Role Of Mitochondria Protein Tom70/MICU1 In Myocardial Ischemia/reperfusion Injury

Background:With the progress of science and technology, economic industry transformation, gradually increase the level of people life, travel more convenient, diet quality improve continuously, at the same time the simple physical activity decrease, the body heat accumulation caused by fat body, consequence is that cardiovascular events incidence. The mortality of patients continue to rise. For the effective prevention, timely diagnosis, reduce the complications of cardiovascular research has become the focus of the field. Percutaneous coronary intervention(PCI) for the timely and smooth criminal angiogenesis of acute ischemic events, save dying myocardium will undoubtedly bring revolutionary hope; but the ischemic myocardium blood reflow after reperfusion injury, will be produced, which has become the obstacle of a gully coronary heart disease treatment. Studies have demonstrated that myocardial ischemia/reperfusion(I/R) injury, the phenomenon of Ca2+ overload can make the mitochondria appeared, Ca2+ overload is one of the important factors that induce the apoptosis of myocardial cells. Mitochondrial calcium uptake 1(MICU1) is a mitochondrial inner membrane protein,can regulate the transport of Ca2+, its main role is to prevent theCa2+ overload can occur within the mitochondria. The mitochondria of eukaryotic cells contain more than 1000 kinds of proteins, about 99% is composed of cytoplasmic ribosomal synthesis after the outer mitochondrial membrane translocase of the outer mitochondrial membrane(TOM) complexes of transport, transport to the mitochondria inside, were localized in the stoma, inner and outer membrane or membrane gap components following exert its physiological function. Translocase of the outer membrane 70(Tom70) is one of the major receptor protein TOM complexes, the main identification with integrated positioning precursor protein signal; a new study has found that protein kinase A may regulate the phosphorylation of Tom70, thereby inhibiting and affect its receptor activityand transport function, at the same time with Parkinson disease, hepatitis C diabetic retinopathy, etc. the pathogenesis.More and more studies have found, inner and outer mitochondrial membrane protein plays an important role in cardiac ischemic injury, by adjusting thesemitochondrial membrane proteins could increase ischemic tolerance of mitochondria, prevent the damage of progress, protect myocardial cellsurvival, maintenance of myocardial morphology and function, and improvethe patientprognosis, improve the quality of life.Expression of myocardialmitochondrialwhether inhibition of MICU1 and Tom70 in I/R injury; whether it is possible to inhibit mitochondrial Ca2+ uptake by regulating the expression of MICU1, reduce the I/R myocardial injury degree; the expression of Tom70 is able to regulate MICU1 protein; whether Tom70 can be achieved by increasing the mitochondrial translocation of MICU1,inhibition of mitochondrial Ca2+ increased, so as to achieve the purpose ofreducing MI/R injury, the mechanism is not clear. This research will be through animal experiments to elucidate the role of mitochondrialfunction of protein Tom70/MICU1 in MI/R and to explore its mechanism, provide the basis for research on the mechanism of new effective treatment for coronary heart disease. Objectives:1. To establishMI/R injury mouse models; observe MI/R injury to themitochondrial function of protein MICU1 and Tom70;2. Effect of studies of MICU1 in MI/R injury in mice;3. Study to regulation the relationship between Tom70 and MICU1;4. Study the influence of Tom70 on MI/R injury and its internal mechanism. Methods:1. To establish the function of myocardial mitochondria protein Tom70 and MICU1 of decreased expression and/or increased mouse models: the use of 2% volatileanesthetics isoflurane anesthetized mice, fixed on the operating table, the mouseleft fourth and 5 intercostal fully exposed theheart, then myocardial injection of 20μg Tom70 siRNA, MICU1 siRNA or Scrambled siRNA, preparation of Tom70, MICU1 expression in mouse model of reduced; in addition, by the same method, Tom70 lentivirus myocardial injection, the establishment of Tom70 and MICU1 expression in mouse model of elevated;2. To establish MI/R mouse models: myocardial injection of 72 hours after left coronary artery ligation in mice through the heart of the anterior descending branch of the 30min; after opening the slipknot reanalyzed vascular blood reflow3 h, to establish MI/R animal models, and then treated mice;3. 24 h after reperfusion of the ischemic myocardium,(1) Western blots were used to determine the expression of Tom70 and MICU1 protein in myocardial mitochondria;(2) To detect the expression of Tom70 protein of myocardial tissue in miceaccording to the operation steps of immunehistochemistry;(3) To detect the expression of MICU1 protein of myocardial tissue in miceaccording to the operation steps of immunefluorescence;(4) Using TUNEL staining and Caspase-3 detection kit for the determination ofmyocardial cell apoptosis;(5) Myocardial mitochondria injury in mice was observed by transmission electron microscope morphology;(6) By using the ATP kit for detecting ATP level, to understand the changes of myocardial mitochondrial function;(7) Determination of the levels of mitochondrial membrane potential using JC-1 kit, to understand the changes of myocardial mitochondrial function;(8) To detect changes of mice in myocardial mitochondria Ca2+ volume using atomic flame absorption method;4. 