Fibroblast GSK-3α Promotes Fibrosis via RAF-MEK-ERK Pathway in the Injured Heart

Abstract

Background: Heart failure is the leading cause of mortality, morbidity, and health care expenditures worldwide. Numerous studies have implicated GSK-3 (glycogen synthase kinase-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms seem to play overlapping, unique and even opposing functions in the heart. Previously, we have shown that of the 2 isoforms of GSK-3, cardiac fibroblast GSK-3β acts as a negative regulator of myocardial fibrosis in the ischemic heart. However, the role of cardiac fibroblast-GSK-3α in the pathogenesis of cardiac diseases is completely unknown.

Methods: To define the role of cardiac fibroblast-GSK-3α in myocardial fibrosis and heart failure, GSK-3α was deleted from fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or Postn-promoter-driven Cre recombinase. Control and GSK-3α KO mice were subjected to cardiac injury and heart parameters were evaluated. The fibroblast kinome mapping was carried out to delineate molecular mechanism followed by in vivo and in vitro analysis.

Results: Fibroblast-specific GSK-3α deletion restricted fibrotic remodeling and preserved function of the injured heart. We observed reductions in cell migration, collagen gel contraction, α-SMA protein levels, and expression of ECM genes in TGFβ1-treated KO fibroblasts, indicating that GSK-3α is required for myofibroblast transformation. Surprisingly, GSK-3α deletion did not affect SMAD3 activation, suggesting the profibrotic role of GSK-3α is SMAD3 independent. The molecular studies confirmed decreased ERK signaling in GSK-3α-KO CFs. Conversely, adenovirus-mediated expression of a constitutively active form of GSK-3α (Ad-GSK-3α^S21A) in fibroblasts increased ERK activation and expression of fibrogenic proteins. Importantly, this effect was abolished by ERK inhibition.

Conclusions: GSK-3α-mediated MEK-ERK activation is a critical profibrotic signaling circuit in the injured heart, which operates independently of the canonical TGF-β1-SMAD3 pathway. Therefore, strategies to inhibit the GSK-3α-MEK-ERK signaling circuit could prevent adverse fibrosis in diseased hearts.

Keywords: cardiovascular diseases; fibroblasts; fibrosis; heart failure; myofibroblasts.

Figure 1:. Deletion of CF-GSK-3α from resident cardiac fibroblasts prevents pressure overload-induced cardiac dysfunction

(A) Experimental design. Two-month-old mice were fed tamoxifen (TAM) chow diet. After 2 weeks of TAM treatment, mice were subjected to TAC surgery and are maintained on the TAM diet till the end of the study. (B) Western blot analysis of GSK-3α protein levels in cardiac fibroblasts after TAM treatment. N=4 per group. Data were analyzed using the Mann-Whitney test and represented as mean ± SEM. Evaluation of cardiac function by m-mode echocardiography; (C) Ejection fraction (EF), (D) Fractional shortening (FS), For C and D – At BL, (N=6) per group. At 4 weeks, CTL-SHAM & CTL-TAC (N=6), KO-SHAM (N=8), and KO-TAC (N=7). At 8 weeks, CTL-SHAM (N=7), KO-SHAM (N=8), CTL-TAC & KO-TAC (N=6). (E) LV end-diastolic interior dimension (LVID;d) and (F) LV end-systolic interior dimension (LVID;s). For E and F – At BL, (N=6) per group. At 4 weeks, CTL-SHAM & CTL-TAC (N=6), KO-SHAM & KO-TAC (N=8). At 8 weeks, CTL-SHAM & CTL-TAC (N=6), KO-SHAM (N=8), and KO-TAC (N=7). Data (1C-1F) were analyzed using Two-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM. BL: Baseline

Figure 2:. Deletion of CF-GSK-3α from resident cardiac fibroblasts prevents pressure overload-induced adverse cardiac remodeling

