Surface morphology and cell size measurement of isolated rat cardiac myocytes

Abstract

We studied the morphologic features of cardiac myocytes isolated from 240 g rats by perfusion with enzyme containing media. Hearts obtained from ether-anesthetized animals were perfused on a modified Langendorff apparatus with Ca 2+-free Hanks' solution, 3% fetal calf serum, 95% O 2, 5% CO 2, pH 7.4 for 12 min, the same media with 0.1% collagenase for 12 min, and finally with enzyme-free media containing EGTA for 12 min. Hearts perfusion fixed at various stages of the cell preparation procedure with 2% glutaraldehyde for light and electron microscopic study of in situ cells revealed that most cellular damage was due to action of the enzyme. Up to 80% of isolated myocytes excluded trypan blue immediately after isolation, but numerous cells in the non-purified preparations exhibited various degrees of membrane blebs, cytoplasmic vacuolization, supercontraction (rounded cells) and trypan blue uptake. Poor preparations were obtained if perfusion pressure exceeded 100 mmHg, Ca 2+ was included, or if pH, perfusion flow rates and times were not carefully controlled. By scanning electron microscopy, elongated cells excluding trypan blue had a uniformly corrugated surface with depressions at the sarcomere ends corresponding to Z-line attachment to the sarcolemma. Irregularly located holes in the Z-line depression corresponded in size and location to T-tubule openings. Cells had irregular elongated shapes due to many transverse intercalated disc areas not only at cell ends but also along lateral portions of the cell. Hematoxylin and Eosin stained smears of isolated myocytes revealed 85% to contain two nuclei. Mean cell length was 94 ± 18 ( s.d.) μm and cell width was 18 ± 4 μm measured in 150 cells from each of six rats. Calculated cell volume for double nucleated cells was nearly two times that for single nucleated cells indicating a constant nuclear to cytoplasmic ratio in the two cell populations. Isolated cardiac myocytes are a useful model to study structural aspects of heart muscle.