Connexin-43-Mediated Gap Junction Coupling Between Adipocytes Regulates Norepinephrine-Induced Ca2+ Responses in Perivascular Adipose Tissue

Figures

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  Figure 1. Norepinephrine (NE) stimulates Ca2+ responses among adipocytes in perivascular adipose tissue (PVAT). (A) Model depicting AdipoqCre+;GCaMPtdtf/Wt mice expressing the genetically encoded calcium indicator, GCaMP5g, and reporter protein, tdTomato, under control of the adipocyte-selective driver, adiponectin. (B) Quantification of adipocyte Ca2+ response amplitude (peak ∆F/Fo) following focal stimulation with NE in aortic perivascular adipose tissue (aPVAT), mesenteric perivascular adipose tissue (mPVAT) and white adipose tissue (WAT) (n = 30–280 from 3 animals per experimental group). Data are presented as mean ± SEM. (C) EC50 values calculated from NE concentration–response curves. (D) Representative traces of NE-evoked Ca2+ responses in mPVAT. Scale bar = 50 µm.

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  Figure 2. Expression of connexin-43 (Cx43) in PVAT from WT and Adipo∆Cx43 KO mice. Immunofluorescent labeling and quantification of Cx43 (magenta) and Caveolin-1 (green) in aPVAT (A), mPVAT (B), WAT (C). Images are representative of labeling in n = 3–4 mice. Scale bar = 50 µm.

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  Figure 3. Gap junctions, but not Cx43 hemichannels, modulate NE-evoked Ca2+ responses in PVAT. (A) Effect of carbenoxolone (Cbx; 50 µmol/L) on the amplitude of adipocyte Ca2+ responses to NE in aPVAT, mPVAT, and WAT. NE was applied at 100 µmol/L in aPVAT, 500 µmol/L in mPVAT, and 1000 µmol/L in WAT. These concentrations were determined based on differing sensitivities to NE calculated in earlier experiments. (B) Representative traces of NE-evoked Ca2+ responses in adipocytes in the presence or absence of Cbx. (C) Effect of blocking Cx43 hemichannels with the mimic peptide 43Gap26 (100 µmol/L) on the amplitude of adipocyte Ca2+ responses to NE in aPVAT, mPVAT, and WAT. Data are presented as mean ± SEM, n = 62–161 from 3 animals per experimental group, **** p < 0.0001, two-tailed t test.

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  Figure 4. The gap junction blocker carbenoxolone (Cbx) reduces dye transfer between adipocytes. (A) The mobile fraction percentage values in mPVAT treated with of 50 µmol/L Cbx or vehicle control (n = 14 from 5 animals, data are presented as mean ± SEM, * p = 0.0363, two-tailed t test). (B) Representative traces of Calcein AM fluorescence recovery after photobleaching in the presence or absence of Cbx. (C) Representative confocal photomicrographs of mPVAT adipocytes. Cell (circle) is shown before photobleaching (left) and immediately after photobleaching (right).

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  Figure 5. Effects of adipocyte-specific Cx43 deletion on NE-induced mesenteric resistance artery constriction. (A) Model depicting Adipo∆Cx43 KO mice with loxP sites flanking exon 2 of Cx43 (Cx43 f/f) and Cre expression under control of the adipocyte driver, adiponectin. (B) EC50 values calculated from NE concentration–response curves normalized to maximum response from within each curve (n = 4–5 from a minimum of 4 animals per experimental group, data are presented as mean ± SEM, * p < 0.05, two-tailed t test, with PVAT vs. without PVAT). (C) The presence of PVAT significantly attenuated NE-induced constriction in both WT and Adipo∆Cx43 KO mice, indicating preservation of the anticontractile effect. (D) Representative constriction curves in response to potassium physiological salt solution (KPSS, including 60 mmol/L of KCl), and norepinephrine (0.1–40 µmol/L) in samples from WT mice with (bottom) and without (top) PVAT. The net beneficial anticontractile effect of PVAT on NE contraction is represented in yellow.

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  Figure 6. mRNA expression of connexin isoforms in wild type and Adipo∆Cx43 KO mice. (A) Relative mRNA expression of Cx26 and Cx43 (n = 6–8 animals per experimental group, data are presented as mean ± SEM, * p = 0.05, two-tailed t test). (B) Relative mRNA expression of Cx32, Cx37, Cx40, Cx45 and Cx50 (n = 6–8 animals per experimental group, data are presented as mean ± SEM). Values were normalized to the housekeeping gene RPS6, and relative Cx expression was quantified using the 2−∆∆Ct method.