No abstract

Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3− and Cl−, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

Ankyrin-B is a multifunctional adapter protein responsible for localization and stabilization of select ion channels, transporters, and signaling molecules in excitable cells including cardiomyocytes. Ankyrin-B dysfunction has been linked with highly penetrant sinoatrial node (SAN) dysfunction and increased susceptibility to atrial fibrillation. While previous studies have identified a role for abnormal ion homeostasis in ventricular arrhythmias, the molecular mechanisms responsible for atrial arrhythmias and SAN dysfunction in human patients with ankyrin-B syndrome are unclear. Here, we develop a computational model of ankyrin-B dysfunction in atrial and SAN cells and tissue to determine the mechanism for increased susceptibility to atrial fibrillation and SAN dysfunction in human patients with ankyrin-B syndrome. Our simulations predict that defective membrane targeting of the voltage-gated L-type Ca(2+) channel Cav1.3 leads to action potential shortening that reduces the critical atrial tissue mass needed to sustain reentrant activation. In parallel, increased fibrosis results in conduction slowing that further increases the susceptibility to sustained reentry in the setting of ankyrin-B dysfunction. In SAN cells, loss of Cav1.3 slows spontaneous pacemaking activity, whereas defects in Na(+)/Ca(2+) exchanger and Na(+)/K(+) ATPase increase variability in SAN cell firing. Finally, simulations of the intact SAN reveal a shift in primary pacemaker site, SAN exit block, and even SAN failure in ankyrin-B-deficient tissue. These studies identify the mechanism for increased susceptibility to atrial fibrillation and SAN dysfunction in human disease. Importantly, ankyrin-B dysfunction involves changes at both the cell and tissue levels that favor the common manifestation of atrial arrhythmias and SAN dysfunction.

Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl − or HCO 3 − was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl − /HCO 3 − -free conditions and in CF. Single-cell RNA sequencing revealed a single epithelial cell type in non-CF gallbladders that co-expressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.