Cadherin-11-Interleukin-6 Signaling between Cardiac Fibroblast and Cardiomyocyte Promotes Ventricular Remodeling in a Mouse Pressure Overload-Induced Heart Failure Model

Figures

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  Figure 1. Increased Cad-11 expression was found in the left ventricles of heart from human heart failing patients and TAC-mice. (A–C) Representative mRNA and protein levels of Cad-11 in heart samples from normal donors and DCM patients. (D–F) Representative mRNA and protein levels of Cad-11 in the left ventricles of WT mice at 4 weeks after sham or TAC operation. (G) qRT-PCR analyses of ANP, BNP, and β-MHC mRNA levels in the left ventricles of normal donors and DCM patients (n = 6). (H) qRT-PCR analyses of ANP, BNP, and β-MHC mRNA levels in the left ventricles of WT-Sham and WT-TAC mice. Data were presented as mean ± SEM. ** p < 0.01 vs. Donor’s heart or WT-Sham group.

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  Figure 2. Loss of Cad-11 alleviated pressure overload-induced LV structural remodeling. (A) Representative images showing gross cardiac morphology used for heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) calculation (top); transverse sections stained with H&E and WGA respectively (middle); Images of representative adult cardiomyocytes used for calculation of cell surface area (bottom). (B) Quantitative assessment of myocyte cell area calculated from isolated cardiac myocytes. (C,D) The ratio of heart weight/body vs. weight or heart weight/tibia vs. length were calculated from WT and Cad-11-knockout mice (KO) after sham or TAC operation. (E–G) Expression levels of cardiac hypertrophy–related genes ANP, BNP, and β-MHC were measured using quantitative reverse transcription polymerase chain reaction from heart samples of Sham- or TAC-mice 4 weeks after surgery. (H) Masson trichrome staining of representative heart sections from wild-type (WT) or Cad-11−/− mice after sham or TAC operation. (I) Statistical analysis of differences in cardiac fibrosis. (n = 4 in each group). (J,K) Protein levels of fibrosis genes in the hearts from indicated mice determined by Western…

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  Figure 3. Cad-11 deficiency alleviated pressure overload-induced LV electrical remodeling. (A–D) Representative action potential curves for indicated mouse heart (A). (B–D) Statistical analysis of 20%, 50%, and 90% action potential durations (APDs) of LV myocytes isolated from Cad-11−/− and WT mice that underwent sham or TAC surgery (n = 6 mice each group). (E) Representative chart of L-type calcium current tracings in indicated mice. (F) Representative I-V curve of Ica,L. Current density-voltage (I–V) relationship of L-type calcium current were measured using patch clamp. (G) Maximum L-type calcium current density in the LV myocytes of WT or Cad-11 KO mice 4 weeks after sham or TAC operation (n = 6 mice in each group). (H) Representative chart of voltage-dependent outward potassium current tracings. (I) Summary graph of the current density of outward potassium currents of WT and Cad-11 KO mice 4 weeks after sham or TAC operation (n = 6 mice in each group). (J–L) The mRNA levels of the calcium channel subunit Cav1.2, potassium channel subunits Kv4.2, Kv4.3 (for Itof), and Kv1.5, Kv2.1 (for IKslow) determined by qRT-PCR. N = 4 mice in each group. Data were presented as mean ± SEM, ** p <…

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  Figure 4. Loss of Cad-11 decreased IL-6 production in transverse aortic constriction (TAC) mice. (A–C) Relative mRNA levels of inflammation-related factors MIF, TNF-α, and IL-6 in samples obtained from WT and Cad-11−/− mice LV tissues following 4 weeks of sham or TAC operation (n = 4 in each group). (D,E) Representative immunohistochemical staining of IL-6 using IL-6 antibody. Quantification of IL-6 expression in the LV of heart sections from the indicated mouse groups (n = 5 in each group) were summarized in (E). (F) Representative images of immunofluorescent staining of heart sections with anti-Vimentin (red), anti-IL-6 (green), and DAPI (blue) from WT-Sham, WT-TAC, Cad-11−/−-sham, and Cad-11−/−-TAC mice. (G) Average IL-6 concentrations (determined by ELISA) in serum in the indicated mouse groups. (n = 6 in each group). Data were presented as mean ± SEM, * p < 0.05, ** p < 0.01 vs. WT-Sham group. ## p < 0.01 vs. WT-TAC group.

