The Role Of Lysosomal Acid Alpha-glucosidase In Coronary Heart Disease

Background:Coronary artery disease (CAD) is a common complex disease caused by genetic factors and environmental factors interact with each other, which is one of the leading causes of death among the crowd, genetic causes for which remain largely unclear. Autophagy is a cellular degradation process of highly conservative, which utilizes lysosome to decompose proteins in cells and their organelles and recycle some of the material they produced. Autophagy has been demonstrated to be involved in the CAD by influencing lipid metabolism and inflammation. We have previously shown that expression of autophagic genes is altered in CAD patients indicating dysfunctional autophagy.Objective:we aimed to explore the expression of a lysosomal hydrolase gene, acid alpha-glucosidase (GAA) gene, in CAD patients and healthy controls to research the function and mechanism of acid lysosome hydrolase in coronary atherosclerotic heart disease occurrence and development, at last provide a new theoretical basis for the diagnosis and treatment of coronary heart disease and research.Methods:GAA gene expression was examined in large cohorts of CAD patients (n=248) and age - and sex - matched healthy controls (n=208). Collect the peripheral venous blood from the people after overnight fasting aseptically, isolate the peripheral leukocytes and blood plasma. Alpha-glucosidase activities were measured with high-sensitivity fluorogenic substrates:the standard curve was made by different concentration gradient of pure 4-methylumbelliferone (4-MU), then use miniature fluorescence spectrometer of Turner Biosystems TBS-380 to measure the fluorescence intensity, at last use the linear regression equation produced by SPSS statistical software to calculate enzyme activity. The relative transcript level of GAA was analyzed by RT-PCR:total RNA was isolated from leukocytes with TRIzol reagent, the cDNAs were reverse transcribed with reverse transcription kits and then amplified GAA gene 30 cycles and Beta-actin gene 25 cycles by PCR. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide, then used gray scale scanning to quantitative them and bands intensities of GAA were normalized to β-actin signals. All the RT-PCR experiments were repeated twice and the averages were used for comparison. The GAA protein levels were detected by Western blot method:Cellular proteins from leukocytes were prepared with cell lysis buffer, protein concentrations were measured with nucleic acid protein analyzer, took 30μg protein using protein separation glue to SDS-PAGE, and then transfer it to the nitrocellulose membrane; then put nitrocellulose membrane and the GAA antibodies or beta actin antibody together to incubate, then incubated with second antibodies which were labeled by the HRP, finally performed imagings using chemiluminescence reagent kit, all the Western blot experiments were repeated twice and the averages were used for comparison. Protein signal data analysis method was same as the RT-PCR experiments. GAA enzymatic activity, protein and transcript levels were determined and compared between CAD patients and controls.Results:1. GAA activities in CAD patients were significantly elevated (P<0.05), compared to controls.2. GAA transcription levels were also significantly increased in CAD patients (P<0.01).3. Multivariate logistic regression analyses revealed that GAA transcript levels were strongly associated with CAD (OR 5.93,95% CI 2.98-11.78, P=3.89×10-7).4. GAA protein levels were increased in CAD patients, but not significant (P>0.05), likely due to insensitive assays.Conclusions:Compared to controls, GAA gene expression was altered in CAD patients. Our data suggested that GAA may be involved in the CAD pathogenesis.