| Objective:Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide. Despite the fact that recent advances in coronary reperfusion strategies effectively improve short-and-long term prognosis following MI, ischemia reperfusion (I/R) injury could hamper the clinical benefit by inducing myocardial stunning, reperfusion arrhythmia, microcirculation dysfunction and no-reflow (NR) phenomenon. Neutrophils play important role in myocardial I/R injury. In response to microbes and other inflammatory stimuli and with the help of reactive oxygen species (ROS), neutrophils casting out neutrophil extracellular traps (NETs). NETs are composed of chromatin, histones and antimicrobial proteins. Many findings show that NETs can bind and kill microbes effectively, and were thought to be a new strategy of neutrophils to defend against invading pathogens and limit microbial spread. Recently, NETs has been demonstrated to participate in the process of thrombosis. Using I/R models, this study was designed to investigate the existence of NETs in I/R myocardium, and the therapeutic potential of NETs clearance based treatment on I/R induced myocardial NR phenomenon, and ventricular remodeling.Methods:After adaptive feeding for one week, six-week-old male Wistar rats were randomized into groups. According to the different types of intervention programs using deoxyribonuclease I (DNase) and recombinant tissue plasminogen activator (rt-PA), rats were divided into I/R Control group (physiological saline), DNase group (DNase), rt-PA group (rt-PA), DNase+rt-PA group (combination of DNase and rt-PA). All groups’rats for short term (Sham group underwent all procedures expect of occlusion of coronary artery) were subjected to45minutes of coronary occlusion followed by3h reperfusion. Drugs for intervention were used5min before reperfusion. Using confocal microscopy, histone2B (H2B), myeloperoxidase (MPO) and deoxyribonucleic acid (DNA) were used to investigate whether NETs formed in rats myocardium suffered from I/R. Triphenyltetrazolium chlorid (TTC) and Evans blue staining were used to explore the influence of intervention drugs on the infarct size. Thioflavin S and Evans blue staining were used to explore the influence of intervention drugs on the no-reflow size. For long term, Sham groups were divided into Sham Control group (physiological saline), Sham DNase group (DNase), Sham rt-PA group (rt-PA), Sham DNase+rt-PA group (combination of DNase and rt-PA). All groups’ rats for short term (Sham group underwent all procedures expect of occlusion of coronary artery) were subjected to45minutes of coronary occlusion followed by reperfusion. Drugs for intervention were used5min before reperfusion. Echocardiographic and hemodynamics measurements were performed in all groups’ rats45days after operation to evaluate myocardial functions and hemodynamic parameters. Masson trichrome staining was used to evaluate left ventricular free wall thickness (LVWT), septum thickness (SPT), index of expansion (IE). And wheat germ agglutinin (WGA) staining was performed to calculate myocardial cross-sectional area (MCSA).Results:1) We confirmed the co-localization of H2B, MPO and DNA in the myocardium received45min ischemia and3h reperfusion, indicating the existence of NETs.2) The coronary ligation area was similar among all sub-groups when evaluate proportion of infarct and no-reflow. Compared with I/R Control group, the infarct area/area at risk of infarction(IA/AAR) was significantly decreased (P<0.001) in the DNase+rt-PA group, and no reflow area/area at risk of infarction(NR/AAR) was also decreased (P=0.033) in this group.3) Compared with I/R Control group, the ejection fraction (EF) and fractional shortening (FS) of rats in DNase+rt-PA group was significantly increased (P=0.024, P<0.001), while LV internal diameter at end-systole and LV internal diameter at end-diastole (LVIDs and LVIDd) were all decreased (P<0.001, P<0.001), and the end-systolic volume and end-diastolic volume (ESV and EDV) were decreased significantly (P=0.002, P=0.002). In addition, the maximal slope of systolic pressure increment and the maximal slope of diastolic pressure increment (P=0.036, P=0.015) in DNase+rt-PA group were also increased when compared to I/R Control group, the left ventricular end diastolic pressure (LVEDP) decreased (P=0.019). Compared with I/R Control group, both the LVWT and IE of DNase+rt-PA group increased (P<0.001; P=0.006), in addition, the MCSA of DNase+rt-PA group decreased significantly (P=0.034)。Conclusions:This work provides the first experimental evidence that NETs are involved in myocardial I/R injury induced microcirculation obstruction, and demonstrates a therapeutic potential of DNase based treatment (DNase plus rt-PA) in ameliorating I/R induced left ventricular functional and structural remodeling. |
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