Visualizing cell death in the developing heart


During embryonic development, the heart maintains its critical function of pumping blood while undergoing rapid and dramatic morphological remodeling. In the mammalian embryo, the heart transforms in a matter of days from a linear tube with one-way blood flow to a dual-flow four-chambered organ. Not only is there a great deal of cell proliferation, differentiation, and growth during this transition, there is also a high frequency of cell death that sculpts the organ. Due to the rapidity of this process, it is not easy to catch all the events in this dynamic progression at a fixed time point in histological tissue sections.

The mechanism of apoptosis for sculpting of tissue structures such as the digits of the paw has been well documented with TdT-mediated dUTP nick end labeling (TUNEL) and other assay methods. However, the rapidly contorting, stretching, pulsating heart does not easily lend itself to such straightforward documentation. In the developing paw, the intercalating tissue between the digits can be labeled with programmed cell death (PCD) assays such as TUNEL or lysosomal and macrophage labeling dyes to document the process of apoptosis and the subsequent removal of the dying cells by macrophage cleanup crews. However, these cleanup crews of macrophages do not seem to be as numerous in the heart as in other rapidly remodeling tissues. One reason for their absence may be that the neighboring myocardial cells seem to be doing a fine job of cleanup and recycling on their own without the help of those scavenging blood cells (Hurle and Ojeda, 1979).

It is hard even to catch glimpses of the cells going through PCD as they seem to be gobbled up before their presence can be detected by assay methods such as TUNEL. Thus, the careful analysis of programmed cell death documented by Sharma et al. (2004) in the April 2004 issue of The Anatomical Record, Part A (view this article online at, during mouse cardiogenesis provides a much needed look at apoptosis in the developing mouse heart by compiling the results of TUNEL assays from several mouse embryo hearts during this critical period in heart remodeling. Due to the rapidity of the removal of the apoptotic cells apparently by myocardial cells surrounding the dying cells, it is difficult to get a good handle on just how many cells are going through this process. Sharma et al. (2004) used a method of templating data or summating TUNEL-positive cells from the hearts of several mouse embryos of equivalent somite stage development. Thus, by compiling data from several embryos, they were able to quantify the amount of cell death occurring in the mouse heart relative to the number of cells present in the structure.

Another way that the apoptotic cells can be recorded before their total removal from the tissue has been documented in chick heart remodeling using a different type of apoptotic assay that labels not only the apoptotic bodies containing DNA fragments, but most apoptotic bodies in the surrounding tissue as well (Schaefer et al., 2004). The LysoTracker dyes (Molecular Probes, Eugene, OR) label acidic lysosomal compartments whether they contain condensed DNA or not, whereas the TUNEL assay is limited to labeling only the subset of compartments with condensed DNA fragments. The LysoTracker assays can also be used in whole mount or on thicker tissue sections where it is easier to see accumulations of LysoTracker dye particles through the entire thickness of myocardium, rather than the comparatively few TUNEL particles in 7–10 μ sections.

Spatiotemporal analyses of cell death during cardiac remodeling provide important baselines to understand the contribution of normal PCD in the sculpting of mature heart structure. From these studies, abnormal PCD in heart development can be assessed. The Sharma et al. (2004) study is significant in that due to the limitations and difficulty in documentation, there have not been many comprehensive studies of mammalian cell death in the developing heart.

The Sharma et al. (2004) study is significant in that due to the limitations and difficulty in documentation, there have not been many comprehensive studies of mammalian cell death in the developing heart.

Due to the accessibility of the embryonic avian heart its development and associated cell death has been extensively studied, more so than mammalian models. Specifically there have been numerous studies documenting foci of PCD in the avian embryonic heart that heretofore have not been observed in the mouse. However, as the Sharma study illustrates, there is cardiomyocyte death in the mammalian outflow tract myocardium just as there is in the chicken heart. However, it does not look as dramatic because the apoptotic bodies are either smaller, as suggested by Pexieder (1975), or just not as numerous. When compared to embryonic heart apoptosis studies done in chick, the mouse may have fewer cells dying at any one time with a longer period of cell death or the apoptotic bodies could be more rapidly cleared. Analysis of apoptosis patterns in the developing mammalianheart may require the summation of data as shown by Sharma et al. (2004), the simultaneous use of multiple assays, or a sensitive assay such as LTR when analyzing the embryo hearts in section.