Differential effect of intrauterine hypoxia on caspase 3 and DNA fragmentation in fetal guinea pig hearts and brains

Abstract

The aim of this study is to quantify the effect of intrauterine hypoxia (HPX) and the role of nitric oxide (NO) on the apoptotic enzyme, caspase 3, and DNA fragmentation in fetal heart and brain. Hypoxia and NO are important regulators of apoptosis, although this has been little studied in the fetal organs. We investigated the effect of intrauterine HPX on apoptosis and the role of NO in both fetal hearts and brains. Pregnant guinea pigs were exposed to room temperature (N = 14) or 10.5% O₂ (N = 12) for 14 days prior to term (term = 65 days) and administered water or L-N6-(1-iminoethyl)-lysine (LNIL), an inducible nitric oxide synthase (iNOS) inhibitor, for 10 days. Fetal hearts and brains were excised from anesthetized near-term fetuses for study. Chronic HPX decreased pro- and active caspase 3, caspase 3 activity, and DNA fragmentation levels in fetal hearts compared with normoxic controls. L-N6-(1-iminoethyl)-lysine prevented the HPX-induced decrease in caspase 3 activity but did not alter DNA fragmentation levels. In contrast, chronic HPX increased both apoptotic indices in fetal brains, which were inhibited by LNIL. Thus, the effect of HPX on apoptosis differs between fetal organs, and NO may play an important role in modulating these effects.

Figure 2.

Immunohistochemistry of caspase 3 in normoxic (NMX) and hypoxic (HPX) fetal heart ventricles untreated (control) and in the presence of l-N6-(1-iminoethyl)-lysine (LNIL). Representative figures (magnification ×200) illustrate caspase 3 expression (brown stain) and nuclei of cardiomyocytes (blue stain).

Figure 1.

Western analysis of caspase 3 in normoxic ([NMX] N = 4) and hypoxic ([HPX] N = 4) left ventricles. Density values of each band were analyzed relative to its loading control (a-actin). Values are mean ± standard error. *Indicates P < .05 versus NMX. N indicates number of fetuses.

Figure 3.

Effect of chronic hypoxia (HPX) and l-N6-(1-iminoethyl)-lysine (LNIL) on caspase 3 activity of fetal guinea pig cardiac ventricles (left) and forebrains (right). Activity levels were measured as picomoles AMC/minute per milligram protein in normoxic ([NMX] N = 4), hypoxic ([HPX] N = 5), and in the presence/absence of LNIL (NMX ± LNIL, N = 4; HPX ± LNIL, N = 4). Values are mean ± standard error. *Indicates P < .05 versus NMX. **Indicates P < .05 versus HPX. Fetal hearts and forebrains were obtained from the same animals. N indicates number of fetuses; AMC, 7-amino-4-methylcoumarin.

Figure 4.

Effect of chronic hypoxia (HPX) and l-N6-(1-iminoethyl)-lysine (LNIL) on DNA fragmentation of fetal guinea pig cardiac ventricles (left) and forebrains (right). DNA fragmentation was quantified as OD_{405 nm}/mg protein in normoxic ([NMX] N = 8), hypoxic ([HPX] N = 6), and in the presence/absence of LNIL (NMX ± LNIL, N = 5; HPX ± LNIL, N = 8). Values are mean ± standard error. *Indicates P < .05 versus NMX. **Indicates P < .05 versus HPX. Fetal hearts and forebrains were obtained from the same animals. N indicates number of fetuses. OD, optical density.