Connexin 43 Plays a Role in Pulmonary Vascular Reactivity in Mice

Abstract

Pulmonary arterial hypertension (PAH) is a chronic condition characterized by vascular remodeling and increased vaso-reactivity. PAH is more common in females than in males (~3:1). Connexin (Cx)43 has been shown to be involved in cellular communication within the pulmonary vasculature. Therefore, we investigated the role of Cx43 in pulmonary vascular reactivity using Cx43 heterozygous (Cx43^+/−) mice and ^37,43Gap27, which is a pharmacological inhibitor of Cx37 and Cx43. Contraction and relaxation responses were studied in intra-lobar pulmonary arteries (IPAs) derived from normoxic mice and hypoxic mice using wire myography. IPAs from male Cx43^+/− mice displayed a small but significant increase in the contractile response to endothelin-1 (but not 5-hydroxytryptamine) under both normoxic and hypoxic conditions. There was no difference in the contractile response to endothelin-1 (ET-1) or 5-hydroxytryptamine (5-HT) in IPAs derived from female Cx43^+/−mice compared to wildtype mice. Relaxation responses to methacholine (MCh) were attenuated in IPAs from male and female Cx43^+/− mice or by pre-incubation of IPAs with ^37,43Gap27. N_ω-Nitro-L-arginine methyl ester (l-NAME) fully inhibited MCh-induced relaxation. In conclusion, Cx43 is involved in nitric oxide (NO)-induced pulmonary vascular relaxation and plays a gender-specific and agonist-specific role in pulmonary vascular contractility. Therefore, reduced Cx43 signaling may contribute to pulmonary vascular dysfunction.

Keywords: connexin43 (Cx43); endothelin-1; gap junction; isoprenaline; nitric oxide; pulmonary arterial hypertension (PAH); serotonin; vascular reactivity.

Figure 1

Connexin gene expression in pulmonary arteries from male and female mice. Cx43 gene expression is reduced in pulmonary arteries from Cx43 heterozygous (Cx43^+/−) mice (A). Female mice have increased levels of Cx43 compared to males (A). Cx43 is the predominant vascular connexin in female mice while male mice show similar levels of Cx43, Cx40, and Cx37. Cx45 is expressed in lower levels than Cxs 43, 40, and 37 in both male and female mice (B). Data are presented as mean ± S.E.M. and were analyzed by two-way ANOVA. n = 6 with each sample run in triplicate. A: * p < 0.05, B: ** p < 0.01, **** p < 0.0001 compared to Cx43.

Figure 2

Gene expression of Cx37 (A), Cx40 (B), Cx45 (C), and Panx1 (D) in pulmonary arteries from male and female wildtype (WT) and Cx43 heterozygous (Cx43^+/−) mice. Data are presented as mean ± S.E.M. and were analyzed by two-way ANOVA. * p < 0.05, ** p < 0.01, n = 6 per group with each sample analyzed in triplicate.

Figure 3

Pulmonary vascular contractility to ET-1 and 5-HT in intralobar pulmonary arteries (IPAs) from male and female wildtype (WT) and Cx43 heterozygous (Cx43^+/−) mice. ET-1 was more potent in IPAs from male Cx43^+/− mice than WT mice (A). There was no difference in contractile response to 5-HT in IPAs from male WT and Cx43^+/− mice (B). There was no difference in ET-1 (C) or 5-HT (D) induced contractile response in IPAs from female WT or Cx43^+/− mice. Data are shown as mean ± S.E.M. Global differences in concentration response curves were compared by two-way ANOVA. Changes in logarithm of median effective concentration (Log EC₅₀) and maximal contractile responses (E_max) between two different groups were analyzed by using the Student’s t-test. * EC₅₀ is significantly (p < 0.05) reduced in male Cx43^+/− mice, n = 5–7 per group.

