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F9. ANALYSIS OF TLR4 IN MYOCARDIAL ISCHEMIA-REPERFUSION INJURY
Ani J Fleisig, Christine L Rothnie, Denise J Spring, Erzsebet Toth, Matthew L Agnew, Timothy H Pohlman, Edward D Verrier; Seattle, WA
Objective: Toll-like receptor 4 (TLR4), is a transmembrane receptor, expressed on leukocytes, that plays a key role in innate immunity and inflammation. TLR4 is also expressed on non-myeloid cells, such as cardiac myocytes, but the functional significance of TLR4 expression (and its downstream myeloid differentiation factor or MyD88) in the heart is not understood. We have previously found resistance to myocardial I/R injury in TLR4 deficient mice, using an in vivo murine model of myocardial ischemia-reperfusion (I/R) injury. We believe that TLR4 activation elicits an inflammatory response which mediates myocardial I/R injury, therefore, deletion of TLR4 affords protection in the heart. To further elucidate the functional significance of TLR4 we have examined myocardial I/R in TLR4-/- and MyD88 -/- mice, as well as, developed an in vitro model that mimics in vivo I/R.
Methods: Wild type, TLR4-/- and MyD88-/- mice on a C57BL/6 genetic background were subjected to regional myocardial I/R injury by transient occlusion of the left anterior descending artery for thirty minutes, followed by a two-hour reperfusion period. Infarct size was measured at the conclusion of the reperfusion period. Frozen-tissue samples of the left ventricle were also obtained for molecular analysis. Cardiomyocyte cultures (HL-1 cells), endothelial cells (HUVEC), and primary cardiomyocytes (isolated from wild type, TLR4-/- and MyD88-/- mice) were exposed to “ischemia” (hypoxia, acidosis, depleted energy sources), followed by a two-hour “reperfusion” period. Cultures were assayed for viability using MTT. Relative activation of signal transduction molecules (MAPKs) was measured. NF-κB activation was measured by EMSA on nuclear proteins from cultured cells.
Results: Compared to wild type, infarct size was smaller in both TLR4-/- (22.63 vs. 38.09, p=0.001) and MyD88-/- mice (25.06 vs. 38.09, p=0.003). TLR4 was expressed on all cell types studied, except TLR4-/-. A dose dependent reduction in the number of viable cells was observed in all cell types exposed to in vitro I/R, except those cell types lacking TLR4 and MyD88. Significant activation differences were seen between the MAPKs (JNK, ERK, p38). Interestingly, we found constitutive activation of NF-κB in MyD88-/- mice.
Conclusions: We conclude that TLR4 signaling through MyD88 contributes to myocardial I/R. Constitutive activation of NF-κB in MyD88-/- may indicate an alternate level of pathway regulation.
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