A method to quantitate cell numbers of muscle cells and non-muscle cells in homogenised heart cell cultures.

Abstract

Neonatal rat heart cell cultures are popular research models in cardiovascular investigations. A major disadvantage is the variable contribution of non-muscle cells to the cultures. As biochemical and pharmacological quantities are generally measured in homogenised cultures, we looked for a method to calculate numbers of muscle cells and non-muscle cells per culture after homogenisation. By means of a model based on the presence of one diploid nucleus per myocyte and per non-muscle cell, a nuclear DNA content of 7.5 pg, and a constant ratio of DNA content and sum of the lactate dehydrogenase isoenzymes LDH-4 and LDH-5 (DNA/LDH4+5 = 11.6 +/- 1.5 micrograms.U-1) in non-muscle cells, we calculated that in 21 neonatal rat heart cell cultures, cultured for 0-6 days, the number of muscle cells was 1.5 X 10(6) per culture, independent of time; and the number of non-muscle cells was low at day 0 (1.6 X 10(5) per culture), increasing to 4 X 10(6) per culture at day 6. Based on a time dependent increase in lactate dehydrogenase content per muscle cell we showed that muscle cells in culture underwent progressive hypertrophy: in 6 days myocyte volume increased fourfold. Thus, by measurement of DNA content and the activities of lactate dehydrogenase and its isoenzymes in a homogenised culture the cellular composition of the culture can be assessed quantitatively.