Background—: Patients with congenital heart disease (CHD) and heterotaxy show high postsurgical morbidity/mortality, with some developing respiratory complications. Although this finding is often attributed to the CHD, airway clearance and left-right patterning both require motile cilia function. Thus, airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD) may contribute to increased respiratory complications in heterotaxy patients. Methods and Results—: We assessed 43 CHD patients with heterotaxy for airway CD. Videomicrocopy was used to examine ciliary motion in nasal tissue, and nasal nitric oxide (nNO) was measured; nNO level is typically low with PCD. Eighteen patients exhibited CD characterized by abnormal ciliary motion and nNO levels below or near the PCD cutoff values. Patients with CD aged >6 years show increased respiratory symptoms similar to those seen in PCD. Sequencing of all 14 known PCD genes in 13 heterotaxy patients with CD, 12 without CD, 10 PCD disease controls, and 13 healthy controls yielded 0.769, 0.417, 1.0, and 0.077 novel variants per patient, respectively. One heterotaxy patient with CD had the PCD causing DNAI1 founder mutation. Another with hyperkinetic ciliary beat had 2 mutations in DNAH11 , the only PCD gene known to cause hyperkinetic beat. Among PCD patients, 2 had known PCD causing CCDC39 and CCDC40 mutations. Conclusions—: Our studies show that CHD patients with heterotaxy have substantial risk for CD and increased respiratory disease. Heterotaxy patients with CD were enriched for mutations in PCD genes. Future studies are needed to assess the potential benefit of prescreening and prophylactically treating heterotaxy patients for CD.

Previous studies have indicated an intimate linkage between gap junction and adherens junction formation. It was suggested this could reflect the close membrane-membrane apposition required for junction formation. In NIH3T3 cells, we observed the colocalization of connexin43 (Cx43α1) gap junction protein with N-cadherin, p120, and other N-cadherin-associated proteins at regions of cell-cell contact. We also found that Cx43α1, N-cadherin, and N-cadherin-associated proteins were coimmunoprecipitated by antibodies to either Cx43α1, N-cadherin, or various N-cadherin-associated proteins. These findings suggest that Cx43α1 and N-cadherin are coassembled in a multiprotein complex containing various N-cadherin-associated proteins. Studies using siRNA knockdown indicated that cell surface expression of Cx43α1 required N-cadherin, and conversely, N-cadherin cell surface expression required Cx43α1. Pulse-chase labeling and cell surface biotinylation experiments indicated that in the absence of N-cadherin, Cx43α1 cell surface trafficking is blocked. Surprisingly, siRNA knockdown of p120, an N-cadherin-associated protein known to modulate cell surface turnover of N-cadherin, reduced N-cadherin cell surface expression without altering Cx43α1 expression. These observations suggest that in contrast to the coregulated cell surface trafficking of Cx43α1 and N-cadherin, N-cadherin turnover at the cell surface may be regulated independently of Cx43α1. Functional studies showed gap junctional communication is reduced and cell motility inhibited with N-cadherin or Cx43α1 knockdown, consistent with the observed loss of both gap junction and cadherin contacts with either knockdown. Overall, these studies indicate that the intracellular coassembly of connexin and cadherin is required for gap junction and adherens junction formation, a process that likely underlies the intimate association between gap junction and adherens junction formation.

Connexin 43 knockout (Cx43α1KO) mice have conotruncal heart defects that are associated with a reduction in the abundance of cardiac neural crest cells (CNCs) targeted to the heart. In this study, we show CNCs can respond to changing fibronectin matrix density by adjusting their migratory behavior,with directionality increasing and speed decreasing with increasing fibronectin density. However, compared with wild-type CNCs, Cx43α1KO CNCs show reduced directionality and speed, while CNCs overexpressing Cx43α1 from the CMV43 transgenic mice show increased directionality and speed. Altered integrin signaling was indicated by changes in the distribution of vinculin containing focal contacts, and altered temporal response of Cx43α1KO and CMV43 CNCs to β1 integrin function blocking antibody treatment. High resolution motion analysis showed Cx43α1KO CNCs have increased cell protrusive activity accompanied by the loss of polarized cell movement. They exhibited an unusual polygonal arrangement of actin stress fibers that indicated a profound change in cytoskeletal organization. Semaphorin 3A, a chemorepellent known to inhibit integrin activation, was found to inhibit CNC motility, but in the Cx43α1KO and CMV43 CNCs, cell processes failed to retract with semaphorin 3A treatment. Immunohistochemical and biochemical analyses suggested close interactions between Cx43α1,vinculin and other actin-binding proteins. However, dye coupling analysis showed no correlation between gap junction communication level and fibronectin plating density. Overall, these findings indicate Cx43α1 may have a novel function in mediating crosstalk with cell signaling pathways that regulate polarized cell movement essential for the directional migration of CNCs.