E. J. Charles1, Y. Tian2, J. H. Mehaffey1, D. Wu1, I. L. Kron1, Z. Yang1 1University Of Virginia,Surgery,Charlottesville, VA, Virgin Islands, U.S. 2Tianjin Medical University General Hospital,Cardiovascular Surgery,Tianjin, , China
Introduction: Damage-associated molecular patterns such as high mobility group box 1 (HMGB1) and mitochondrial DNA (mtDNA) may play critical roles in mediating myocardial ischemia-reperfusion (IR) injury. We hypothesized that HMGB1 and cell-free mtDNA collectively released from ischemia myocardium would lead to activation of splenic leukocytes and cause reperfusion injury.
Methods: Levels of HMGB1 and cell-free mtDNA were measured in plasma and cardiac perfusate obtained from C57BL6 mice (n=4-8/group) that underwent sham surgery or 10, 20, or 40-minute occlusions of the left coronary artery (LCA) without reperfusion. Perfusate was obtained via antegrade coronary perfusion with phosphate-buffered saline. Levels were measured using SYTOX® Green florescence and Western blot. Separate C57BL6 mice (n=4-8/group) underwent 20 minutes of LCA occlusion followed by 60 minutes of reperfusion. Resultant myocardial infarct size was measured with TTC and Phthalo blue staining. Treated groups received recombinant HMGB1 (0.1μg/g/2μl; rtHMGB1), mtDNA obtained from naive C57BL6 mouse livers (0.5ug/g/2μl; mtDNA), or both rtHMGB1 and mtDNA (H+D) 5 minutes prior to reperfusion via external jugular vein injection. An additional group of mice underwent splenectomy prior to LCA occlusion and received both rtHMGB1 and mtDNA (H+D+Splx).
Results: Plasma levels of HMGB1 and mtDNA were low and not significantly different between mice undergoing sham or LCA occlusion without reperfusion. However, cardiac perfusate levels were significantly increased in ischemic hearts after 40 minutes of LCA occlusion (p<0.05 vs. other groups). Infarct size as a percentage of left ventricular risk region after 40 minutes of LCA occlusion without reperfusion was 23.9±5.4%, compared to 0.0% after 20-minute occlusion (p<0.05). In mice undergoing 20 minutes of LCA occlusion followed by 60 minutes of reperfusion, injection of rtHMGB1 or mtDNA did not exacerbate infarct size compared to control mice (p>0.99). However, H+D mice had significantly larger infarct size (21.2±4.9%), compared with control, rtHMGB1, and mtDNA (all p<0.01, Figure 1). This increased infarct size was attenuated by splenectomy (H+D+Splx: 5.3±2.1%, p=0.02 vs H+D). There were no significant differences between groups in size of risk region as a percentage of left ventricular mass.
Conclusion: The release of both HMGB1 and mtDNA together from ischemic myocardium is critical to cause reperfusion injury and leads to increased infarct size. Blocking the effects HMGB1 and/or mtDNA on splenic leukocyte activation may provide a therapeutic option for attenuating myocardial IR injury.
