The myocardial infarct-exacerbating effect of cell-free DNA is mediated by the high-mobility group box 1–receptor for advanced glycation end products–Toll-like receptor 9 pathway

摘要

IntroductionDamage-associated molecular patterns, such as high-mobility group box 1 (HMGB1) and cell-free DNA (cfDNA), play critical roles in mediating ischemia-reperfusion injury (IRI). HMGB1 activates RAGE to exacerbate IRI, but the mechanism underlying cfDNA-induced myocardial IRI remains unknown. We hypothesized that the infarct-exacerbating effect of cfDNA is mediated by HMGB1 and receptor for advanced glycation end products (RAGE).MethodsC57BL/6 wild type mice, RAGE knockout (KO), and Toll-like receptor 9 KO mice underwent 20- or 40-minute occlusions of the left coronary artery followed by up to 60?minutes of reperfusion. Cardiac coronary perfusate was acquired from ischemic hearts without reperfusion. Exogenous mitochondrial DNA was acquired from livers of normal C57BL/6 mice. Myocardial infarct?size (IS) was reported as percent risk region, as measured by 2,3,5-triphenyltetrazolium chloride and?Phthalo blue (Heucotech, Fairless Hill, Pa) staining. cfDNA levels were measured by Sytox Green assay (Thermo Fisher Scientific, Waltham, Mass) and/or spectrophotometer.ResultsFree HMGB1 and cfDNA levels were increased in the ischemic myocardium during prolonged ischemia and subsequently in the plasma during reperfusion. In C57BL/6 mice undergoing 40′/60′ IRI, deoxyribonuclease I, or anti-HMGB1 monoclonal antibody reduced IS by approximately half to 29.0%?±?5.2% and 24.3%?±?3.5% (P?P?P?=?not significant vs control). However, combined administration of recombinant HMGB1?+?mitochondrial DNA significantly increased IS (P?Conclusions:Both HMGB1 and cfDNA are released from the heart upon reperfusion after prolonged ischemia and?both contribute importantly and interdependently to?post-IRI by a common RAGE-Toll-like receptor 9–dependent mechanism. Depleting either of these 2 damage-associated molecular patterns suffices to significantly reduce IS by approximately 50%.