Connexin37‐Dependent Mechanisms Selectively Contribute to Modulate Angiotensin II‐Mediated Hypertension

Figures

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  Figure 1. Loss of Cx37−/− alters the increase in blood pressure in models of renin‐dependent hypertension. A, WT mice receiving daily 1 mg Ang II/kg body weight displayed a 50% increase in systolic blood pressure (SBP). Under the same conditions, Cx37−/− mice displayed a significantly lower increase of SBP. WT NaCl N=5; Cx37−/− NaCl, WT Ang II (angiotensin II), Cx37−/− Ang II N=6. B, Similar observations were made in mice receiving daily 0.25 mg Ang II/kg body weight WT NaCl, Cx37−/− NaCl N=7; WT Ang II, Cx37−/− Ang II N=8. C, Two weeks after the 2K1C (2‐kidney, 1‐clip) procedure, Cx37−/− mice also showed a lower increase in SBP than WT controls. WT sham (sham‐operated controls) WT 2K1C, N=5; Cx37−/− sham, Cx37−/− 2K1C N=6. D, In contrast, both types of mice became similarly hypertensive after a treatment with l‐NAME (N‐nitro‐l‐arginine‐methyl‐ester); N=6. ***P≤0.001 vs corresponding controls (NaCl‐infused or sham‐operated animals); °°° P≤0.001 vs WT mice, as given by 2‐way ANOVA of AUC (areas under the SBP curve). Ang II indicates angiotensin II; Cx37, connexin37; WT, wild type.

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  Figure 2. Cx37−/− mice become rapidly normotensive after the 2K1C procedure. A, One month after the 2K1C surgery, WT mice were significantly hypertensive, compared with their sham‐operated controls, whereas Cx37−/− mice featured a control SBP (systolic blood pressure). WT sham (sham‐operated controls) N=15; Cx37−/− sham N=14; WT 2K1C (2‐kidney, 1‐clip) N=19; Cx37−/− 2K1C N=17. B, Compared with controls infused with NaCl, both WT and Cx37−/− mice showed a significant increase in SBP after a daily infusion of 0.25 mg Ang II/kg body weight during 1 month. However, this increase was significantly lower in Cx37−/− than in WT mice. WT NaCl, Cx37−/− NaCl N=7; WT Ang II (angiotensin II); Cx37−/− Ang II N=8. C, In contrast, the mice of these 2 genotypes showed a similar increase in blood pressure, after a daily treatment with l‐NAME (N‐nitro‐l‐arginine‐methyl‐ester, 500 mg/L in the drinking water) for 1 month. N=6. Results are means+SEM. *P≤0.05 and ***P≤0.001 vs respective control mice; °°° P≤0.001 vs WT mice, as given by 2‐way ANOVA. Ang II indicates angiotensin II; Cx37, connexin37; n.s., not significant; WT, wild type.

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  Figure 3. Renin expression and secretion are increased by the 2K1C surgery in both WT and Cx37−/− mice. A, Four weeks after surgery, the basal levels of the renin transcript increased in the clipped, hypoperfused kidney of both WT and Cx37−/− mice. WT sham (sham‐operated controls) N=14; Cx37−/− sham N=13; WT 2K1C N=16; Cx37−/− 2K1C N=12. B, The plasmatic concentration of renin (PRC) was also significantly increased in the 2 types of mice. However, Cx37−/− mice had basal and 2K1C‐induced levels of PRC significantly higher than WT animals. WT sham N=8; Cx37−/− sham N=6; WT 2K1C (2‐kidney, 1‐clip) N=9; Cx37−/− 2K1C N=6. Results are means+SEM. *P≤0.05, ***P≤0.001 vs respective control mice; ° P≤0.05, °°° P≤0.001 vs WT mice, as given by 2‐way ANOVA. C, Renin immunostaining (green) increased in the hypoperfused left kidney of both WT and Cx37−/− mice, while decreasing in their contralateral right kidney, and was higher in Cx37−/− than in WT mice. Cx40 (Connexin40). Immunostaining (red) localizes the renin‐secreting cells of the afferent arterioles. Bar=50 μm. AngI indicates angiotensin I; WT, wild type.

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  Figure 4. The ACEs were similarly expressed in the kidneys of WT and Cx37−/− mice. The levels of the ACE (A) and the ACE2 (B) transcripts were comparable in the hypoperfused kidneys after the 2K1C (2‐kidney, 1‐clip) surgery and in the sham‐operated controls of both WT and Cx37−/− mice. WT sham N=8; Cx37−/− sham N=7; WT 2K1C N=8; Cx37−/− 2K1C N=10. Results are means+SEM. Statistical analysis was performed using 2‐way ANOVA. ACE and ACE2 indicates angiotensin‐converting enzymes; WT, wild type.

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  Figure 5. Loss of Cx37 selectively alters the expression of some Ang II‐stimulated signals. The levels of transcripts coding for Angiotensinogen (Agt), Angiotensin receptor 1a (Agtr1a), and Angiotensin receptor 1b (Agtr1b) were similar in the aortas of WT and Cx37−/− mice. In contrast, the transcripts coding for Angiotensin receptor 2 (Agtr2), Connector enhancer of kinase suppressor of Ras 1 (Cnksr1), SH2 domain‐containing tyrosine phosphatase 2 (Ptpn6), and Glutamyl aminopeptidase A (Enpep), which all contribute to control the Ang II pathway, were increased in the aortas of Cx37−/− mice. N=9. Results are means+SEM. *P≤0.05, ***P≤0.001 vs WT mice, as given by t test. Ang II indicates angiotensin II; Cx37, Connexin37; WT, wild type.

