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. 2020 Aug;24(15):8505-8517.
doi: 10.1111/jcmm.15469. Epub 2020 Jun 24.

Cardiac β-adrenergic receptor activation mediates distinct and cell type-dependent changes in the expression and distribution of connexin 43

Yi Zhang  1 , Meng-Chen Hou  1   2 , Jing-Jing Li  3 , Ying Qi  3 , Yu Zhang  1 , Gang She  1 , Yu-Jie Ren  2 , Wei Wu  1 , Zheng-Da Pang  1 , Wenjun Xie  3 , Xiu-Ling Deng  1   4 , Xiao-Jun Du  1   5
Affiliations

Cardiac β-adrenergic receptor activation mediates distinct and cell type-dependent changes in the expression and distribution of connexin 43

Yi Zhang et al. J Cell Mol Med. 2020 Aug.

Abstract

Activation of the sympatho-β-adrenergic receptors (β-ARs) system is a hallmark of heart failure, leading to fibrosis and arrhythmias. Connexin 43 (Cx43) is the most abundant gap junctional protein in the myocardium. Current knowledge is limited regarding Cx43 remodelling in diverse cell types in the diseased myocardium and the underlying mechanism. We studied cell type-dependent changes in Cx43 remodelling due to β-AR overactivation and molecular mechanisms involved. Mouse models of isoproterenol stimulation or transgenic cardiomyocyte overexpression of β2 -AR were used, which exhibited cardiac fibrosis and up-regulated total Cx43 abundance. In both models, whereas Cx43 expression in cardiomyocytes was reduced and more laterally distributed, fibroblasts exhibited elevated Cx43 expression and enhanced gap junction communication. Mechanistically, activation of β2 -AR in fibroblasts in vitro elevated Cx43 expression, which was abolished by the β2 -antagonist ICI-118551 or protein kinase A inhibitor H-89, but simulated by the adenylyl cyclase activator forskolin. Our in vitro and in vivo data showed that β-AR activation-induced production of IL-18 sequentially stimulated Cx43 expression in fibroblasts in a paracrine fashion. In summary, our findings demonstrate a pivotal role of β-AR in mediating distinct and cell type-dependent changes in the expression and distribution of Cx43, leading to pathological gap junction remodelling in the myocardium.

Keywords: IL-18; cardiac fibrosis; connexin 43; gap junction remodelling; β-AR activation.

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Conflict of interest statement

None declared.

