Mechanism Of Mechanical Trauma Induces Cardiomyocyte Apoptosis In Mice

BackgroundMechanical trauma (MT, such as that induced by motor vehicle crashes) represents an important medical and social problem in the world. It is well recognized that chest MT causes direct heart damage (i.e., cardiac contusion and vascular lacerations). However, several clinical groups have reported that blunt chest MT causes myocardial infarction (MI) and cardiac dysfunction even in the absence of coronary artery damage or direct mechanical cardiomyocyte injury. These results strongly suggest that besides the primary injury to the heart that occurs immediately after MT, it may cause secondary heart injury through yet to be identified mechanisms.More and more evidents demonstrated that cardiomyocytes apoptosis induce cardiac dysfunction. Administration of a nonselective caspase inhibitor immediately after MT not only blocked cardiomyocyte apoptosis but also attenuated posttraumatic cardiac dysfunction. These results demonstrated that traumatic injury causes cardiomyocyte apoptosis, which contributes to post-trauma cardiac dysfunction. However, the origin of the pathological mediator(s) and molecular mechanisms responsible for post-trauma cardiomyocyte apoptosis remain unknown. Identifying the mechanisms responsible for post-traumatic cardiac dysfunction and searching for therapeutic strategies to prevent secondary cardiac injury after trauma is critical in order to reduce overall trauma-related organ injury.Aim1. To determine the origin [i.e., traumatic cardiomyocytes (TC) vs. traumatic plasma (TP)] of the pathological mediator(s) that cause cardiomyocyte apoptosis after MT;2. To find out pathological mediator(s) responsible for initiating cardiomyocyte apoptosis after MT;3. To delineate the intracellular signaling pathway by which cardiomyocyte apoptosis is initiated by pathological mediator(s) following MT for identifying the relationships of pathological mediator(s), apoptosis with oxidative/nitrative stress.Methods1. Non-lethal MT model. After anesthetized, mice were placed in a Noble-Collip drum (12-in.-diameter) and subjected to 200 revolutions at a rate of 40 rpm to induce a non-lethal MT.2. To determine the origin [i.e., traumatic cardiomyocytes (TCs) vs. traumatic plasma (TP)] of the pathological mediators that cause cardiomyocyte apoptosis after MT, normal cardiomyocytes (NCs, isolated from sham traumatic mice) or TCs (immediately isolated from traumatic mice after the trauma procedure) cultured in vitro for 12 h with medium containing 10% plasma obtained from normal plasma (NP) or TP (isolated from different traumatic mice 1.5 h after MT).3. To identify the specific factor(s) responsible for initiating cardiomyocyte apoptosis after MT, cardiomyocytes isolated from traumatized mice were incubated with one of the following factors for 12 h: 1) NP; 2) TP; 3) Cytomix [a mixture of Interferon-γ(IFN-γ), Interleukin-1β(IL-1β), and Tumor necrosis factor-α(TNF-α)]; 4) individual components of Cytomix; 5) TP plus anti-TNF- antibody; or 6) TNF- knockout mice TP.4. To delineate the role of oxidative/nitrative stress in cardiomyocyte apoptosis initiated by MT, cardiomyocytes isolated from traumatized mice were subjected to one of the following treatments for 12h: 1) NP; 2) TP; 3) TP plus 1400W (a highly selective iNOS inhibitor); 4) TP plus apocynin (a NADPH oxidase inhibitor); or 5) TP plus FP15 (a peroxynitrite decomposition catalyst).5. Wild type mice were subjected to MT as described above and treated with vehicle, etanercept (a TNF- antagonist), 1400W, apocynin and FP15 individually; TNF- knockout mice were subjected to traumatic injury without receiving any treatment. Mice were sacrificed 12h after trauma.6. Plasma IFN- , IL-1 , and TNF- concentration were determined by using Enzyme linked immunosorbent assay (ELISA) kits.7. Cardiomyocyte apoptosis was determined by caspase-3 activity, DNA fragmentation or Terminal deoxynucleotidyl