Comparative Proteomics Study On Myocardial Mitochondrial Proteins In Rats Following Severe Burn

Ischemia/hypoxia is one of the key scientific issues following severe burns,which may induce cell death and organic damages for the disturbance of energy metabolism. Myocardium is a highly oxidative tissue that produces more than 90% of its energy from mitochondrial respiration. It has been demonstrated that mitochondria dysfunction in oxidative phosphorylation plays an important role in the inducement and aggravation of myocardial damage during severe burn or following ischemia/hypoxia. As heart is the power organ of circulation, myocardial damages would undoubtedly aggravate ischemia and hypoxia of other organs, resulting in the forming of vicious circle.Disturbance of mitochondrial energy metabolism is the common result of multiple complex factors, especially when the body suffered from burn, trauma, stress or hypoxia. There is little knowledge on the entire mitochondria injury and compensation response during the development of energy metabolism disturbance, so few methods was found effectively in alleviating the disturbance of mitochondrial energy metabolism. Comparative proteomics gives us a global description of the differential expression of myocardial mitochondria proteins in the development of energy metabolism disturbance at the early stage of severe burn, and shed light on the exploration of upstream molecular mechanism, therefore, effective methods alleviating the post-burn energy metabolism disturbance may be found.In this study, comparative proteomics was used to reveal the differential expression of myocardial mitochondria proteins in rats with burn, followed by evaluating the effect induced by changed expression of key proteins, which shed light on the injury and compensation response in myocardial mitochondria when suffered from stress and hypoxia, helping to identify the key protein that playing important roles in the development of energy metabolism disturbance. Those findings will provide new targets and theoretical references in alleviating myocardial injury during early stage after severe burn. I. Materials and methodsBoth in vivo and in vitro experiments were adopted in the present study.1. Purified mitochondria were obtained by Nycodenz density gradient centrifugation, the purity and integrity were identified through electron microscope and western blot analysis.2. A 30% TBSAⅢ°scald rat model was established, 2-DE maps in control and post-burn groups were obtained by comparative proteomics, and then were analyzed with PDQuest software, the differential expression proteins were identified through in-gel digestion and HPLC-chip-MS/MS analysis.3. Both in vivo and in vitro experiments were adopted to verify the expression changes of key protein, and revealed the possible effects through inhibiting the complexⅠactivity by rotenone. At the same time, the ultrastructure damages of myocardial mitochondria were evaluated through electron microscope analysis.II. Results and conclusions1. The purity and integrity of purified myocardial mitochondria isolated by Nycodenz density gradient centrifugation are proved quite well through electron microscope and western blot analysis.2. Well focused and distinct 2-DE maps with good reproducibility were obtained, means of 394.7±5.5, 391.0±13.9, and 385.3±12.6 protein spots were detected from the control, 1 hour post-burn, and 3 hour post-burn groups. All of the maps were analysed with PDQuest software, a total of ten differential expression proteins was successfully identified by HPLC-chip-MS/MS.3. Eight of the ten differential expression proteins show a downregulated expression in post-burn groups, they are TNF receptor-associated protein 1, methylcrotonoyl-Coenzyme A carboxylase 1, acetyl-Coenzyme A dehydrogenase long-chain and short-chain, NADP(+)-dependent malic enzyme 3, ETF-QO, NADH dehydrogenase, EF-Tumt. The decreased expression may result in the disturbance of ATP synthesis, burst of ROS, impairment of the resist ability to ROS injury and apoptosis induction. The protein expression level of LOC498909 and ATP synthaseαsubunit were increased in burn groups, which may be the compensation effect to resist the injury.4. When analyzing the function of differential expression proteins, intrinsic relationship was found between EF-Tumt and NADH dehydrogenase. EF-Tumt is an upstream element that control the protein translation of NADH dehydrogenase, and NADH dehydrogenase is the catalytic core of complex I, so, It can be presumed that EF-Tumt play an important role in the induction of energy metabolism disturbance through inhibiting the activity of complexⅠb y decreasing the protein translation of NADH dehydrogenase.5. To tentatively confirm above presumption, the expressions of EF-Tumt and NADH dehydrogenase subunit 3 were verified in rat myocardium, the results were consistent with those in 2-DE and the reliability of 2-DE was verified; and downregulation of EF-Tumt was found in cultured cardiomyocyte treated with hypoxia. Meanwhile, decreased activity of cardiomyocyte treated with rotenone and aggravated damage of mitochondria ultrastructure in burned rat heart were found. All of the injury may be the effect that induced by the decreased expression of EF-Tumt. However, more studies are needed to conform this effect.