Gender differences in pulmonary vascular connexin expression and vasoreactivity in mice genetically deficient for connexin 43

M. Htet, J. E. Nally, P. E. Martin, Y. Dempsie. School of Health and Life Sciences, Glasgow Caledonian University, Glasgow, United Kingdom.

Introduction: Connexins are transmembrane proteins which serve as a conduit for transfer and exchange of small molecules between adjacent cells. Aberrant connexin-mediated signalling may play a role in increased pulmonary vascular reactivity during pulmonary arterial hypertension (PAH) (1). In its idiopathic and familial forms PAH is more common in women than men (∼3:1) (2). The aim of this study was to investigate i) expression of connexin 43 (Cx43), Cx37, Cx40, Cx45 and pannexin1 (Panx1) and ii) pulmonary vascular reactivity in male and female mice genetically deficient in Cx43.

Methods: Male and female Cx43 heterozygous (Cx43^+/-) and wild type (WT) mice (C57BL6; 5-6 months old) were sacrificed by pentobarbitone injection (60 mg/kg, i.p.). Intra-lobar pulmonary arteries (IPAs∼250μm in internal diameter) were dissected out and mounted (Krebs-Heseleit solution, 37 °C) on wire myographs. Cumulative concentration response curves to endothelin-1 (ET-1) and 5-hydroxytryptamine (5-HT) were constructed. Gene expression in pulmonary arteries (main and branch) was studied by real time quantitative polymerase chain reaction (qRT-PCR) and analysed by ΔΔCT method. Statistical analysis was done by two-way ANOVA with Bonferroni’s multiple comparison test or Student’s t-test as appropriate. Data are presented as mean ± SEM.

Results: Downregulation of Cx43 gene expression was confirmed in both male and female Cx43^+/- mice. Gene expression of Cx43 was significantly lower in male mice than in female mice for both genotypes (P<0.05; n=6/ group). Male, but not female, Cx43^+/- mice also displayed significant reduction in gene expression of Cx37, Cx40, Cx45 and Panx1 (P<0.05, n=6/ group). The potency of ET-1 was significantly (P<0.05) greater in IPAs from male Cx43^+/- mice (pEC₅₀: 8.77±0.12; n=5) when compared to those from male WT mice (pEC₅₀: 8.46±0.79; n=6) and female Cx43^+/- mice (pEC₅₀: 8.48±0.04; n=7). Female mice showed no difference in contractile responses to ET-1 between any of the groups studied. No differences in the contractile responses to 5-HT were observed in any groups of mice studied.

Conclusion: Male Cx43^+/- mice had reduced gene expression of Cx37, Cx40, Cx43, Cx45 and Panx1 compared to female Cx43^+/- mice and this correlated with increased pulmonary vascular reactivity to ET-1.

References:

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

2. Peacocok et al., (2007) Eur Respir J. 30:104-9