Construction And Phenotype Analysis Of Roundlet - Specific Expression Of Transgenic Mice With PID1 Cardiac Tissue

PID1 (phosphotyrosine interaction domain containing 1) gene, also known as NYGGF4. In our earlier studies, we isolated and characterized PID1, a novel hunman gene that was expressed at a high level in the omental adipose tissue of obese patients. Amino acid sequence analysis revealed NYGGF4 was localized in the cytoplasm, there are several serine and threonine phosphorylation sites, and there is a phosphotyrosine-binding (PTB) domain in the C-terminus of the protein, suggesting that the protein may be involved in intracellular signal transduction. This gene was primarily high expressed in heart, skeletal muscle and adipose tissue. Our further studies revealed that overexpression of PID1 may inhibit the PI3K-AKT signaling pathway. Gene expression profiling is an important clue to reveal the gene function, if the gene is highly expressed in certain tissues, it is likely that the gene plays an important role in this tissues. The PID1 gene highly expressed in heart tissue, while the PI3K-AKT signaling pathway is closely related with cardiac development, cardiac hypertrophy and heart protection function, suggesting that the gene may play an important role in heart development, cardiac hypertrophy and heart protection. Therefore we generated a PID1 heart-specific expression transgenic mouse model to further study the effect of PID1 on the heart development, cardiac hypertrophy, and heart protection.Part ⅠGeneration of Heart-Specific PID1 Expression Transgenic MiceObjective:Establishment of heart-specific PID1 expression vector PBS ⅡSK-aMHC-PID1, generation of heart-specific PID1 expression transgenic mice.Methods:PID1 gene was cloned from mouse with RT-PCR and identified with sequencing analysis. PID1 gene was inserted into the down stream of αMHC promoter to construct the vector which expresses at heart specially to construct the vector PBS Ⅱ SK-aMHC-PID1. The PBS Ⅱ SK-aMHC-PID1 construct was linearized, using Notl digestion and injected into pronuclei of fertilized eggs. The genotype of transgenic line was identified by PCR and the expression level of the gene PID1 was determined by Western blot.Results:1) Heart-specific PID1 expression vector PBS Ⅱ SK-aMHC-PID1 was successfully constructed; 2) Two independent lines with PID1 overexpression in heart were obtained.Conclusion:The heart-specific PID1 expression transgenic mice were obtained.Part Ⅱ Analysis the phenotype of the heart-specific PID1 expression transgenic mice under normal circumstancesObjective:To observe the general condition of the heart-specific PID1 expression transgenic mice under normal circumstances and the change of heart weight index, the overall appearance and function.Methods:The heart-specific PID1 expression transgenic (TG) mice and the same age and sex wild-type (WT) mice were observed about the growth and development, daily life and activity from birth under normal circumstances; we weighed the heart weight and body weight to calculate the heart weight index (HW/BW) in 2 months after birth; light microscopic was used to observe the overall change of the heart; echocardiography was used to observe the change of the morphpholoical and functional.Results:1) The growth and development, daily life and activity of the transgenic mice were similar with the wild type mice, they can survive for more than one year and a half even longer; 2) the heart weight index was no significantly difference; 3) The appearance of the heart was no significantly difference; 4) The heart function by echocardiography was also; echocardiography found the morphpholoical and functional of heart were no significant change.Conclusion:Compared with WT mice, the heart-specific PID1 expression transgenic mice had no significantly changes about the the growth and development, daily life, activity and the morphpholoical and functional of the heart.Part Ⅲ Analysis the phenotype of the heart-specific PID1 expression transgenic mice by the induction of ISO experimentObjective:To observe the change of heart weight index of the heart-specific PID1 transgenic mice by the induction of ISO.Methods:Two months old female heart-specific PID1 transgenic mice and wild-type mice were used as experimental subjects. They were randomly divided into two groups, experimental group and control group. Isoproterenol was administered intraperitoneally with 0.06 mg/g/weight in experimental groups daily, the control mice were injected with equal volume of physiological saline, for 6 days. We measure the heart weight, body weight and calculate the heart weight index.Results:1) The heart weight index of the TG mice and the WT mice on isoproterenol-induced were respectively 6.14±0.39 and 6.96±1.13, there were no significantly differences (P>0.05); 2) the heart weight index of the TG mice and the WT mice on saline were respectively 4.60±0.46 and 4.83±0.29, there were also no significantly differences; 3) compared with WT mice and TG mice in the control group (physiological saline group), WT mice and TG mice on ISO were respectively increased by 33.5% and44.1%, there was significantly difference (P<001).Conclusions:The heart weight index of the heart-specific PID1 transgenic mice and WT mice were no significant differences by the induction of ISO.Part IV Analysis the phenotype of the heart -specific PID1 expression transgenic mice by the induction of TAC experimentObjective:To observe the changes of the cardiac structure and function of the heart-specific PID1 expression transgenic mice by the induction of TAC experiment, and explore the mechanism of the changes.Methods:Two months old female heart-specific PID1 transgenic mice and wild-type mice were used as experimental subjects. They were randomly divided into two groups, experimental group (TAC) and control group (Sham). The cardiac function was detected before operation and one week, four weeks after surgery by echocardiography; we measured the heart weight, body weight and calculated the heart weight index after one week and four weeks surgery; H&E staining was used to observe the morphology of heart; Massion staining was used to observe myocardial fibrosis and cardiac remodeling.Results:1)1) After one week,the volume of the heart increased significantly in TG mice; H&E staining showed that the left ventricle of TG mice in TAC group increased significantly, but the wall thickness was no significantly difference; compared with WT mice,the heart weight index of the heart-specific PID1 transgenic mice increased significantly; Massion staining showed that there were no myocardial fibrosis after one week surgery;The results of the echocardiography revealed that there were no significantly differences,the indicators that response the cardiac function of the echocardiography were no significantly differences.2)After four weeks,the volume of the heart increased significantly in TG mice;H&E staining showed that the left ventricle of TG mice in TAC group increased significantly and the wall was thinning and the myocardial cells increased and disorganized;Massion staining showed that there were myocardial fibrosis seriously,there was fibrosis around the myocardial tissue, surrounding the myocardial cells, normal myocardial cells replaced by fibrous tissue, the myocardial cells became compensatory hypertrophy;the heart weight index of PID1 increased significantly;The results of the echocardiography revealed that there were significantly differences,the analysis of data revealed that there were significantly changes about intraventricular septum systole(IVS; s), left ventricular internal diameter systole (LVID; s), left ventricular volume, systole (LV vol; s), ejection fraction (EF) of fractional shortening (% FS) and other indicators.Conclusion:1) The cardiac structure of TG mice has changed significantly after, while the cardiac function has no change.2) The cardiac structure and function of the heart were significantly impaired after four weeks surgery.