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  Figure 5. IL-6 participates in Cad-11-induced cardiac fibroblast activation. (A) The IL-6 protein levels determined by ELISA in primary cardiac fibroblasts treated with 5 μg/mL hCad-11-Fc, hIgG1, or PBS for 48 h. (B) Primary cardiac fibroblasts were cultured with 20 μg/mL hIgG1 or the indicated concentrations (μg/mL) of hCad-11-Fc for 48 h and the supernatants were analyzed for IL-6 by ELISA (n = 5 in each group). (C,D) Representative images of wound-healing assay in CFs captured at 0 and 24 h with or without the stimulation of hCad-11-Fc and anti-IL-6. Scale bar, 100 μm. The relative speed of fibroblast migration was measured by its mean linear movement over wound edges at 24 h (n = 5 in each group). (E,F) Representative immunoblots and statistics of fibrosis-related protein levels with or without the stimulation of hCad-11-Fc and anti-IL-6 measured by western blotting. Data were presented as mean ± SEM, * p < 0.05, ** p < 0.01 vs. the hIgG group, IgG+Ctrl group. ## p < 0.01 vs. hCad-11-Fc+Ctrl group.

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  Figure 6. IL-6 mediates Cad-11-induced cardiomyocyte hypertrophy in NMVMs through a fibroblast-cardiomyocyte paracrine effect. (A) Schematic diagram of cardiac myocytes stimulated by conditioned medium collected from CFs infected with IgG or hCad-11-Fc. (B) Group information (C) Representative image of α-actinin staining for NMCMs from the indicated four mice groups. (D) Quantification of myocytes area for α-actinin staining of NMCMs in the indicated four mice groups (n = 5 in each group). (E–G) mRNA levels of cardiac hypertrophy–related genes ANP, BNP, and β-MHC using quantitative reverse transcription polymerase chain reaction from the indicated four groups of cells (n = 4 in each group). Data were presented as mean ± SEM, ** p < 0.01 vs. IgG conditional medium+Ctrl group. ## p < 0.01 vs. hCad-11-Fc conditional medium+Ctrl group.

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  Figure 7. IL (interleukin)-6-activated MAPK and CaMKII–STAT3 pathways participate in Cad-11-induced cardiac fibroblasts activation and paracrine effect-induced cardiomyocyte hypertrophy. (A–D) Representative immunoblots and statistics showing phosphorylated ERK, JNK, P38 and phosphorylated CaMKII, STAT3 protein levels in neonatal mouse ventricular fibroblasts (CFs). The cells were pretreated for 30 min with or without anti-IL-6 antibody followed by incubation with IgG or hCad-11-Fc for 48 h (n = 3 in each group). (E–H) Representative immunoblots and statistics showing phosphorylated ERK, JNK, P38, and phosphorylated CaMKII, STAT3 protein levels in neonatal mouse ventricular myocytes (NMVMs) pretreated for 30 min with or without anti-IL-6 antibody followed by incubation with the conditioned medium collected from CFs stimulated with IgG or hCad-11-Fc. Data were presented as mean ± SEM, * p < 0.05, ** p < 0.01 vs. IgG+Ctrl group, IgG conditional medium+Ctrl group. # p < 0.05, ## p < 0.01 vs. hCad-11-Fc+Ctrl group, hCad-11-Fc conditional medium+Ctrl group.

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  Figure 8. Loss of Cad-11 alleviates pressure overload-induced LV remodeling partially through MAPK and CaMKII–STAT3 pathways. (A,B) Representative western blotting and quantitative data of phosphorylation protein levels of ERK, JNK, and P38 (n = 3 in each group). (C,D) CaMKII, STAT3 in wild-type (WT) and Cad-11−/− mice LV tissues following a 4-week sham or TAC operation (n = 3 in each group). Quantification of each protein expression level was normalized to GAPDH levels. Data were presented as mean ± SEM, * p < 0.05, ** p < 0.01 vs. WT-Sham group. ## p < 0.01 vs. WT-TAC group.

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  Figure 9. Model illustrating that Cad-11-IL-6 signaling pathway mediated cardiac fibrosis and myocardial hypertrophy in TAC-Induced Heart Failure.