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TRPM2 Inhibition Increases Cardiomyocyte Viability Following Oxygen-Glucose Deprivation (OGD)

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

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TRPM2 Inhibition Increases Cardiomyocyte Viability Following Oxygen-Glucose Deprivation (OGD)

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  1. TRPM2 Inhibition Increases Cardiomyocyte Viability Following Oxygen-Glucose Deprivation (OGD) Matthias J Merkel, MD PhD1,2; Liu Lijuan, DVM2; Cao Zhiping, PhD2; Kent Thornburg, PhD1,3; Paco S Herson, PhD1; Donna M Van Winkle, PhD1,2 1Anesthesiology, OHSU, Portland, OR; 2Anesthesia & Research Services, OHSU, Portland, OR and 3Heart Research Center, OHSU, Portland, OR RESULTS BACKGROUND 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). 1A 1B 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 HYPOTHESIS 2B 2A • TRPM2 inhibition preserves viability of cardiomyocytes when given following oxygen and glucose deprivation METHOD • Animals • IACUC approved • Adult mouse cardiomyocytes (CMa) were isolated and cultured from 10-12 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 Ca2+) • 10 min perfusion with Ca2+-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 Ca2+ 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 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) 3B 3A 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 CONCLUSION merkelm@ohsu.edu • 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: 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, 13,1815-1826 O’Connell et al.: Isolation and culture of adult mouse cardiac myocytes. Methods Mol Biol 2007, 237, 271-296

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