Toll-like Receptor 4 on Leukocytes is Necessary for Cardiomyocyte Hypoxia - Reoxygenation Injury
Heather-Marie P. Wilson, Denise J. Spring, Christine Rothnie, Erzsebet Toth, Edward D. Verrier; Surgery, University of Washington, Seattle, WA
Comment on this Abstract
Objective: Cardiac ischemia reperfusion (I/R) injury occurs after most heart surgery. Previous work in our laboratory suggests that in response to ischemic stress, Toll-like receptor 4 (TLR4) mediates myocardial damage. Our overall objective is to determine the cellular and molecular mechanisms by which the innate immune system contributes to myocardial ischemic damage. The specific objective of this study is to determine the role of TLR4 on leukocytes and cardiomyocytes in mediating I/R injury in an in vitro model.
Methods: An in vitro I/R model was developed to determine the effect of leukocytes (WBCs) on cardiomyocyte viability following hypoxia/reperfusion. Primary WBCs were isolated from the buffy coat of peripheral blood collected from wildtype and TLR4-null mice. Murine primary adult cardiomyocytes or HL-1 cells (a murine cardiomyocyte cell line) were plated on laminin or gelatin-coated plates 24 hr prior to experiment. Cardiomyocytes were exposed to the following conditions: 1) 1 hr normoxia (control: constant pH, 21% O2) or 1 hr hypoxia (0.05% O2) in the presence of WBCs (wildtype or TLR4-null) followed by 1 hr reoxygenation with addition of fresh media; 2) 1 hr normoxia (control) or 1 hr hypoxia followed by 1 hr reoxygenation with primary WBCs (wildtype or TLR4-null). At the conclusion of the experiment, the cardiomyocytes were collected, stained with trypan blue, and counted for viability using a hemocytometer.
Results: In vitro studies indicate that the presence or later addition of wildtype primary WBCs to hypoxia-treated cardiomyocytes during reperfusion resulted in a 20% decrease of viability in primary and cell line-derived murine cardiomyocytes compared to untreated control cardiomyocytes. TLR4-null WBCs had no effect on hypoxia-stressed cardiomyocyte viability. These preliminary data suggest that immune cells significantly contribute to the extent of myocardial damage from I/R and TLR4 may mediate this damage. WBCs do not need to be exposed to hypoxia for this effect to occur indicating that hypoxia-stressed cardiomyocytes release signal(s) to activate the leukocytes.
Conclusion: We provide evidence for the role of TLR4-expressing leukocytes in ischemic damage of cardiomyocytes. We have developed an in vitro model of I/R for defining the role of individual cell populations (specifically cardiomyocytes and circulating leukocytes) in this process, which will allow us to further explore the TLR4 signaling pathways as targets for therapeutic intervention.
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