Vascular Reactivity of Isolated Pulmonary Artery and Thoracic Aorta in Mice Genetically Deficient in Connexin 43

Pulmonary arterial hypertension (PAH) is a terminal disease, associated with both constriction and remodelling of the pulmonary vasculature. Cellular communication is thought to be critical to both the constriction and remodelling processes. Connexins are gap junction forming transmembrane proteins which allow the exchange of small signalling molecules (<1kDa) between adjacent cells. Recent evidence suggests dysregulation of connexin signalling plays a role in the development of PAH (1-3). The aim of this study was to assess the role of connexin 43 (Cx43) in vascular reactivity in isolated intra-lobar pulmonary arteries (IPAs) using mice genetically deficient in connexin 43 (Cx43+/- mice). In order to compare the pulmonary and systemic circulations, contractile responses were also investigated in isolated thoracic aorta from Cx43+/- mice. As there is a female gender bias associated with PAH, we assessed vascular responses in both male and female mice.

Male and female Cx43 +/- mice (4) and wild type (WT) littermates (20 to 28 weeks old) were euthanized by administration of pentobarbitone (60 mg/kg, ip.). IPAs (~350µm id) and thoracic aorta (~600µm id) were dissected free and mounted (Krebs-Heseleit solution, 37 ˚C) on wire myographs. Cumulative contractile response curves (CRCs) to 5-hydroxytryptamine (5-HT; 1nM-0.3mM) and endothelin-1 (ET-1; 0.1nM-0.1µM) were constructed. Statistical analysis was performed by two-way ANOVA with Bonferroni’s multiple comparison test or Student’s t-test as appropriate. Data are presented as mean±s.e.m.

ET-1 was more potent in IPAs derived from male Cx43+/- mice than those from male WT mice (pEC50: 8.77±0.12, n=5 cf 8.46±0.08, n=6, p<0.05). However, there was no significant difference in the contractile response to 5-HTbetween IPAs from male WT and Cx43+/- mice. In addition, there were no significant differences between female WT and Cx43+/- mice in contractile responses to 5-HT or ET-1. In studies using thoracic aorta, there was no significant difference in the contractile response to 5-HT between Cx43+/- and WT female mice. 5-HT showed increased efficacy(P<0.05) in aortic rings from male Cx43+/- mice when compared with male WT mice (Emax: 95.67±14.31% of KCl maximal contraction cf 68.47±9.03% of KCl maximal contraction, n=6). There were no significant differences in contractile responses to ET-1 in aortas from male or female Cx43+/- mice compared to WT controls.

In conclusion, our results show a role for Cx43 in ET-1 mediated contraction of IPAs in male mice only. This study does not support a role for Cx43 in 5-HT mediated contraction of IPAs. In the thoracic aorta, Cx43 was shown to play a role in 5-HT mediated contraction but not ET-1 mediated contraction. Since genetic manipulation of mice can lead to compensatory changes, these results remain to be verified using pharmacological manipulation of Cx43.

(1) Dempsie et al., (2015) Biochem Soc Trans 43: 524-9

(2) Billaud et al., (2011) Respir Res 12: 30

(3) Tan & He (2009) Pathol Res Pract 205: 473-82

(4) Reaume et al., (1995) Science 267: 1831-1834