Proteomic Changes Of Myocardial Mitochondrial Proteins In Aging Rats And Functional Analysis Of Differential Protein

Recently, aging process is a hot research spot now worldwide. A strong link between mitochondrial function and aging process: firstly, mitochondria are known to be strong producers of reactive oxygen species (ROS) in heart and myocardial mitochondria are susceptible targets of oxidative damage. Increased ROS production in aging myocardium could result in mitochondrial membrane potential loss and cardiac myocyte apoptosis; secondly, mitochondrial DNA (mtDNA) deletions and mutations had been reported to occur in various aging tissues such as heart, skeletal muscle and liver, mtDNA damage could also induce alterations on polypeptides in the respiratory complexes with subsequent electron transfer decrease leading to further ROS production; thirdly, both mitochondrial transcription and translation activities were found to be impaired in aging hearts. Powerful comparative proteomic techniques with high resolution, high throughput, realtime express analysis can provide effective methods for detecting more key proteins simultaneously during complex pathological progress of multi-gene and multifactor diseases. Unlike traditional ways, more new biomarks and key molecules were found by this strategy to benefit for diagnosis and elucidation of pathological mechanism. Until now there are no data on the association between mitochondrial proteomics expression pattern changes and aging. In the present study, we, therefore, investigated the mitochondrial proteomics expression patterns of neonatal, adult and aged SD rats by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) techniques. We got a group of significant differential proteins including ALDH2 which may be involved in aging. We also drew a positive conclusion through further analysis and validation of the biological function of ALDH2 with Western Blot, RT-PCR, gene transfection and some other cellular and molecular techniques. Additionally, the relationship between ALDH2 polymorphism and longevity was studied. PartⅠComparative proteome analysis of myocardial mitochondrial proteins from rats at different developmental stagesAim: To establish and optimize 2-DE technique for proteomics of mitochondrial proteins from heart of rats. Myocardial mitochondrial proteins related to the aging will be identified. Methods: Mitochondria were isolated from the neonatal(2-days), adult(10-weeks) and old(12-months) hearts of rats. Total proteins extracted from mitochondria were applied to the 2-DE. Sample separation, protein loading, electrophoresis parameters, fixation styles, staining methods and chaotropic agent were optimized. The differentially expressed protein spots evaluated by a software were identified by MALDI-TOF-MS. ATP content in the hearts of three groups were also measured. Results: 45% alcohol and 5% acetic acid was found to have the best protein fixation effect. Both colloidal Coomassie Brilliant Blue and fast silver staining method were good. Chaotropic agent including 9M urea could get the most protein spots. The 2-DE electrophoretogram with high resolution and reproducibility of heart mitochondrial proteins have been successfully established. By comparing and analyzing the 2-DE images, over 1100 protein spots were detected on the gel at three states. In total, we identified 84 differentially expressed protein features, corresponding to 43 different genes. most of these proteins are associated with the mitochondrial respiratory chain, energy metabolism, transport and stress response. ATP content was lowest in the neonatal groups(15.49±1.33.g ATP/g heart tissue), and that in the old rat groups were reduced as compared to adult rat groups(41.48±2.99vs 59.64±2.68. gATP/g heart tissue, p＜0.01). These biochemical tests for energy metabolism in mitochondria confirmed the proteomic resul ts. Conclusions: The 2-DE techniques for proteomics of mitochondrial proteins from heart of SD rats have been established. Significant changes in the mitochondrial protein expressions of the aging heart were identified by the 2-DE electrophoretogram with high resolution and reproducibility. These findings provide clues for understanding the mechanism of aging. PartⅡVerification of differential protein ALDH2 and effect of ALDH2 on human embryonic lung diploid fibroblasts (MRC-5) on the aging processAim: Validation of differential proteins again is essential for next analysis. The role of ALDH2 in replicative senescence of normal human embryonic lung diploid fibroblasts (MRC-5) need to be investigated. Methods. Western blot and RT-PCR were done to further confirm the downregulation of ALDH2 in the mitochondria of aging hearts. ALDH2 gene was introduced into MRC-5 cells through infection with reconstructed adenovirus vector and the expression of ALDH2 was detected. Then the effects of ALDH2 on replicative cellular senescence of MRC-5 cells were examined by SA—.—gal staining, ROS and cell cycle. Results: Compared with the control cells, MRC-5 cells transfected with ALDH2 cDNA showed the decreased positive rate of SA—.—gal staining and ROS(p＜0.01). The cell cycle was no changed. Conclusions: ALDH2 gene may contribute to delay the process of cellular senescence of MRC-5.PartⅢThe study of relationship between ALDH2 polymorphism and longevityAbstract Aim: To analyze the genotype frequency of ALDH2 in population of long life-span, the relationship between ALDH2 polymorphism and longevity was evaluated. Methods: Epidemiological data were collected from 397 persons of long life-span and 161 control subjects in Zhangqiu city of Shandong province in China, and ALDH2 genotypes were detected by denaturing high-performance liquid chromatography(DHPLC). Results: The frequencies of ALDH2 genotypes in longevity residents were ALDH2~*1/ALDH2~*1 65.7%, ALDH2~*1/ALDH2~*2 32.7% and ALDH2~*2/ALDH2~*2 1.5% respectively; the allele frequency of ALDH2~*1 was 82.12% and 17.88% for allele frequency of ALDH2~*2. It had significant difference(p＝0.001, longevity vs control). Conclusions: ALDH2 polymorphism was significantly correlated with longevity.Conclusions1. To establish a stable and standard 2-DE technique platform. Through analysis and optimization of 2-DE associated techniques, we found(1) Both colloidal Coomassie Brilliant Blue and fast silver staining were good; (2) 45% alcohol and 5% acetic acid was found to have the best protein fixation effect and it was recommended for complex protein sample; (3)The number of protein spots was affected distinctly by various chaotropes, chaotropic agent including 9M urea could get the most protein spots.2. The protein profile of myocardial mitochondria from different developmental stages displayed obviously difference. 43 distinct differential proteins were identified and this implied these differential proteins may be involved in aging. To our best knowledge, some of identified proteins such as ALDH2 were firstly reported.3. Compared with the control cells, MRC-5 cells transfected with ALDH2 cDNA showed that the positive rate of SA—.—gal staining and ROS were all decreased. ALDH2 gene may contribute to delay the process of cellular senescence of MRC-5 cells.4. ALDH2 polymorphism was significantly correlated with longevity. The potential application and novelty of this project1. Recent technological advances in proteomics allow us for the first time to find the decreased ALDH2 expression profiles in aging rat hearts. ALDH2 gene may contribute to delay the process of cellular senescence of human diploid fibroblasts.2. ALDH2 polymorphism was significantly correlated with longevity. ALDH2 might be new possible targets which could delay the aging process.