Morphological analysis of the trachea and pattern of breathing in βENaC-Tg mice

Abstract

Cystic fibrosis (CF) is caused by mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) gene which is a Cl⁻ channel and a regulator of the epithelial Na⁺ channel (ENaC). We have recently shown that newborn CFTR-deficient mice exhibit abnormalities of the tracheal cartilage leading to altered ventilation (Bonvin et al., 2008). However, the mechanism by which a lack of CFTR causes tracheal cartilage defects remains unknown.

The main goal of the present study was to determine whether the development of airway cartilage defects is related to ENac channel dysfunction.

We thus performed macroscopic analysis of the trachea and explored ventilatory function in adult βENaC-overexpressing (βENaC-Tg) mice with airway Na⁺ hyperabsorption and “CF-lung” lung disease, at 2 and 5 month of age.

Only minor cartilaginous abnormalities were observed in 8 out of 16 βENaC-Tg mice and in 2 out of 20 littermate controls. Breathing pattern was progressively altered in βENaC-Tg mice as evidenced by a significant decrease in respiratory frequency.

Our results suggest that Na⁺ hyperabsorption alone is not a major contributor to the development of tracheal malformation observed in CF mice and that breathing pattern changes in βENaC-Tg mice likely reflect airflow limitation due to airway mucus obstruction.

Introduction

Cystic fibrosis (CF) results primarily from mutations inducing inadequate functioning of CFTR (cystic fibrosis transmembrane conductance regulator) which is a cAMP-activated Cl⁻ channel and a regulator of the epithelial Na⁺ channel (ENaC). In the CF airway, the failure of CFTR causes decreased Cl⁻ secretion and increased fluid absorption leading to airway surface liquid (ASL) volume depletion and impaired mucus clearance (Mall, 2009).

In the last decades, several mouse models harbouring distinct Cftr mutations have been generated. Although they fail to develop classical lung disease similar to human CF, minor functional and histopathological changes have been reported in the upper airways (Cohen et al., 2004, Kent et al., 1997). In addition, we have recently demonstrated that newborn Cftr knockout and F508del-CFTR mice exhibit tracheal cartilage ring abnormalities that affect mechanical properties of the trachea and limit respiratory airflow (Bonvin et al., 2008). It indicates that tracheal ring malformations should be caused by molecular mechanisms governing tracheal development related to ion transport defect or by other CFTR-related function.

The hypothesis that increased Na⁺ absorption and ASL depletion may play a key role in pathogenesis of CF airway disease led to the development of mice that overexpress the β-subunit of ENaC to increase airway Na⁺ absorption (Mall et al., 2004). These animals do develop spontaneous CF-like lung disease including impaired mucociliary clearance, airway mucus obstruction and chronic airway inflammation (Mall et al., 2008).

The aim of the present study was to determine whether the cartilage malformations of the trachea are related to increased Na⁺ absorption and ASL depletion or to a CFTR- related cellular defect. We, thus, performed morphological analyses of the trachea and explored ventilatory function in adult βENaC-Tg mice and littermate control mice.