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  Figure 6. Loss of Cx37 selectively alters the expression of the AT2 receptors in the aortas of Cx37−/− mice. A, Immunofluorescence showed a similar staining for AT1R (upper panels) in the aortas of all WT and Cx37−/− mice. In contrast, the staining for AT2R was more intense in the aortas of Cx37−/− than in WT mice. Bar=20 μm. L indicates lumen; M, media. B, Western blot showed that the aortas of WT and Cx37−/− mice expressed similar levels of the AT1R protein. C, In contrast, the basal levels of the AT2R protein were higher in Cx37−/− than in WT mice, and were not increased after the 2K1C surgery. WT sham N=21; Cx37−/− sham N=17; WT 2K1C N=19; Cx37−/− 2K1C N=17. D and E, Comparable observations were made for both AT1R (D) and AT2R (E) in mice infused daily with 1 mg Ang II/kg body weight WT NaCl N=4; Cx37−/− NaCl, Cx37−/− Ang II N=3, WT Ang II N=6. Results are means+SEM. ° P≤0.05 vs WT mice, as given by 2‐way ANOVA. 2K1C indicates 2‐kidney, 1‐clip; AngII, angiotensin II; AT1R, Ang II type 1 receptors; AT2R, Ang II type 2 receptors; Cx37, Connexin37; DAPI, 4′,6‐diamidino‐2‐phenylindole; Sham, sham‐operated controls; WT, wild type.

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  Figure 7. Loss of Cx37 does not alter the expression of several signals involved in the Ang II effects. The levels of transcripts coding for the chymase (Cma1), which converts Ang I to Ang II, the Regulator of G‐protein signaling 2 (Rgs2), which negatively regulates AT1R function by modulating its Gαq subunit activity, the Dual specificity protein phosphatase 1 (Dusp1), an AT2R‐associated phosphatase that contributes to AT2R‐mediated inactivation of the ERK cascade, and Mas1, the receptor for Ang‐(1–7) that promotes vasodilatation, were similar in the aortas of WT and Cx37−/− mice. WT N=9; Cx37−/− N=8. Results are means+SEM. Statistical analysis was performed using t test. Ang indicates angiotensin; AT1R, Ang II type 1 receptors; AT2R, Ang II type 2 receptors; Cx37, Connexin37; WT, wild type.

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  Figure 8. Loss of Cx37−/− alters the activation of downstream key signaling proteins to the Ang II signaling pathway. A through C, After a 2‐hour exposure to 40 μmol/L Ang II, the levels of phosphorylated ERK (A), MLC2 (B), and AKT (C) increased in the aortas of WT but not of Cx37−/− mice. WT Ctl, WT Ang II N=3; Cx37−/− Ctl, Cx37−/− Ang II N=5. Results are means+SEM. *P≤0.05 vs respective control mice and ° P≤0.05, °° P≤0.01 vs WT mice, as given by 2‐way ANOVA. Ang II indicates angiotensin II; Ctl, control; Cx37, Connexin37; MLC2, myosin light chain 2; WT, wild type.

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  Figure 9. Loss of Cx37 alters the basal expression of aortic Cx43, and protects against the overexpression of Cx40 and Cx45 after the 2K1C procedure. A, Four weeks after the 2K1C procedure, Western blots showed a marked increase of Cx37 levels in the aortas of the WT mice that had become hypertensive. WT sham N=9; Cx37−/− sham N=7; WT 2K1C N=9; Cx37−/− 2K1C N=11. B, The levels of Cx40 were also increased in the aortas of WT made hypertensive, but not in Cx37−/− mice. WT sham N=18; Cx37−/− sham N=15; WT 2K1C N=16; Cx37−/− 2K1C N=14. C, The levels of Cx43 were enhanced in response to a renin‐dependent model of hypertension (WT 2K1C). Cx37‐deficient mice also displayed elevated Cx43 protein levels, which decreased after the 2K1C procedure. WT sham N=17; Cx37−/− sham N=13; WT 2K1C N=14; Cx37−/− 2K1C N=17. D, Comparable levels of Cx45 were evaluated in the aortas of control WT and Cx37−/− mice. After the 2K1C surgery, these levels did not change in the aortas of WT mice, but decreased in those of Cx37−/− mice. WT sham N=9; Cx37−/− sham N=7; WT 2K1C N=9; Cx37−/− 2K1C N=11. Results are means+SEM. *P≤0.05, **P≤0.01 vs respective control mice; °° P≤0.01, °°° P≤0.001 vs WT mice, as given by 2‐way…

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  Figure 10. Loss of Cx37 did not alter the expression and activation of aortic eNOS. Western blots showed similar levels of the eNOS protein (A) and of its activated, phosphorylated form (B) in the aortas of all groups of mice. WT sham N=21; Cx37−/− sham N=15; WT 2K1C N=17; Cx37−/− 2K1C N=15. Results are means+SEM. Statistical analysis was performed using 2‐way ANOVA. Cx37 indicates Connexin37; 2K1C, 2‐kidney, 1‐clip; eNOS, endothelial nitric oxide synthase; Sham, sham‐operated controls; WT, wild type.