Morphologic Variation in Isolated Neonatal and Adult Mouse Cardiac Myocytes 117

Abstract

Studies of cardiac cellular physiology in transgenic mouse models have been limited in part by an inability to use a single technique that yields freshly isolated neonatal and adult cardiac myocytes. The goal of this project is to develop a uniform cell isolation technique applicable to mice from all age groups for studies of single cell morphology, contractility, and electrophysiology. Excised, intact hearts, harvested from two-day-old and adult mice, are perfused via an aortic cannula using a modification of the Langendorff technique. Hearts are perfused with enzyme solution containing collagenase L and protease, and washed free of enzyme by perfusion with 0.1mM calcium Tyrode's solution before separation of the cells. Trypan blue staining indicates 70% viability for neonatal cells and 85% viability for adult cells. Two populations of neonatal cells, both spindle and round shaped, are apparent; adult cells are uniformly rod shaped. Electrical field stimulation of the myocytes results in consistent cell contractions and release of intracellular calcium stores in both the neonatal and adult cardiac myocytes. Field stimulation-induced calcium transients are present in both spindle and round shaped neonatal myocytes. Thus, this single technique provides high-yield, physiologically-intact, isolated cardiac myocytes from both neonatal and adult mouse heart. In contrast to previous reports using minced tissue preparations, our perfusion technique results in the isolation of a population of elongated neonatal cardiac myocytes. This technique provides a means for future studies of cardiac myocyte morphology and physiology using transgenic mouse models.