Molecular Mechanisms Of Myocardial Injury After Non-lethal Trauma And The Role Of Adiponectin

BackgroundNon-lethal trauma (e.g. traffic accidents-induced mechanical trauma) has always been one of the most concerned issues in the medical world. With the development of medical care provisions, direct hazards rendered by trauma per se have been substantially reduced, whereas secondary organ failure induced by trauma is increasingly recognised. Recent multi-hospital evidence showed that the prevalence of myocardial infarction (MI) and secondary heart failure was significantly increased in post-traumatic patients even in the absence of coronary artery dissection and direct myocardial injury. However, the mechanisms remain elusive.In a recent study employing a mouse traumatic shock model we found that the intensity of traumatic injury leathal to rats failed to induce multi-organ injury in mice. Mice fully recovered from trauma (7 days after trauma) sustained far more severe myocardial injury when subjected to myocardial ischemia. Studies have shown that trauma can induce the elevation of plasma tumor necrosis factorα(TNF-α) and other inflammatory cytokines. However, the elevation of these inflammatory cytokines are only transient and are unlikely to directly enhance myocardial ischemic injury long after trauma. This prompted us to speculate that a secondary change might be induced by these cytokines, which can serve as a more direct injury-inducing factor that enhances post-traumatic myocardial ischemic injury. However, it still remains an enigma as to the validity and nature of this secondary change induced by inflammatory cytokines that increases myocardial ischemic injury.Adiponectin is a cytokine secreted by adipocytes and is present in high abundance in the circulation. Studies showed that plasma adiponectin is conversely related to the morbidity of ischemic cardiac injury, and adiponectin can protect myocardium against ischemia/reperfusion injury (MI/R) by attenuating oxidative/nitrative stress. Basic and clinical studied both confirmed that reciprocal regulations exist between adiponectin and TNF-α(and possibly several other inflammatory cytokines). The elevation of inflammatory cytokines can inhibit the secretion of adiponectin and decrease its plasma level. Therefore, we hypothesized that the decrease in the plasma adiponectin level secondary to the temporary elevation of TNF-αand other inflammatory cytokines induced by non-lethal trauma might be an important reason that sensitizes post-traumatic myocardium to ischemic injury. In the present study, we aimed to test this hypothesis and further to elucidate the molecular mechanisms responsible for the aggravation of post-traumatic myocardial ischemia injury. Our study might shed light to trauma-induced pathological changes and further provide experimental basis for the mechanisms underlying trauma-induced secondary ischemic cardiac injury.Objectives(1) To investigate the effects of non-lethal trauma on the the serevity of MI/R injury (myocardial infarction, cardiac function and cardiomyocyte apoptosis).(2) To ascertain the time-course changes of plasma TNF-αafter trauma and to verify the possible existance of a modulatory effect between TNF-αand adiponectin.(3) To observe the effects of the changes in plasma adiponectin level on myocardial oxidative/nitrative stress and the serevity of MI/R injury. Meanwhile, to further define the role of the changes in plasma adiponectin level in the aggravation of post-traumatic MI/R injury and to elucidate the underlying mechanisms.Methods(1) Mice traumatic injury model: Male C57B16/J mice or adiponectin knockout mice (Adp-/-, 20 - 25g) were fully anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and placed in a Noble-Collip drum apparatus. Whole-body non-lethal trauma was induced by a total of 200 revolutions at a rate of 40 rpm. Sham traumatized mice were subjected to the same revolution but the animals were taped on the inner wall of drum, thus avoiding traumatic injury.(2) Mice MI/R model: A slipknot was placed at the halfway point of the left anterior descending artery (LAD) with 6-0 silk sutures and ischemia was induced by ligation of the slipknot. Reperfusion was performed by releasing the slipknot 30 min after the ligation. Cardiomyocyte apoptosis and oxidative/nitrative stress were determined at 3 h, and myocardial infarction and cardiac function determined at 24 h, after the conclusion of the reperfusion. Sham operation was performed in a same manner except that the LAD will be left unligated.(3) Determination of cardiac function by intraventricular catheterization: A 1.4F Millar-tip catheter transducer was inserted to the left ventricular cavity through the left carotid artery. Heart rate, left ventricular end diastolic pressure (LVEDP), maximal positive and negative values of the instantaneous first derivative of LVP (+dP/dtmax and -dP/dtmax) were obtained using computer algorithms and an interactive videographics program (Po-Ne-Mah Physiology Platform P3 Plus, Gould Instrument Systems, Inc.).