Differentiation-dependent expression of heart type fatty acid-binding protein in C2C12 muscle cells

Abstract

The aim of the present work was to establish a cell culture model for the investigation of the influence of heart type fatty acid-binding protein (H-FABP) on differentiation and lipid metabolism. Up to now no data have been reported on H-FABP in cell lines of skeletal muscle, one of the major sources of this protein in vivo. For this purpose mouse C2C12 cells were chosen, because these cells can be stimulated to differentiate in vitro from myoblasts to spontaneously contracting, multiply nucleated myotubes expressing muscle-specific proteins like creatine kinase. Analysis of the cellular proteins by two-dimensional gel electrophoresis and ELISA demonstrated that the expression of H-FABP is differentiation dependent as well in these cells. Furthermore, immunofluorescent labeling with H-FABP-specific antibodies revealed that induction of this protein occurred mainly in myotubes. Myoblasts contained only 7.1 +/- 3.1 ng H-FABP/mg soluble protein, however, upon differentiation, this value increased about 60-fold to 420 +/- 90 ng/mg (n = 4) in a mixture of myoblasts and myotubes. H-FABP from C2C12 cells was subsequently cloned and shown to be identical to the known mouse H-FABP. The induction of H-FABP during differentiation was also detected at mRNA level by probing with H-FABP-cDNA. Insulin, a known stimulator of in vitro muscle cell differentiation, led to an increased differentiation as referenced by creatine kinase activity, which is paralleled by an increased H-FABP expression. The enhancement of H-FABP expression by insulin was found to be time- and dose-dependent. The increasing H-FABP content may relate to an increasing fatty acid oxidation that has been reported for differentiated L6 cells, a related muscle cell line from rat. Such a correlation would favor a role of H-FABP in lipid metabolism.