APPLICATION OF STEREOLOGICAL ANALYSIS OF CELL-VOLUME TO ISOLATED MYOCYTES IN CULTURE WITH AND WITHOUT ADRENERGIC-INNERVATION

A three-dimensional analysis to evaluate structural changes in cultured cardiac myocytes following adrenergic innervation was performed using stereological techniques formerly limited to cells in tissue and organs. Cell volumes were calculated for two groups of cells at 96 hours in culture: isolated myocytes and myocytes innervated with adrenergic neurons. Relative and absolute volumes of the nucleus, cytoplasm, and cell were quantified by systematically sampling sections throughout the cell and by point count sampling techniques. Volumetric estimates were similarly determined for the mitochondria, sarcomeres, and other cellular components in the cytoplasm. Data were analyzed with ANOVA and randomized block design to control for variation among the cultures. Adrenergic innervation produced a 44% increase in cell volume, X +/- SEM, (3,344 +/- 196-mu-m3 to 4,816 +/- 400-mu-m3, P = 0.007). The absolute volume of mitochondria significantly increased after innervation (521 +/- 42-mu-m3 to 744 +/- 54-mu-m3, P < 0.01). Absolute sarcomere volume did not change significantly (750 +/- 92-mu-m3 to 642 +/- 1061-mu-m3, P = 0.14). Other cellular components, defined as all cytoplasmic components except mitochondria and sarcomeres, significantly increased with innervation (1,739 +/- 166-mu-m3 to 3,097 +/- 338-mu-m3, P = 0.02). The relative volume of the nucleus and the cytoplasm in the cell remained unchanged following innervation. However, the relative volume of mitochondria decreased by 6%, the percent of the cytoplasm occupied by the sarcomeres decreased by 44%, and the volume occupied by the other cellular components increased by 22%. These findings support the use of stereological analysis as a means to quantify cell volumes of cultured myocytes. Significant increases in cell size, as well as changes in intracellular composition, accompany in vitro adrenergic innervation in the developing cardiac myocyte.