Studies on heart. XVIII. Heart component influencing the maintenances of spreading and beating of rat myocardial cells in serum-free culture.

Abstract

The heart component which maintains rat myocardial cell spreading and beating in serum-free culture was investigated. Spreading and well-spreading percentage of single cells cultured in Eagle minimum essential medium (MEM)-0.5% bovine serum albumin for 2 days at 37.degree. C and relative beating percentage and rate of single well-spreading cells incubated with sample in Eagle MEM for 1 h at 37.degree. C were used as parameters of the maintenance activities. Filtrates of heart ventricle fragments cultured in Eagle MEM for 4 days at 37.degree. C significantly promoted all 4 parameters. Precipitates salted out with 30-50% saturated ammonium sulfate from aqueous alkaline extracts of rat, bovine and rabbit ventricles had promoting effects similar to culture filtrate. Extracts prepared from brain, liver, spleen, pancreas, small intestine, kidney and skeletal muscle did not affect or inhibit spreading and beating. Desalted culture filtrates and extracts of ventricles did not possess trypsin inhibitory activity. Bovine ventricle extract was fractionated using successive chromatography on DEAE-cellulose, Sephadex G-100 and CM-cellulose column. The isolated principle, Fr. BVP (bovine ventricle protein), was a protein with MW 100,000 and 18 amino acids. Fr. BVP significantly maintained spreading and beating of myocardial cells in serum-free culture in a concentration of 100 .mu.g/dish. This protein differed biologically and chemically from Fr. A, inotropic protein of bovine heart, which was effective for promoting the beating of myocardial cells cultured with serum.