Apoptosis in the developing mouse heart

Abstract

Apoptosis occurs at high frequency in the myocardium of the developing avian cardiac outflow tract (OFT). Upor down-regulating apoptosis results in defects resembling human conotruncal heart anomalies. This finding suggested that regulated levels of apoptosis are critical for normal morphogenesis of the four-chambered heart. Recent evidence supports an important role for hypoxia of the OFT myocardium in regulating cell death and vasculogenesis. The purpose of this study was to determine whether apoptosis in the outflow tract myocardium occurs in the mouse heart during developmental stages comparable to the avian heart and to determine whether differential hypoxia is also present at this site in the murine heart. Apoptosis was detected using a fluorescent vital dye, Lysotracker Red (LTR), in the OFT myocardium of the mouse starting at embryonic day (E) 12.5, peaking at E13.5-14.5, and declining thereafter to low or background levels by E18.5. In addition, high levels of apoptosis were detected in other cardiac regions, including the apices of the ventricles and along the interventricular sulcus. Apoptosis in the myocardium was detected by double-labeling with LTR and cardiomyocyte markers. Terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick endlabeling (TUNEL) and immunostaining for cleaved Caspase-3 were used to confirm the LTR results. At the peak of OFT apoptosis in the mouse, the OFT myocardium was relatively hypoxic, as indicated by specific and intense EF5 staining and HIF1␣ nuclear localization, and was surrounded by the developing vasculature as in the chicken embryo. These findings suggest that cardiomyocyte apoptosis is an evolutionarily conserved mechanism for normal morphogenesis of the outflow tract myocardium in avian and mammalian species.

Temporal–spatial ablation of neural crest in the mouse results in cardiovascular defects

“…The embryos harvested at E13.5 and E14.5 have an elongated muscular OFT. This OFT seems to recede between E14.5 and E15.5, consistent with the pattern of OFT shortening seen in WT murine embryos (Barbosky et al, 2006;compare Fig. 4A-C with 4D-F).…”

Cathepsin D gene expression outlines the areas of physiological cell death during embryonic development

“…In addition, the myocardial layer of the proximal segment of the outflow tract also undergoes a dramatic cell death process ( Fig. 2A-C; Hurle et al, 1977;Sugishita et al, 2004;Barbosky et al, 2006). All the mentioned areas of cell death appeared clearly outlined by the expression domain of the cathepsin D gene in our study .…”

“…The expression pattern of cathepsin D observed here indicates that lysosomes are intensely activated in most areas of PCD, suggesting they are responsible for the high affinity of the apoptotic areas to vital staining. Consistent with this interpretation, the lysosomal marker LysoTracker Red has been successfully introduced as vital staining marker to map areas of cell death (Zucker el al., 1999;Barbosky et al, 2006).…”

Cardiac Vasculature: Development and Pathology

“…Lysotracker Red staining indicates that most of the apoptotic cells are within the myocardium not in the vasculature. The aorta (ao) and pulmonary artery (pa) are indicated at a low magnification (A, 100x) and higher magnifications are in (B, 200x) and (C, 400x) (Barbosky et al, 2006). (Albrecht and Christofori, 2011;Schulte-Merker, 2011;Witte et al, 2011).…”

“…(Nanka et al, 2008;Naňka et al, 2006;Sugishita et al, 2004c;Tomanek et al, 2003;Wikenheiser et al, 2006)) discovered by the use of hypoxia indicators that there is a pattern of differential hypoxia within the embryonic heart that may explain in part why vessels form or are more concentrated at particular places. The spatiotemporal pattern of outflow tract (OFT) myocardial hypoxia as measured by the hypoxia indicator EF5 correlates with HIF-1a nuclear localization, cardiomyocyte apoptosis, and morphogenesis of the OFT in both avian and mouse embryos (Sugishita et al, 2004A, B, C;Barbosky et al, 2006;Wikenheiser et al, 2006). Incubation of embryos under hyperoxic conditions reduced tissue hypoxia, HIF-1a nuclear localization and cell death in the OFT myocardium and resulted in abnormal conotruncal morphologies.…”