Abstract Body: Introduction: Ischemia/reperfusion (I/R) injury of the heart releases pro-inflammatory factors, including mitochondrial DNA (mtDNA). MtDNA can act as Damage Associated Molecular Patterns (DAMPs), mainly recognized by Toll-like receptor 9 (TLR9), which initiates pro-inflammatory pathways. We have shown that in ST-elevation myocardial infarction patients, the mtDNA DAMPs level is highly elevated. Prior studies showed that loss of TLR9 reduces infarct size in mouse hearts subjected to I/R ex vivo. However, the role of TLR9 in regulating inflammatory response in cardiac I/R injury in vivo is unclear.
Hypothesis: Loss of TLR9 ameliorates cardiac I/R injury by suppressing inflammation in vivo.
Methods: C57BL/6J wild-type (WT) and TLR9 germline KO (TLR9 gKO) mice were subjected to closed-chest I/R surgery. For WT mice, either ODN2088 (TLR9 antagonist) or vehicle were given at reperfusion. Cardiac systolic function and infarct size were evaluated by echocardiography and TTC staining (respectively), 24 hours after the reperfusion. In a separate group, immune cells were isolated from left ventricles and spleens; live cells were stained with immune cell markers and detected by flow cytometry. Furthermore, adult mouse ventricular myocytes (AMVMs) isolated from WT and TLR9 gKO mice were subjected to I/R in vitro.
Results: Circulating mtDNA DAMPs level was significantly increased after I/R. TLR9 gKO mice exhibit significantly reduced infarct size by ~40% (p=0.0063) and preserved the systolic function, with no sex difference. In WT mice subjected to I/R, ODN2088 treatment reduced the total number of cardiac leukocytes (CD45⁺) (p=0.0306), myeloid cells (CD45⁺, CD11b⁺) (p=0.0233), neutrophils (CD45⁺, CD11b⁺, Ly6G⁺) (p=0.0386) and TNF-α⁺ cells (p=0.0262), and a trend of reduced IL-1β⁺ cells (p=0.0638). There were no differences in IL-6⁺ cells, total macrophages, or resident macrophages. In AMVMs, loss of TLR9 suppressed the transcription of inflammatory markers TNFα and IL1β.
Conclusions: Blocking TLR9 during reperfusion significantly reduces infarct size by suppressing subsequent cardiac inflammatory response. The mtDNA-DAMP-TLR9 signaling pathway may be a viable target to mitigate cardiac I/R injury.