24 h after reperfusion of the ischemic myocardium,(1) By transthoracic echocardiography, detection of functional changes in miceheart condition;(2) To detect cardiac function after the mouse coronary artery ligation of left anterior descending again, using TTC double staining method in accordance with the requirements of the operation, the steps of detecting myocardial infarctionarea changes of mice. Results:1.The changes of membrane and cristaestructure change, mitochondrial ATP generation level, the membrane potential and the amount of Ca2+ as the detection index evaluation of mitochondrial morphology and function. The results found that, compared with the normal mice, the morphology of mitochondria in myocardium of mice after I/R treatment is not the same size, mitochondrial membrane rupture, mitochondrialcristae, disorder offuzzy not clear; level and mitochondrial membrane potential of ATP generation than normal mice decreased, mitochondrial Ca2+ levels than normal mice; at the same time, the expression of I/R after treatment myocardialmitochondrial MICU1 and lower Tom70 protein. After I/R treatment, inhibition of myocardial mitochondrial function, disrupted mitochondrial morphology and Ca2+ overload appears mitochondria, that appeared in the myocardial cell injury in the sub organ level, and inner and outer membraneprotein MICU1 and Tom70 function associatedwith the material transport internalmitochondrial expression also decreased.2.Compared with normal group: MICU1 expression decreased in the myocardial tissue of mice and immunefluorescence detection of myocardial mitochondrial Western blot display, MICU1 protein expression decreased obviously. Compared with the MI/R in the normal group, the blood 3h after reperfusion, MICU1 expression decreased in mice myocardium tissue by TUNEL staining positive indextechnology, as green cell number increased, Caspase-3 activity determination showed that the detected value increased, increased myocardial cell apoptosis; injury of myocardial mitochondrial structure form, ATP emissions reduced, levelmembrane potential decreased, atomic absorption method was used to detect the mitochondrial Ca2+ increased, that decreased expression of MICU1 protein inmicemyocardium injuryincreases in sub cellular organ level; after reperfusion24 h, decreased expression of MICU1 protein increased the area of myocardial infarctionin I/R mice, and the heart function also appears to fall further.3. Compared with normal group: Tom70 expressiondecreased / increased inmyocardium of mice by immunehistochemistry and myocardial mitochondrial Western blot analysis showed that Tom70 protein expression appearscorrespondingly decrease / increase. Compared with the normal MI/R group, the blood 3h after reperfusion, Tom70 expression decreased mice showed decreasedexpression of MICU1 protein was detected in Western blot; Western blot in detecting myocardial mitochondriaof mice increased the expression of MICU1 protein increased the expression of Tom70 protein increased, but this effect can be reduced by low MICU1 protein expressionwasinhibited. This indicates that themitochondrial outer membrane transport protein receptor associated with Tom70 and mitochondrial MICU1 protein; furtherdetectedby immuneprecipitation, found that the direct interaction of Tom70 and MICU1 two proteins, suggesting that MICU1 protein is in combination withmitochondrialTOM complex receptor Tom70 protein translocated to mitochondria, and play its role.4. Compared with MI/R in the normal group, Tom70 expression decreased in micefrom macroscopic to microscopic detection display, heart function is restrained, myocardial infarction area increased, increased levels of myocardial cell apoptosis, mitochondrial cristae disappeared, and morphological damage degreeincreases, mitochondrialmembrane potential and reduced levels of ATP, Ca2+ content in mitochondria increased; Tom70 protein increased expression of mice compared with MI/R in normal group mice, can improve the detection results of the above indexes, make heart function enhancement, reduces myocardial infarct size, myocardial cell apoptosis levels decreased, mitochondrial membrane rupture andthe cristae disappeared, the degree of injury is reduced, the increased level ofmitochondrial ATP and membrane potential of mitochondria, the amount of Ca2+decreased; but the detection indexresults improved, can reduce the expression by MICU1 was inhibited. Show that Tom70 can regulate the expression of mitochondrial MICU1, to reduce the degree of MI/R injury. Conclusions:1.I/Rincreasethe injury of myocardial mitochondria, inhibition of mitochondrial function of Tom70 protein and MICU1 expression;2.MICU1 inhibited mitochondrial Ca2+ overload, thus alleviate the MI/R injury;3. Tom70 MICU1 can adjust to the mitochondrial translocation;4.Tom70 may reduce MI/R injury by increasing the mitochondrial translocation of MICU1 inhibition of mitochondrial Ca2+ overload.