Transferase mediated dUTP nick endlabelling (TUNEL) staining.8. Myocardial superoxide content was determined by lucigenin-enhanced luminescence.9. Cardiac nitrotyrosine content (a footprint of in vivo peroxynitrite formation) was determined by an ELISA method.10. Myocardial gp91phox (a major component of NADPH oxidase) or iNOS content were measued using Western blots.11. NO and its metabolic products (NO2 and NO3), collectively known as NOx, were determined using a chemiluminescence NO detector. Results1. The addition of TP to TCs caused increase in caspase-3 activity (2.18±0.24 folds over NC+NP, p<0.01, n=9). No significant caspase-3 activation was observed when TCs were cultured with NP (1.13±0.09 folds over NC+NP, P>0.05, n=10). However, significant caspase-3 activation was observed when TP was added to NCs (1.88±0.18 folds over NC+NP, P <0.01, n=9). These results provide clear evidence that preapoptotic mediators are present in the plasma of traumatic animals, not within TCs themselves.2. Compared with control, plasma IL-1 , IFN- , and TNF- levels were all significantly elevated in traumatic animals (2.19±0.16 vs. 1.11±0.02 pg/mg protein, 1.22±0.14 vs. 0.68±0.02 pg/mg protein, 0.48±0.02 vs. 0.19±0.01 pg/mg protein, P <0.01, n=7-9), although individual cytokines exhibited different time courses after trauma. Noticeably, all three cytokines peaked at or before 1.5 h after traumatic injury.3. Incubation of TCs with NP supplemented with Cytomix resulted in significant caspase-3 activation (1.71±0.18 folds over NP, P <0.01, n=9) that was comparable with that seen in cardiomyocytes treated with TP, suggesting that cytokines are likely to be responsible for TP-induced cardiomyocyte apoptosis.4. Compared with NP, the addition of IFN- or IL-1 alone had no significant effect on caspase-3 activity (0.88±0.15 and 1.11±0.12 vs. 1.00±0.06 folds over NP, P>0.05, n=7-9). However, the addition of TNF- alone at the concentration used in the Cytomix significantly increased cardiomyocyte caspase-3 activity (1.64±0.14 folds over NP, P <0.01, n=9), indicating that TNF- is the cytokine that is responsible for the cardiomyocyte apoptosis induced by Cytomix.5. Neutralization of TNF- with anti-TNF- antibody almost completely blocked TP-induced cardiomyocyte caspase-3 activation (1.13±0.17 vs. 1.88±0.18 folds over NP,P < 0.01, n=7). Compared with wildtype mice TP, the addition of TP from TNF-–/– mice failed to induce significant caspase-3 activation (1.10±0.19 vs. 1.88±0.18 folds over NP,P < 0.01, n=9). These results provide evidence that TNF- present in the TP is the primary factor that causes the cardiomyocyte apoptosis induced by TP.6. iNOS and gp91phox protein expression were significantly upregulated (2.44±0.42 folds over NP and 1.63±0.18 folds over NP,P < 0.01, n=7-9), NO and superoxide production were significantly increased (2.37±0.30 folds over NP and 1.54±0.17 folds over NP,P < 0.01, n=7-9), and the production of peroxynitrite was significantly increased (8.02±1.23 vs. 1.87±0.25 nmol/g protein,P < 0.01, n=7-9) in cardiomyocytes exposed to TP, compared with NP. More importantly, neutralization of TNF- with anti-TNF- antibody almost completely blocked TP-induced oxidative and nitrative stress. iNOS and gp91phox protein expression were significantly inhibited (1.34±0.32 vs. 2.44±0.42 folds over NP, 1.15±0.10 vs. 1.63±0.18 folds over NP,P < 0.01, n=7-9), NO and superoxide production were significantly decreased (1.32±0.12 vs. 2.37±0.30 folds over NP, 1.11±0.09 vs. 1.54±0.17 folds over NP,P < 0.01, n=7-9), and the production of peroxynitrite was significantly decreased (2.77±0.72 vs. 8.02±1.23 nmol/g protein,P < 0.01, n=7-9) in cardiomyocytes exposed to TP, compared with vehicle. These results provide clear evidence that TNF- present in the TP is the primary factor that causes significant oxidative/nitrative stress in TCs.7. Compared with TP, treatment with 1400W significantly reduced NO production (1.04±0.13 vs 2.26±0.11 folds over NP, P < 