Correlation Analysis Of The Expression Of Gene With The Morphologic Changes Of Cardiac Hypertrophy

Cardiac hypertrophy is one of the main ways in which cardiomyocytes respond to the pathological stimuli which include mechanical , neurohormonal stimuli and cell factor etc. Through which improves cardiac pump function compensationally. However, this compensatory mechanism can become overwhelmed by biomechanical stress of long standing, finally resulting in heart failure.TNNI3K(cTnI-interacting kinase) is cardiac-specific gene which is undetectable in other tissues. it could interect with a series of sarcomeric regulating proteins, among which the most frequent candidate was cTnI and then it was named TNNI3K. It may participate in the development of cardiac hypertrophy by regulation of contraction proteins through an unknown signaling pathway.PartⅠExpression of TNNI3K during the development of pressure overload hypetrophyObjective:To study the relationship between cardiac hypetrophy and TNNI3K by observing the morphologic changes during the pressure overload hypetrophy and the expression of TNNI3K gene.Methods:Male SD rats weighting 190-200g were divided randomly into the sham operation control group(Con) and the model group(Mod).Each of the groups were subdivided into two weeks(2W),four weeks(4W),six weeks(6W)groups, respectively. Cardiac hypertrophy models were made through partial ligation of abdominal aorta as described by Anversa P. Con group only underwent operating procedure without ligating abdominal aorta. 1 Histochemical and immunohistochemical stainingAt every experimental point, the left ventricle of the rats was taked out and fixed with 4% paraformaldehyde 48houres, and then embedded in paraffin which were sliced up into 5 micron thick sections. The sections were used for HE staining or immunohistochemical staining, then observed by light microscope and analyzed by(ImageProPlus 6.0)..2 The fixation for transmission electron microscopeThe mixture of 3% paraformaldehyde and 1%glutaraldehyde was used in order to fixed the left ventricle samples for transmission electron microscope.Results:The hemodynamic parameter and heart mass index of model group were changed respectively after the abdominal aorta constrictive operation two to six weeks. The mean diameters and the thickness of left ventricle of the Mod4W and Mod6W groups are 7.38±0.43mm and 5.89±0.23mm. But in contral group they are 7.14±0.33mm and 3.8±0.45mm. The ratio of the thickness to the diameter in the two modal groups is bigger than it in the contral group. It show the heart is centrality hypertrophy In the mordal groups .1 The histological changes of left ventricle.The myocardial cells of control groups arrange tidily and the nucleus are clear unter the light microscope. The mean diameters of the cells and their nucleus are 15.19±2.24μm and 2.23~2.39μm. The myocardial cells of the Mod2w group become hypertrophy extensively. Its mean diameters are 15.19±2.24μm and the nucleus are 3.03±1.2μm. The arrangement of the myocardial cells of the Mod4w group become tangly. The myocardial hypotrophy is aggravated and its mean diameters are 15.72±1.62μm, the nucleus are 3.45±0.82μm. Further more the space among the cells becomes wider, and the fibrosis can be observed in extracellular matrix. The myocardial hypotrophy of the Mod6w group is more aggravating than that of the Mod4w group. The mean diameter of the cells and their nucleus are 16.08±2.69μm and 3.65±1.02μm. The arrangement of the myocardia become more inordinate and the interruption appears among them. The fibrosis is high.2 The ultrastructure change of left ventricle.The myocardial myofibrils arrange tidily, and its light band, dark band, Z line and intercalated disc can be observed clearly in every contral group. Its sarcomere is 1.63μm. Most of the mitochondrions are ellipse and the arrangement of them is like a string of beads. The mitochondrial crista are clear and present as a shutter. The shape of the myofibrils of the Mod2w group is similar to that of the contral group, but the sarcomeres are 1.83±0.08μm long. During this period, the mitochontrions get together, its shape is nearly round mostly, its crista are dense, and the number of them is increased. Beside this, the microvascular vessels in the matrix are enlarged. The chromatin of the pericyte is agglomerated, and appears at the edge of nuceus. It indicate that the cell is prometaphase apoptosis. The length of the sarcomeres of the myocardial myofibrils is 1.85±0.01μm in the Mod4w group, the conglomeration of the mitochondrions is enhanced, and the number of mitochondrion is larger than that of the Mod2w group in the conglomeration. But the shape of the mitochondrial crista appears like that of the Mod2w group. Beside the enlarged microvascular vessels and prometaphase pericyte apoptosis, the anaphase apoptosis cells is also discovered. Its nuclear is crinkly, chromatin is condensation under the nuclear membrance, and the apoptotic body is formed. The length of sarcomere is inceased to 1.94±0.09μm in the Mod6w group. Sparse area is presented in the conglomeration of the mitochondrions. The mitochondrial crista become thicker and concentrated than that of the other groups. The died myocardial cell could be observered, its cell membrane is disaggregated, the most of organelles are dissolved, but some little round mitochondriions with intact shape could be seen. The nucleus is shrinking, chromatin is condensation under the nuclear membrance. 3 Immunohistochemical staining of left ventricle.The results of the immunohistochemical analysis show that the levels of the TNNI3K gene expression are similar in each contral group. Its mean density is 0.16 OD. But the mean density of the Mod2w group is 0.15±0.04OD, is slightly lower than that of the contrial group. The differentiation between them has no signification in statistics. Along with the development of the cardiac hypetrophy TNNI3K gene expression is gradually increased. The mean density are 0.17±0.02 OD and 0.20±0.04 OD respectively in the Mod2w and Mod2w groups.Conclusion:1 The rat model of pressure overload hypetrophy can be made successfully through partial ligation of rat abdominal aorta.2 The fibrosis of the mitrx and the cellular apoptosis are aggaravated gradually with the occurrence and development of cardiac hypertrophy.3 The TNNI3K may be bringed into fullplay during the metaphase and anaphase of the cardiac hypetrophy.PartⅡThe expression of TNNI3K gene in the cardiomyocytes of neonatal ratObjective: To study the effect of AngⅡon the myocardial cells and the crosstalk between AngⅡand TNNI3KMethods: The hearts of the neonatal rats, which were born 24h to 72 h, were used for primary cell culture. To Prepare the cover slip with adherent cells , cells were seeded at six-well plates and cultured for 24 h in DMEM containing 10% fetal bovine serum. The cells were washed with serum-free medium and then treated with angiotensin II (10μmol/L) or PBS (contral group) for another 4 days. After washing with PBS , adherent cells were fixed with 95% alcohol for 20 min and stained with immunocytochemical method by using the antibody of TNNI3K. Images were obtained using a digital camera attached to a microscope for analysis. Cell size was analyzed using Image- Pro PLUS 6.0 softerware. The data was analyzed using SPSS V13.0 softerware. Results: It is hypertrophy that the cardiomyocytes of neonatal rat are induced by AngⅡ, their mean diameter is 42.48±1.91μm and the area is 1546.428±633.61μm2. But the mean diameter of the normal cardiomyocytes of neonatal rat is 35.81±2.36μm, the area is 1032.672±334.35μm2. It indicated that the hypertrophy model of cardiomyocyte is formed. In the model, the expression of TNNI3K increased. The integrated optical density is 202.86±74.40 OD.Conclusion: The TNNI3K was involved in the development of cardiomyocytes hypertrophy induced by AngⅡin positive regulation manner.