Disruption of copper-dependent signaling pathway in the nitrofen-induced congenital diaphragmatic hernia

Normal development of the fetal diaphragm requires muscularization of the diaphragm as well as the structural integrity of its underlying connective tissue components. Developmental mutations that inhibit the formation of extracellular matrix (ECM) have been shown to result in congenital diaphragmatic hernia (CDH). Copper (Cu) is an important element during diaphragm morphogenesis by participating in cross-linking of collagen and elastin fibers. Cu transport is strictly regulated by two membrane proteins: Cu-uptake transporter 1 (CTR1) and the Cu-efflux pump ATP7A. Animals lacking Cu-dependent enzymes exhibit abnormal connective tissue with diaphragmatic defects. However, the molecular basis of disruptions in Cu-mediated ECM formation in CDH remains unclear. We designed this study to investigate the hypothesis that diaphragmatic expression of CTR1 and ATP7A is decreased in the nitrofen-induced CDH model.

Timed-pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on selected time-points D15 and D18. Microdissected fetal diaphragms (n = 48) were divided into control and nitrofen-induced CDH samples (n = 12 per experimental group and time-point). Diaphragmatic gene expression levels of CTR1 and ATP7A were analyzed by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed to evaluate CTR1 and ATP7A protein expression in fetal diaphragms, which was combined with specific rhodanine staining to determine diaphragmatic Cu content.

Relative mRNA levels of CTR1 and ATP7A were significantly reduced in diaphragms of nitrofen-exposed fetuses on D15 (0.06 ± 0.02 vs. 0.18 ± 0.08; p < 0.05 and 0.04 ± 0.02 vs. 0.08 ± 0.02; p < 0.05) and D18 (0.10 ± 0.03 vs. 0.17 ± 0.02; p < 0.05 and 0.09 ± 0.03 vs. 0.16 ± 0.04; p < 0.05) compared to controls. Immunoreactivity of CTR1 and ATP7A was markedly decreased in the malformed diaphragmatic ECM of nitrofen-exposed fetuses on D15 and D18, which was associated with a significantly decreased diaphragmatic Cu content on D15 (7.22 ± 2.91 vs. 17.50 ± 3.09; p < 0.05) and D18 (17.60 ± 3.54 vs. 28.20 ± 4.63; p < 0.05) compared to controls.

Reduced diaphragmatic expression of CTR1 and ATP7A during morphogenesis may impair the activity of Cu-dependent enzymes and thus contribute to defective ECM during diaphragmatic development.