanne-Dorigny, Switzerland 5 Department of Pathology and Immunology, University of Geneva, Rue Michel-Servet 1, 1211 Geneva, Switzerland Corresponding author: Sara Sabatasso, M.D. University Centre of Legal Medicine Lausanne-Geneva (CURML) Unit of Forensic Medicine, CMU Rue Michel-Servet 1 1211 Geneva Tel: +41 (0)79 556 9118 Switzerland E-mail: [email protected] TABLE OF CONTENTS SUPPLEMENTARY MATERIAL .......................................................................................................................2 Supplementary Methods ...................................................................................................................................2 Animals and ex-vivo Langendorff heart perfusion ....................................................................................2 Immunohistochemistry and immunofluorescence.....................................................................................2 Confirmation of ischemia in the ex-vivo Langendorff model ...................................................................3 Evans blue and TTC staining ......................................................................................................................3 H&E ...............................................................................................................................................................3 Immunohistochemistry for dCx43 ..............................................................................................................3 Changes in left ventricular developed pressure (LVDP) and coronary flow ..........................................4 Supplementary Figures .....................................................................................................................................5 Figure S1 – diagram of the experimental design and workflow ....................................................................5 Figure S2 – Evans blue and TTC staining .....................................................................................................6 Figure S3 – H&E staining .............................................................................................................................7 Figure S4 – immunohistochemistry for dephosphorylated Cx43 (dCx43) ....................................................7 Figure S5 – LVDP and heart rate ..................................................................................................................8 Figure S6 – MS/MS .......................................................................................................................................9 Supplementary Tables ....................................................................................................................................10 1 Aljakna Khan et al., Early Myocardial Ischemia Markers by MALDI IMS SUPPLEMENTARY MATERIAL Supplementary Methods Animals and ex-vivo Langendorff heart perfusion All animal experiments were performed according to the approved protocol by the Swiss veterinary authorities (GE/83/16). Lewis male rats (195-275 g) were housed in controlled, conventional conditions (20-24°C and 30-70% humidity) in an authorized facility under veterinary supervision with free access to food and water. All rats were allowed a minimum one-week adaptation period after arrival to the local animal facility and were randomly assigned into control or ischemic groups. The rats were premedicated by subcutaneous injection of buprenorphine (0.05 mg/kg, Temgesic, Reckitt Benckiser AG, Switzerland). After 20 minutes, the rats were deeply anesthetized by one intraperitoneal injection of ketamine and diazepamum mix (100 mg/kg, Ketasol, Graeub AG, Switzerland and 5 mg/kg, Valium, Roche Pharma AG, Switzerland, respectively). This anesthetic mix was chosen to minimize the adverse cardiovascular effect. After confirmation of deep anesthesia by absence of reflex in the posterior paws, the hearts were rapidly isolated, cannulated via the aorta to the ex-vivo Langendorff system, and retrogradely perfused under constant pressure at 37°C with oxygenated Krebs-Henseleit buffer solution (NaCl 118 mM, KCl 4.7 mM, MgSO4 1.19 mM, KH2PO4 1.2 mM, CaCl2 1.36 mM, NaHCO3 25 mM, Glucose 11 mM) (Fig. S1A). Euthanasia was performed by rapid excision of the heart under deep anesthesia. Throughout the experiments, the temperature was monitored. The left ventricular developed pressure (LVDP) and heart rate were measured via a balloon, which was inserted into the left ventricle, and connected by a pressure transducer to a computer with appropriate software (Lab Chart, ADInstruments). LVDP was fixed around 6 mmHg for all of the hearts at the beginning of the stabilization period. The hearts were stabilized for 20 min and local ischemia was induced by complete ligation of LAD using silk suture (6-0 Perma Hand, BV-1, ETHICON). Hearts were exposed to 15 min, 30 min, 1 h, 2 h, and 4 h ischemia (n = 5 per time point: 5 biological replicates at each time point, 30 samples in total) and were immediately frozen. The use of optimal cutting temperature (OCT) polymer was intentionally omitted because OCT can lead to analyte ion suppression. Control hearts were subjected to the same procedure, except the suture was not tied (it was only passed under LAD). After 20 min of stabilization, control hearts were maintained by Langendorff perfusion for 1h. Success of LAD ligation was confirmed by staining with Evans blue and triphenyltetrazolium chloride (TTC), H&E, immunohistochemistry for connexin43 (Cx43) as well as by monitoring the changes in LVDP and coronary flow (see below, Figs. S2-S5 and Table S1). Immunohistochemistry and immunofluorescence For immunohistochemistry, cryosections from frozen hearts (12 m) were fixed for 5 min in pre-chilled methanol, permeabilized in 0.2% Triton X-100, neutralized with 0.5 M NH4Cl in PBS, incubated with 3% H2O2, blocked with bovine serum albumin (BSA) and avidin/biotin blocking kit (Abcam), probed with primary antibody that recognizes multiple phospho-forms of Cx43 (rabbit polyclonal, Alpha Diagnostic Intl. Inc. Cx43B12-A, 1:100), incubated with biotinylated secondary antibody (anti-rabbit, Vector Laboratories, 1:200) and streptavidin- HRP (1:300), visualized with colorimetric detection kit (Vector AEC, Vector Laboratories), and counterstained with hematoxylin (Fig.1 p-y in the main text of the article). For immunofluorescence, cryosections (7 m) were dried for 15 min at RT, fixed for 10 min in 2 Aljakna Khan et al., Early Myocardial Ischemia Markers by MALDI IMS pre-chilled acetone, blocked and permeabilized with blocking buffer (1% BSA, 10% goat serum, and 0.5% TritonX-100 in PBS), probed with primary antibody for Nav1.5 (custom-made rabbit polyclonal for amino acids 493-511 of rat Nav1.5, gift from Prof. Hugues Abriel, 1:200), and incubated with secondary DyLight 488 antibody (goat anti-rabbit IgG H&L, Abcam, 1:200). Coverslips were mounted with FluorSave (Merck Millipore) (Fig.6 in the main text of the article). Negative controls (the primary antibody
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