Experimental Study Of Rat Cardiomyocytes Isolated And Gene Expression

Background:The collection of gene expression data from myocardium is important for understanding the cellular mechanisms of cardiac diseases. Many clinical and basic researchers have conducted gene expression analysis using RNA from the whole cardiac biopsies. However previous analyses of the normal ventricular tissues have demonstrated that myocardial cells only represent a part of it. The abundance of fibroblasts, smooth muscle cells and other cell types in whole tissue can complicate the gene expression analysis and the interpretation of results. Therefore, it is necessary for us to purify myocardial cells from cardiac tissue and then conduct downstream application. Many researchers have already developed cell purification methods for extracting RNA. But there are some disadvantages, such as low output, complex and technically difficult procedures that require professional training, expensive equipments and long duration etc. Enzymolysis method is a regular method to purify myocardial cells, especially in the rat, with different technical details used by different operators. And to the best of our knowledge there has not been an exhaustive assessment of the effect of this method that is used for following RNA extraction and gene expression analysis. We hope our research could be a proof for this application.Methods:The whole hearts were obtained from male SD rats aged8weeks. We used the enzymolysis method with Langendorff perfusion system to purify myocardial cells. The purified samples were observed through optical microscope and manual cell counting was conducted. The samples were also observed through fluorescence microscope after acridine orange stain. The total RNA extracted from the purified samples was quantified by biophotometer and evaluated by agarose gel electrophoresis. Furthermore RIN (RNA Integrity Number) was measured to assess RNA integrity. Real-time RT-PCR was performed to compare nonpurified and purified samples for the expression of troponin T (TnT; myocardium marker), vimentin (Vim; fibroblast marker), and smooth muscle alpha-actin (a-SMA; smooth muscle marker). Two different housekeeping genes, peptidylprolyl isomerase A (PPIA) and ribosomal protein, large, PO (RPLPO), were used for normalization so as to verify the results. Furthermore, we purified the myocardial cells from the hearts of Wistar rats aged2,3and12weeks, and quantified the expression level of two different isoforms of adenine nucleotide translocase (ANT), ANT-1and ANT-2. PPIA was used as the internal control gene. Results:The purification process which took about30minutes had fairly good output. After purification, most myocardial cells displayed normal morphology, exhibiting rod-like shape and defined striations under optical microscope. Cell imaging using acridine orange staining in which myocardial cells also displayed normal morphology showed bright green nucleus and dark background without fluorescense. The myocardial cells proportion results of three different rats were87.3±3.2%,91.8±5.1%and88.1±3.3%respectively. The A260/280ratios of the total RNA extracted from purified samples were between1.8and2.0. The image of electrophoresis (n=3) and the figures of RIN (n=6), which were between7and8and indicated that RNA was suited for Real-time RT-PCR, both demonstrated no significant degradation of RNA during the purification process (P>0.05). After purification TnT expression significantly increased, and Vim and α-SMA significantly decreased (n=6; P<0.05). The fold change was2.56,0.38, and0.16, respectively, using PPIA as the housekeeping gene, and2.33,0.35, and0.17, respectively, using RPLP0. The expression level of ANT-1was much higher than ANT-2(10±1.3fold, n=6). The fold exceeded the results reported before. And the levels of the transcripts for ANT-1, ANT-2in rat heart during ontogeny (2,3and12weeks, n=6) also had diversity compared with the former literature which used cardiac tissues as samples.Conclusion:1. The purification protocol using enzymolysis method in the Langendorff perfusion system can yield good output, purify myocardium efficiently and exclude other cell types.2. RNA has not been degraded significantly during the process. This method is very suited for gene expression analysis of myocardial cells.3. Considering the diversity between the gene expression levels obtained from myocardial cells and the results of former literatures, we should emphasize that we can gain more reliable gene expression data from myocardial cells than the whole cardiac tissues.