Figure 4

Effects of genetic reduction or pharmacological inhibition of Cx43 on pulmonary vascular relaxation responses. Male (A) and female (B) Cx43 heterozygous (Cx43^+/−) mice show reduced relaxation in response to MCh. Pre-incubation with ^37,43Gap27 also reduced the relaxation response in male (C) and female (D) mice. MCh-induced relaxation was ablated in the presence of l-NAME (E). ^37,43Gap27 partially inhibited isoprenaline-induced relaxation in IPAs from male mice (F) as did l-NAME (G). Global differences in concentration response curves were compared by two-way ANOVA. Changes in logarithm of median effective concentration (Log EC₅₀) and maximal relaxation responses (R_max) between two different groups were analyzed by using the Student’s t-test. Data are shown as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 5–6 per group. Statistical symbols shown on the right hand side of graphs indicate global shifts in the concentration response curves. Statistical symbols underneath curves indicate changes in maximal relaxation (R_max) values.

Figure 4

Effects of genetic reduction or pharmacological inhibition of Cx43 on pulmonary vascular relaxation responses. Male (A) and female (B) Cx43 heterozygous (Cx43^+/−) mice show reduced relaxation in response to MCh. Pre-incubation with ^37,43Gap27 also reduced the relaxation response in male (C) and female (D) mice. MCh-induced relaxation was ablated in the presence of l-NAME (E). ^37,43Gap27 partially inhibited isoprenaline-induced relaxation in IPAs from male mice (F) as did l-NAME (G). Global differences in concentration response curves were compared by two-way ANOVA. Changes in logarithm of median effective concentration (Log EC₅₀) and maximal relaxation responses (R_max) between two different groups were analyzed by using the Student’s t-test. Data are shown as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 5–6 per group. Statistical symbols shown on the right hand side of graphs indicate global shifts in the concentration response curves. Statistical symbols underneath curves indicate changes in maximal relaxation (R_max) values.

Figure 5

Gene expression of BMPRII (encoded by BMPR2) (A), eNOS (encoded by NOS3) (B), and Tph1 (C) in pulmonary arteries of male and female WT and Cx43^+/− mice. No differences were observed in expression of BMPR2, NOS3, or Tph1 between WT and Cx43^+/− mice (either male or female). BMPR2 and NOS3 were significantly downregulated in female WT mice compared to male WTs. Data are presented as mean ± S.E.M. and were analysed by using two-way ANOVA. * p < 0.05, n = 6 per group with each sample run in triplicate.

Figure 6

Assessment of the development of right ventricular hypertrophy by right ventricular weight ratio left ventricle plus septal weight (RV/LV+Septum) (A). Concentration response curves (CRCs) to ET-1 (B) and 5-HT (C) in intra-lobar pulmonary arteries derived from hypoxic wildtype (WT) and hypoxic Cx43 heterozygous (Cx43^+/−) mice. Data are shown as mean ± S.E.M. Data in panel A were analyzed by two-way ANOVA. In panels B and C, global differences in CRCs were compared by two-way ANOVA. Changes in the logarithm of median effective concentration (Log EC₅₀) and maximal contractile responses (E_max) between two different groups were analyzed by using the Student’s t-test * p < 0.05, ** p < 0.01, *** p < 0.001, n = 5–7 per group. The statistical symbol shown on the right hand side of graph B indicates a global shift in the CRC. The symbol underneath the curve indicates changes in the median effective concentration (EC₅₀) while the symbol above the curve indicates changes in the maximal response (E_max).

Figure 7

Cx43 expression in pulmonary arteries of wildtype (WT) and Cx43 heterozygous (Cx43^+/−) mice under normoxic and chronic hypoxic conditions. Cx43 gene expression is reduced by hypoxia in both WT and Cx43^+/− mice (A). Confocal images of Cx43 immunofluorescence staining in normoxic and hypoxic WT and Cx43^+/− mouse lung tissue sections are shown in (B). Green fluorescence punctate findings represent Cx43 immunoreactivity and blue staining represents nuclei. Scale bars represent 10 µm. For panel A, data is presented as mean ± S.E.M. and was analyzed by two-way ANOVA, * p < 0.05, n = 6 with each sample run in triplicate.

Figure 8

Gene expression of Cx37 (A), Cx40 (B), Cx45 (C), Panx1 (D), BMPRII (E), eNOS (F) and Tph1 (G) in pulmonary arteries of wildtype (WT) and Cx43 heterozygous (Cx43^+/−) mice under normoxic and chronic hypoxic conditions. All data are presented as mean ± S.E.M. and were analyzed by two-way ANOVA. * p < 0.05, ** p < 0.01, n = 6 per group with each sample run in triplicate.