Morphometric studies were performed 8 weeks after TAC surgery. Assessment of cardiac hypertrophy; (A) Heart weight (HW) to tibia length (TL) ratio, CTL-SHAM & CTL-TAC (N=6), KO-SHAM & KO-TAC (N=8), and (B) Quantification of cardiomyocyte cross-sectional area (CSA) CTL-SHAM & CTL-TAC (N=6), KO-SHAM (N=8), KO-TAC (N=9) and Representative images of HE staining. Data (2A-2B) were analyzed using Two-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM. Assessment of cardiac fibrosis by Masson’s Trichrome staining; (C) Representative Trichrome-stained LV regions and (D) Quantification of LV fibrosis. CTL-SHAM (N=4), KO-SHAM, CTL-TAC & KO-TAC (N=6) (E) Representative images of α-SMA staining, white arrows indicate α-SMA^+ve non-vascular cells, and, (F) Quantification of α-SMA^+ve non-vascular cells. Scale bar = 30 μm. N=3 per group. RNA was extracted from the left ventricle of experimental animals, and gene expression analysis was carried out by qPCR; The gene expression from each group was normalized to the CTL-SHAM group; (G) ANP, CTL-SHAM, CTL-TAC, & KO-TAC (N=6), KO-SHAM (N=5), (H) BNP, CTL-SHAM (N=6), KO-SHAM, CTL-TAC & KO-TAC (N=5) (I) COL1A1, CTL-SHAM & KO-SHAM (N=4), CTL-TAC & KO-TAC (N=5) and (J) COL3A1, CTL-SHAM & KO-SHAM (N=4), CTL-TAC & KO-TAC (N=6). Data (2D, 2F-2J) were analyzed using Kruskal-Wallis followed by Dunn test and represented as mean ± SEM.

Figure 3:. Deletion of CF-GSK-3α from myofibroblasts prevents pressure overload-induced cardiac dysfunction

(A) Experimental design. Two-month-old mice were fed tamoxifen (TAM) chow diet. After 1 week of TAM treatment, mice were subjected to TAC surgery and are maintained on the TAM diet till the end of the study. (B) Western blot analysis of GSK-3α protein levels in cardiac fibroblasts after TAM treatment. N=4 per group. Data were analyzed using the Mann-Whitney test and represented as mean ± SEM. Evaluation of cardiac function by m-mode echocardiography; (C) Ejection fraction (EF), (D) Fractional shortening (FS), For C and D – At BL, N=6 per group. At 4 weeks, CTL-SHAM, CTL-TAC & KO-TAC (N=6), KO-SHAM (N=7). At 8 weeks, CTL-SHAM & KO-SHAM (N=6), CTL-TAC & KO-TAC (N=7). (E) LV end-diastolic interior dimension (LVID;d) and (F) LV end-systolic interior dimension (LVID;s). For E and F – At BL, N=6 per group. At 4 weeks, CTL-SHAM & KO-TAC (N=6), KO-SHAM (N=7), and CTL-TAC (N=8). At 8 weeks, CTL-SHAM, KO-SHAM & KO-TAC (N=6), CTL-TAC (N=7). Data (3C-3F) were analyzed using Two-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM. BL: Baseline

Figure 4:. Deletion of CF-GSK-3α from myofibroblasts prevents pressure overload-induced adverse cardiac remodeling

Morphometric studies were performed 8 weeks after TAC surgery. Assessment of cardiac hypertrophy; (A) Heart weight (HW) to tibia length (TL) ratio, CTL-SHAM & KO-SHAM (N=8), CTL-TAC (N=9), KO-TAC (N=7). (B) Quantification of cardiomyocyte cross-sectional area (CSA) and Representative images of HE staining. Assessment of cardiac fibrosis by Masson’s Trichrome staining; (C) Representative Trichrome-stained LV regions and (D) Quantification of LV fibrosis. CTL-SHAM (N=3), KO-SHAM & KO-TAC (N=5), CTL-TAC (N=6) (E) Representative images of α-SMA staining, white arrows indicate α-SMA^+ve non-vascular cells and, (F) Quantification of α-SMA^+ve non-vascular cells. Scale bar = 30 μm. N=3 per group. RNA was extracted from the left ventricle of experimental animals, and gene expression analysis was carried out by qPCR; The gene expression from each group was normalized to the CTL-SHAM group; (G) ANP, CTL-SHAM & KO-SHAM (N=6), CTL-TAC & KO-TAC (N=7) (H) BNP, CTL-SHAM (N=7), KO-SHAM, CTL-TAC & KO-TAC (N=6) (I) COL1A1, CTL-SHAM (N=6), KO-SHAM, CTL-TAC & KO-TAC (N=6) and (J) COL3A1, N=6 per group. Data (4B, 4D, 4F) were analyzed using Kruskal-Wallis followed by Dunn test and represented as mean ± SEM. Data (4A, 4G-4J) were analyzed using Two-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM.

