Characterization of Two Populations of Ectopic Cells Isolated from the Hearts of NGF Transgenic Mice

Abstract

We have observed sympathetic hyperinnervation and hyperplasia of an unknown cell population within the base of the hearts from transgenic mice in which nerve growth factor (NGF) expression was targeted to cardiac tissue (Hassankhani et al., 1993, 1995). To characterize this ectopic cell population, cells from the base of hearts of 3- to 4-week-old transgenic mice were dissociated and established in tissue culture, and their properties were analyzed using morphological, immunocytochemical, and physiological techniques. Morphological studies of the ectopic cells revealed the temporal expression of two different cellular phenotypes: (i) a spindle-shaped cell type, present by 1 to 2 days in vitro (DIV) and (ii) cells with a fiber-like morphology, detected by 7 DIV. In contrast to transgenic cardiac myocytes that express immunoreactivity to antibodies against sarcomeric myosin, beat spontaneously (∼60/min), and are electrically excitable, the ectopic cell types did not stain with an antibody against sarcomeric myosin, never showed contractile activity, and did not manifest membrane excitability. Moreover, these two types of ectopic cells demonstrated other distinctive characteristics. The spindle-shaped subpopulation typically formed small clusters of cells that were immunostained by antibodies to GFAP, vimentin, and low-affinity NGF receptor (LNGFR) and demonstrated a low incidence of dye coupling (30%), whereas the fiber-like cells aligned themselves along their long axes, immunostained for gp140 trkA, LNGFR, and anti α-smooth muscle actin and showed extensive (100%) and diffuse dye spread to numerous other contiguous fiber-like cells. Dye spread was not observed between the adjacent fiber-like and the spindle-shaped cells and was reversibly blocked between homologous cells after treatment with halothane, a gap junction channel blocking agent. In comparison to cardiac myocytes, macroscopic junctional conductance of the spindle-shaped cells was more voltage-sensitive. At the single channel level, unitary junctional conductances of approximately 60 pS were predominant. Overall, these results indicate that these ectopic cells are likely derived from neural crest. The spindle-shaped cells appear to be immature Schwann cells, whereas the fiber-like cells may be related to the ectomesenchymal cells contributing to the morphogenesis of the cardiac outflow tract.