0.01, n=10) without affecting superoxide production (1.42±0.07 vs. 1.62±0.15 folds over NP, P >0.05, n=10) in cardiomyocytes exposed to TP. In contrast, treatment with apocynin significantly reduced superoxide production (1.04±0.05 vs.1.62±0.15 folds over NP, P < 0.01, n=10) without affecting NO production (2.20±0.19 vs. 2.26±0.11 folds over NP, P >0.05, n=10). Treatment with FP15 affected neither NO (2.16±0.18 vs.2.26±0.11 folds over NP, P >0.05, n=9) nor superoxide production (1.40±0.06 vs.1.62±0.15 folds over NP, P >0.05, n=9). However, all three treatments significantly reduced myocardial nitrotyrosine content (2.61±0.35, 3.06±0.55, 1.55±0.33 vs. 8.09±1.28 nmol/g protein, P < 0.01, n=11) and reduced TP-induced caspase-3 activation (1.24±0.16, 1.05±0.10, 1.01±0.14 vs. 1.91±0.14 folds over NP, P < 0.01, n=10). These results demonstrated that TNF- -initiated oxidative/nitrative stress plays a crucial role in TP-induced cardiomyocyte apoptosis.8. Compared with sham, MT resulted in wild type mice significant cardiomyocyte apoptosis in vivo as evidenced by increased TUNEL positive cells (8.37±0.57 vs. 0.32±0.09% ,P < 0.01, n=10) and elevated caspase-3 activity (2.9±0.2 folds over control, P < 0.01, n=10). Treatment with etanercept markedly reduced post-traumatic cardiomyocyte apoptosis (TUNEL staining: 1.2±0.14 vs. 8.37±0.57%, P < 0.01, n=10) and decreased caspase-3 activity (1.5±0.45 vs. 2.9±0.2 folds over control, P < 0.01, n=10). Impressively, compared with MT wildtype mice, few cells were TUNEL positive (0.8±0.19 vs. 8.37±0.57%, P < 0.01 , n=10) and no significant caspase-3 activation (1.4±0.16 vs. 2.9±0.2 folds over control, P < 0.01, n=10) was observed in TNFα?/? mice after MT.9. Compared with sham, iNOS (29,610±5,093 vs. 1,938±717 AU , P < 0.01,n=10) and gp91phox (104,317±4,361 vs. 10,619±1,522 AU , P < 0.01,n=10) protein expression were upregulated, NO ( 9.9±0.7 vs. 7.1±0.4μmol/g protein, P < 0.01,n=10) and superoxide production (273±13.9 vs. 153±8.3 RLU/mg tissue, P < 0.01, n=10)were increased, and protein nitration was significantly increased (2.86±0.09 vs. 1.67±0.05 nmol/g protein, P < 0.01, n=10) in traumatic wild type mice treated with vehicle.However, compared with vehicle, treatment with etanercept or TNF-αknockout in vivo reduced iNOS protein expression ( 3,380±1,053 ,2,512±1,154 vs. 29,610±5,093 AU, P < 0.01 , n=10) and NO production (7.04±0.56,7.44±0.32 vs. 9.9±0.7μmol/g protein, P < 0.01, n=10), decreased gp91phox protein expression ( 26,478±4,361, 6861±2,239 vs. 104,317±4,361 AU, P < 0.01 , n=10 ) and superoxide generation (163.1±12.70, 146.1±22.18 vs. 273.2±14.93 RLU/mg tissue, P < 0.01, n=10), and reduced protein nitration (2.09±0.12, 1.54±0.19 vs. 2.86±0.08 nmol/g protein, P < 0.01 , n=10).10. Compared with vehicle, blocking iNOS/NADPH oxidase activity with 1400W/apocynin or increasing peroxynitrite decomposition with FP15 in vivo markedly reduced post-traumatic cardiomyocyte apoptosis as determined by TUNEL staining (2.50±0.49, 1.51±0.28 and 1.62±0.12 vs. 8.23±0.48%, P < 0.01 , n=9-11) and caspase-3 activation (1.63±0.14, 1.29±0.09 and 1.05±0.10 vs. 2.05±0.15 folds over sham, P < 0.05 or P < 0.01 , n=9-11).Conclusion1. The present study demonstrates for the first time that the pathological mediators that cause cardiomyocyte apoptosis after trauma are present in TP, not within TCs themselves.2. These results provide the first evidence that overproductive TNF-αin TP is the most important pathological mediator that mediate the cardiomyocyte apoptosis induced by non-lethal MT. 3. These results demonstrate for the first time that there exists a TNF--initiated, cardiomyocyte iNOS/NADPH oxidase-dependent, peroxynitrite-mediated signaling pathway that contributes to posttraumatic myocardial apoptosis.The present study provide the experimental evidences to identify the mechanism of MT-induced cardiomyocyte apoptosis on molecular level, and provide a new strategy to prevent cardiomyocytes apoptosis after MT.