Gene expression in rod shaped cardiac myocytes, sorted by flow cytometry.

Abstract

Primary cardiac myocyte cultures are usually contaminated with variable parts of different cell types, such as fibroblasts, endothelial cells and smooth muscle cells. Thus, the objective of our study was to analyse the gene expression in a pure population. To obtain an homogeneous population, cardiac myocytes from adult rats were fixed with ethanol and sorted by flow cytometry. This approach is suitable for isolating either single cells or up to several thousand cells. To measure the messenger ribonucleic acid (mRNA) expression of different genes at the level of a few rod-shaped myocytes, a cDNA library was created by polymerase chain reaction (PCR). Sorting by a fluorescence-activated cell sorter (FACS) resulted in pure rod-shaped cardiac myocytes and isolated RNA from these cells is undegraded, as shown by Northern blotting. We demonstrated both the expression of housekeeping genes, such as beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as well as the myocyte-specific transcripts, alpha-cardiac myosin heavy chain (alpha-MHC) and beta-MHC. Furthermore, we showed the induction of the immediately early gene c-fos at the level of ten sorted cells. This method allows one to study gene expression in different cell types within the heart, in tissue samples or to tackle the problem of heterogeneity within a cell population.