Abstract 11803: Extracellular Mitochondrial DNA is a Putative Toll-Like Receptor 9 Agonist on Cardiac Fibroblasts During Myocardial Infarction
Background Toll-like receptor 9 (TLR9) recognizes CpG-motifs in bacterial DNA. Mitochondrial DNA (mtDNA) resembles bacterial DNA, and has been shown to be an endogenous ligand for TLR9. In such, there is a rationale for hypothesizing that mtDNA is released upon myocardial infarction (MI) with subsequent stimulation of cardiac TLR9. Therefore we investigated the expression and function of myocardial TLR9.
Methods and Results By PCR, we observed that plasma levels of mtDNA displayed a rapid, transient increase during human MI and PCI as compared to patients with stable angina pectoris undergoing the same procedure. We further analyzed TLR9 expression by RT-PCR in viable cardiac tissue at 3, 7 and 28 days after induction of MI in mice. An induction peaking at day 7 post-MI was seen. Furthermore, murine cardiac myocytes, fibroblasts and endothelial cells were isolated and analyzed for the presence of TLR9. Although present in all cellular entities, substantially higher TLR9 levels were observed in fibroblasts. Accordingly, further in vitro studies were performed in adult murine cardiac fibroblasts stimulated with different mtDNA mimicking molecules (i.e. CpG ODN) on activation of two canonical signaling pathways (NFκB and IRF/IFNβ by detection of TNFα and IL-8, and IFNβ, respectively). First, we demonstrated increases of TNFα, IL-8 and IFNβ upon stimulation with CpG A, B and C with varying potency. Using the most potent CpG (B), the dose-response relationship, as well as temporal profile was assessed. Peak expression levels were seen at 5 hours. Furthermore, a robust dose-response relationship was demonstrated with calculated EC50 values being equal for TNFα, IL-8 and IFNβ. We also demonstrated that induction of TNFα and IL-8 depends on internalization of CpG as chloroquine effectively attenuated activation. Finally, we demonstrated that CpG B exclusively signals through cardiac fibroblast TLR9 as a specific TLR9 antagonist (ODN 2088) completely inhibited activation within a narrow dose-inhibition window.
Conclusion TLR9 is both expressed and functional in cardiac tissue with cardiac fibroblasts being the putatively most important cellular source. This suggests that mtDNA released upon MI can function as a ligand mediating activation of cardiac TLR9.
- © 2011 by American Heart Association, Inc.