Figures

Figure 1
Figure 1
β‐AR activation was required for ISO‐induced cardiac remodelling, Cx43 up‐regulation and redistribution. A, Representative HE staining (upper panels), Masson's trichrome staining (middle panels) and Cx43 immunohistochemistry (IHC) staining (lower panels) of LV myocardium from mice treated with saline (Ctrl), ISO and ISO plus BIS or ICI at day‐7 after treatment. Scale bar: 50 µm. Arrows indicate lateralized Cx43 and arrowheads for ID Cx43 localization. Western blotting images and quantification of band intensity for protein levels of (B) Col‐I, (C) Col‐III and Cx43 (D) in the LV myocardium at day 7 after drug treatment (n = 7‐9 mice/group). Quantificational analysis in IHC staining for (E) Cx43‐positive area and (F) the ratio of lateral to ID localized Cx43 area in LV sections of mice treated with saline, ISO and ISO plus BIS or ICI for 7 d (n = 5‐6 mice/group). Data were expressed as mean ± SEM. *P < .05, **P < .01 and ***P < .001 vs control, # P < .05, ## P < .01 and ### P < .001 versus ISO. Statistical significance was determined by one‐way ANOVA followed by Tukey's multiple comparisons test
Figure 2
Figure 2
ISO treatment of cultured cardiac fibroblasts enhanced Cx43 expression and intercellular coupling through β2‐AR/cAMP/PKA pathway. Western blotting images and quantification of band intensity for Cx43 expression in fibroblasts (A) treated with ISO at different concentrations (0.01 ~ 100 μmol/L) for 24 h (n = 5 independent isolation/5 hearts), or (B) treated with ISO (1 μmol/L) for various durations (6 ~ 72 h, n = 5 independent isolation/5 hearts), or (C) treated with ISO (1 μmol/L), ICI (0.1 μmol/L), ICI plus ISO for 24 h (n = 5 independent isolation/5 hearts), and (D) treated with ISO, forskolin (100 μmol/L), H‐89 (0.1 μmol/L) and H89 plus ISO for 24 h (n = 9 independent isolation/5 hearts). E, Representative fluorescence images of Cx43 (red) and α‐SMA (green) in fibroblasts treated with ISO, ICI plus ISO for 48 h. Scale bar: 20 µm. F, Western blotting images and quantification of band intensity for α‐SMA expression in fibroblasts treated with ISO or ICI plus ISO for 48 h (n = 5 independent isolation/5 hearts). G, Representative fluorescence images and (H) quantification of Lucifer yellow transfer area after scrape‐loading by dye transfer assay in fibroblasts treated without or with ISO and ISO plus ICI for 48 h. (n = 15 assays from 5 independent isolation/5 hearts). Scale bar: 50 µm. Data are expressed as mean ± SEM. *P < .05, **P < .01 and ***P < .001 vs control, # P < .05, ## P < .01 and ### P < .001 vs ISO. Statistical significance was determined by one‐way ANOVA followed by Tukey's multiple comparisons test
Figure 3
Figure 3
β2‐AR overexpression in cardiomyocytes led to age‐related fibrosis with altered Cx43 expression and localization. Representative images of HE staining (A), Masson's trichrome staining (B) and (C) Cx43 IHC staining in LV sections from NTG and β2‐TG mice aged at 3 and 5 months (Scale bar: 50 µm in A and C, 100 µm in B). Arrows indicate lateralized Cx43 and arrowheads for ID Cx43 localization. Western blotting images and quantification of band intensity for expression of (D) Col‐I, (E) Col‐III and (F) Cx43 in LV tissue lysate (n = 5 mice/group). Quantificational analysis for (G) total Cx43 positive area (n = 5 mice/group) and (H) the ratio of lateral to ID localized Cx43 (n = 5 mice/group) in LV sections of 5‐month‐old NTG and β2‐TG mice. Data were expressed as mean ± SEM. *P < .05, **P < .01 and ***P < .001 vs NTG; ## P < .01 and ### P < .001 vs β2‐TG mice at 3 months. Statistical significance was determined by one‐way ANOVA followed by Tukey's multiple comparisons test (D‐F) or two‐tailed unpaired Student's t test (G‐H)
Figure 4
Figure 4
Activation of β2‐AR suppressed Cx43 expression and localization in primary adult mouse cardiomyocytes. A, Representative fluorescence images for Cx43 (red) and α‐actinin (green) in cardiomyocytes treated with ISO (1 μmol/L), ISO plus BIS (0.1 μmol/L) or ICI (0.1 μmol/L) for 48 h. Scale bar: 10 µm. Quantitative analysis for (B) the lateralized Cx43 area per image field, (C) the total Cx43‐positive area (include ID localized Cx43) in cardiomyocytes (n = 16‐22 cells/group from 5 hearts). D and E, Western blotting images and quantification of band intensity for Cx43 expression in cardiomyocytes treated with ISO, ISO plus BIS or ICI for 48 h (n = 5 independent isolation/2 hearts). F, Representative fluorescence images for Cx43 (red) and α‐actinin (green) in cardiomyocytes isolated from NTG and β2‐TG mice at 3 and 5 months of age. Quantificational analysis for (G) the lateralized Cx43 fluorescence puncta area per image field, (H) the total Cx43‐positive area in cardiomyocytes isolated from 5‐month‐old NTG and β2‐TG mice (n = 15~17 cells/group from 5 hearts). I and J, Western blotting images and quantification of band intensity for Cx43 expression in cardiomyocytes isolated from 5‐month‐old NTG and β2‐TG mice (n = 5 independent isolation/2 hearts). Data were expressed as mean ± SEM. *P < .05 and ***P < .001 vs Ctrl or NTG; ## P < .01 and ### P < .001 vs ISO. Statistical significance was determined by one‐way ANOVA followed by Tukey's multiple comparisons test (B‐E) or two‐tailed unpaired Student's t test (G‐J)
Figure 5
Figure 5
Cardiomyopcyte‐derived IL‐18 by β‐AR activation contributed to Cx43 up‐regulation in fibroblasts in a paracrine fashion. Quantification of IL‐18 expression in cardiomyocytes by Western blotting analysis (A) (n = 6 mice/group), or concentrations by ELISA (B) in myocardium tissue lysate (n = 6 mice/group) and (C) in plasma (n = 6 mice/group) in 5‐month‐old NTG and β2‐TG mice. D, Immunofluorescence staining of WGA (green), IL‐18 (yellow), α‐SMA (red) and DAPI (blue) in LV myocardium from 5‐month‐old NTG and β2‐TG mice. Scale bar: 20 µm. Concentration of IL‐18 (E) in myocardium tissue lysate and (F) in plasma from control and 7‐day after ISO treatment measured by ELISA (n = 5‐7 mice/group). Concentration of IL‐18 (G) in primary cardiomyocytes in culture or (H) culture media harvested from cardiomyocytes with ISO treatment (1 μmol/L, 48 h) by ELISA (n = 4‐6 independent isolation/2 hearts). I, Western blotting images and quantification of band intensity for Cx43 expression in adult mouse cardiac fibroblasts treated with IL‐18 (10 ng/mL) for 48 h (n = 5 independent isolation/5 hearts). J, Representative images of Cx43 IHC staining in LV sections and (K) quantificational analysis for Cx43 positive area and the ratio of lateral to ID localized Cx43 area in LV sections of mice treated daily with IL‐18 nAb or IgG for 7 d commencing from ISO infusion (n = 7 mice/group). Scale bar: 50 µm. Arrows indicate lateralized Cx43 and arrowheads for ID Cx43 localization. Data were expressed as mean ± SEM. *P < .05, **P < .01 and ***P < .001 vs NTG or control. Statistical significance was determined by two‐tailed unpaired Student's t test
Figure 6
Figure 6
Schematics depicting gap junction remodelling in cardiomyocytes and fibroblasts induced by cardiac β‐AR activation. In both ISO stimulation and β2‐TG mouse models, β‐AR activation in cardiomyocytes suppressed Cx43 expression and shifted Cx43 localization from ID to the lateral side of cardiomyocytes. Meanwhile, Cx43 expression in fibroblasts, when tested in vivo and in vitro, were up‐regulated via direct activation of β2‐AR/cAMP/PKA signalling cascade as well as stimulation by IL‐18 released from cardiomyocytes upon β‐AR activation (curved arrow). These changes would increase the probability of intercellular coupling via gap junctions
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