(4) Determination of myocardial infarction: Myocardial infarct size was determined by Evans blue/2,3,5-triphenyl tetrazolium chloride (TTC) double staining.(5) Determination of myocardial apoptosis: Myocardial apoptosis was determined by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining (TUNEL) and caspase-3 activity assay.(6) Plasma TNF-αand adiponectin assays: Plasma TNF-αand adiponectin levels were determined by enzyme-linked-immunosorbent-assay (ELISA).(7) Expression of inducible nitric oxide synthase (iNOS): Expression of iNOS was determined by Western blotting.(8) Contents of nitric oxide (NO) and its in vivo metabolites NO2 and NO3 (collectively termed as NOx) were determined using nitrate reductase method.(9) ?O2? production was determined by lucigenin-enhanced luminescence and in situ dihydroethidium (DHE) staining.(10) Myocardial ONOO- formation was determined by competitive ELISA and immunohistochemistry.Results(1) Mice in the trauma + MI/R group (MI/R induced 7 d after trauma) showed increased myocardial infarct size as compared to those in sham truam + MI/R group (MI/R induced 7 d after sham trauma) (P < 0.05). Meanwhile, mice in the former group had increased LVEDP (P < 0.01) and decreased±dP/dtmax (P < 0.05). Cardiomyocyte apoptosis index (AI) and caspase-3 activity were also enhanced in mice subjected to trauma + MI/R (P < 0.01 vs. sham truam + MI/R).(2) Plasma TNF-αlevel peaked 3 h after trauma (P < 0.01 vs. time point 0) and swiftly declined thereafter, without showing a second peak. Plasma adiponectin level gradually decreased and reached the lowest level 3 d after trauma (P < 0.01 vs. time point 0) and was quickly restored to basal level. The time-course change of adiponectin was abolished by the administration of TNF-αinhibitor etanercept (16 h and 1 h before trauma, 8 mg/kg×2).(3) Blockade of TNF-αby etanercept alleviated myocardial infarction, cardiac dysfunction and cardiomyocyte apoptosis induced by MI/R 7 d post-truama. Globular domain of adiponectin (gAd, 0.5 mg/kg) intraperitoneally administered at both 10 min before reperfusion at the 7th day post-trauma and at the 3rd day post-trauma markedly alleviated MI/R injury.(4) Myocardial ?O2? production was significantly enchanced in mice subjected to MI/R injury after trauma (P < 0.01 vs. sham trauma). Blockade of TNF-αwith etanercept, or intraperitoneal administration of gAd at both 10 min before reperfusion at the 7th day post-trauma and at the 3rd day post-trauma all markedly decreased myocardial ?O2? production (P < 0.01~0.05 vs. sham trauma).(5) Myocardial iNOS expression was enhanced and NOx production increased (~ 1.5 fold, P < 0.05 vs. sham trauma) in mice subjected to MI/R injury after trauma. Blockade of TNF-αwith etanercept, or intraperitoneal administration of gAd at both 10 min before reperfusion at the 7th day post-trauma and at the 3rd day post-trauma all markedly decreased myocardial iNOS expression and NOx production (P < 0.01~0.05 vs. sham trauma).(6) Myocardial histochemistry signal of nitrotyrosine was enhanced and ONOO- formation increased (~ 5 fold, P < 0.05 vs. sham trauma) in mice subjected to MI/R injury after trauma. Blockade of TNF-αwith etanercept, or intraperitoneal administration of gAd at both 10 min before reperfusion at the 7th day post-trauma and at the 3rd day post-trauma all markedly decreased myocardial histochemistry signal of nitrotyrosine and ONOO- formation (P < 0.01~0.05 vs. sham trauma).(7) Intraperitoneal administration of Mn(III)TBAP (?O2? scavenger, 10 mg/kg) or EUK134(ONOO- scavenger, 5 mg/kg)attenuated myocardial infarction, cardiac dysfunction and cardiomyocyte apoptosis and caspase-3 activity induced by MI/R at the 7th day post-truama.(8) The replenishment of exogenous gAd reduced myocardial infarct size by 37.8% in Adp-/- mice subjected to MI/R injury 7 days after trauma (P < 0.01 vs. vehicle), whereas etanercept only mildly reduced the infarct size by (12.4%, P = 0.046 vs. vehicle). Meanwhile, gAd significantly alleviate the decline in±dp/dtmax in such an animal model (P < 0.01 vs. vehicle), but etanercept failed to alleviate such a decline (P > 0.05 vs. vehicle).Conclusions(1) Non-lethal trauma can aggravate MI/R injury in mice and may serve as a'silent killer'that renders myocardium more susceptible to ischemic injury.(2) Elevation in plasma TNF-αcan induce a secondary decline in adiponectin concentraion. Blockade of TNF-αor direct replenishment of exogenous gAd can reduce the susceptibility of myocardium to post-traumatic MI/R injury, indicating that the decrease in plasma adiponectin level induced by elevated TNF-αmight play a role in the aggravation of MI/R injury after trauma.(3) Decreased plasma adiponectin induced by elevated TNF-αcan enhance myocardial oxidative/nitrative stress after MI/R and, therefore, increase the susceptibility of myocardium to I/R injury.(4) Replenishment of exogenous gAd, but not the administration of etanercept, can attenuate myocardial infarction and cardiac dysfunction induced by post-traumatic MI/R injury in Adp-/- mice, thus implicating the decrease in plasma adiponectin level secondary to elevated TNF-αas an important factor in the aggravation of post-traumatic MI/R injury.