Analysis of TdT-Mediated dUTP Nick End Labeling (TUNEL)-Positive Cells Associated with Cardiac Myogenesis in Mouse Embryo

Abstract

Caspase activation is associated with skeletal muscle differentiation in mouse embryos. We examined the relationship between cardiac myogenesis and cell death using mice hearts at embryonic days (E) 11.5–15.5 and fetal rat heart H9C2 cells. The number of TdT-mediated dUTP nick end labeling (TUNEL)-positive cells increased with fetal age and was much higher than that of single-stranded DNA (ssDNA)- and active caspase-3 (aCasp3)-positive cells. TUNEL and aCasp3 double staining resulted in 3 types of positive cells: TUNEL⁺/aCasp3⁺, TUNEL⁺/aCasp3^- and TUNEL^-/aCasp3⁺. TUNEL⁺/aCasp3^- cells were the most common but lacked morphological features of apoptosis, such as nuclear condensation or fragmentation. The expression of anti-apoptotic factors increased during E11.5–15.5. Furthermore, TUNEL-positive H9C2 cells without nuclear condensation or fragmentation were observed only in myotubes later in the culture period. In this study, the dynamics of TUNEL-positive cardiomyocyte was inconsistent with the activation of apoptosis cascade, and their morphological feature was clearly different from representative apoptosis. From these findings, we concluded that the increased number of TUNEL-positive cardiomyocyte, having the DNA strand breaks, would be associated with the progression of cardiac myogenesis.