Early reperfusion following myocardial ischemia is the main cardioprotective treatment in clinical practice today. However, this results in various degrees of myocardial ischemia-reperfusion (I/R) injury with cell death and cardiac dysfunction. Successful, novel interventions at the time of reperfusion are necessary to reduce myocardial I/R injury in a clinical feasible manner. The transient receptor potential melastatin 2 (TRPM2) receptor, a non-selective cation channel expressed in various cells including cardiomyocytes, is activated following the release of reactive nitrogen and oxygen species (RNS/ROS) and the formation of ADPribose (ADPr). TRPM2 activation results in cell death following oxidative stress (1). TRPM2 Inhibition Increases Cardiomyocyte Viability Following Oxygen-Glucose Deprivation (OGD) Matthias J Merkel, MD PhD 1,2 ; Liu Lijuan, DVM 2 ; Cao Zhiping, PhD 2 ; Kent Thornburg, PhD 1,3 ; Paco S Herson, PhD 1 ; Donna M Van Winkle, PhD 1,2 1 Anesthesiology, OHSU, Portland, OR; 2 Anesthesia & Research Services, OHSU, Portland, OR and 3 Heart Research Center, OHSU, Portland, OR Animals IACUC approved Adult mouse cardiomyocytes (CM a ) were isolated and cultured from week old C57\BL6 male mice hearts. Hearts were rapidly excised under general anesthesia Cell isolation & culture (adapted from (2)): Manual perfusion with 2 ml Krebs-Henseleit solution (+ 1.2 mM Ca 2+ ) 10 min perfusion with Ca 2+ -free Krebs-Henseleit solution and 10mM BDM 25 min perfusion with collagenase type 2 (140 ml total) Add stopping buffer (1%BSA in Krebs-Henseleit solution) Disperse and mince with sterile transfer pipettes Re-suspend in 10 ml stopping buffer Re-introduce Ca 2+ in 3 steps to final concentration of 1.2 mM Plate 30,000 rod-shaped on laminin-coated cells per well (24 well plate) Viability assessment Cell death assessed by trypane blue staining Count > 300 total cells Data are expressed as percentage of dead cells (mean ± SEM) 1-way ANOVA and Newman-Keuls post test Experimental design & timeline: All experiments were accompanied by an oxygenated and vehicle control BACKGROUND METHOD RESULTS CONCLUSION  Our findings suggest that TRPM2 activation is an important cell death mechanism during reoxygenation in cardiomyocytes.  Pharmacologic TRPM2 inhibition during the clinical feasible timepoint of reperfusion represents an attractive novel intervention to protect ischemic myocardium at risk. REFERENCES: 1)Yang et al.: Activation of the transient receptor potential M2 channel and poly(ADP-ribose) polymerase is involved in oxidative stress-induced cardiomyocyte death. Cell Death Differ 2005, 13, )O’Connell et al.: Isolation and culture of adult mouse cardiac myocytes. Methods Mol Biol 2007, 237, HYPOTHESIS TRPM2 inhibition preserves viability of cardiomyocytes when given following oxygen and glucose deprivation Figure 1: Concentration-dependent reduction in cell death following TRPM2 inhibition at time of RGR Maximum reduction in cell death was achieved with 30 uM FFA = Flufenamic acid (A) and 100 uM 2-APB = 2-aminoetoxydiophenyl borate (B); vehicle = ethanol 1:1000, vehicle 2 = 1:100 Figure 2: Improved viability by TRPM2 inhibition at different time points during RGR Pharmacological inhibition of TRPM2 during RGR improved cell viability at all three timepoints tested. A) FFA (30 uM) B) 2-APB (100 uM) Figure 3A: Improved viability following OGD by TRPM2 inactivation TRPM2 –specific shRNA pretreated cells are more resistance to consecutive OGD/RGR compared to untreated (naïve) or GFP shRNA only (GFP) Figure 3B: No additional benefit of shRNA and inhibitor TRPM2 –specific shRNA (shRNA) and TRPM2 inhibition (2-APB) showed similar reduction in cell death alone or in combination 1A 2A 2B 1B 3A 3B