Figure 5:. CF-GSK-3α regulates TGF-β1-induced myofibroblast transformation

WT and GSK-3α KO mouse embryonic fibroblasts (MEFs) were treated with TGF-β1 (10 ng/mL). (A) Western blot analysis of α-SMA protein levels after 48h of TGFβ1 treatment; Representative blot and quantification WT(N=6), WT + TGFβ1, KO & KO + TGFβ1 (N=4). For wound closure assay, WT and GSK-3α KO MEFs were seeded and grown till confluency. A scratch was made followed by TGF-β1 (10 ng/mL) treatment, and wound closure was monitored; (B) Representative images and (C) Quantification of wound closure at 48h, N=3 per group. For gel contraction assay, collagen gels were populated with WT and GSK-3α KO MEFs followed by TGF-β1 (10 ng/mL) treatment. % Gel contraction was calculated after 48h of TGFβ1 treatment; (D) Representative image and (E) Quantification of gel contraction, N = 4 per group. RNA was extracted from cells after 24h of TGFβ1 treatment, and gene expression analysis was carried out by the qPCR method; the gene expression from each group was normalized to the WT group. For F and G, N=3 per group, (F) COL1A1, (G) Fibronectin −1. Data (5A, 5C, 5E-5G) were analyzed using Kruskal-Wallis followed by Dunn test and represented as mean ± SEM.

Figure 6:. CF-GSK-3α mediates pro-fibrotic effects independent of SMAD3

WT and GSK-3α KO MEFs were treated with TGF-β1 (10 ng/mL) for 1h. Western blot analysis of SMAD3 phosphorylation; (A) Representative blot and quantification. Additionally, nuclear-cytoplasmic extraction was carried out and SMAD3 protein levels were analyzed by Western blotting; (B) Representative blot, and quantification. For loss of function studies, CFs were isolated from adult GSK-3α^fl/fl mice and GSK-3α was deleted by adenoviral expression of Cre. After transduction, CFs were treated with TGF-β1 (10 ng/mL) for 1h. Western blotting was carried out; (C) Representative Western blot, and quantification of (D) GSK-3α and (E) SMAD3. For gain-of-function studies, GSK-3α was overexpressed in neonatal rat ventricular fibroblasts (NRVFs), and cells were treated with TGF-β1 (10 ng/mL, 1h). Western blotting was carried out; (F) Representative Western blot, and quantification of (G) GSK-3α and (H) SMAD3. N=3 per group for B & E; N=4 per group for A, D, G & H. Data (6A, 6B, 6E, 6H) were analyzed by Kruskal-Wallis followed by Dunn test and represented as mean ± SEM. Data (6D & 6G) were analyzed using the Mann-Whitney test and represented as mean ± SEM.

Figure 7:. CF-GSK-3α promotes fibrosis through the RAF-MEK-ERK signaling network

At 4 weeks post-TAC, proteins were extracted from CFs of CTL and GSK-3α^FKO mice, and comparative kinome profiling was carried out; (A) A hierarchically-clustered heatmap of kinomic peptide phosphorylation signal intensity (change from mean) demonstrates CTL and GSK-3α KO signatures (B) Table displays the BioNavigator generated Mean Final Score of GSK-3α KO altered kinases (C) that are mapped to the literature annotated ERK centric network model. N=3 per group. Input nodes (kinases) have large blue circles around them, with smaller circles in the top right. Arrowheads denote the direction of interaction, and the color of the lines indicates the type of interaction (green: positive, red: inhibitory, grey: complex). (D) At 4 weeks post-TAC, CFs were isolated from CTL and GSK-3α^FKO mice. ERK levels were analyzed by western blotting. Representative western blot and quantification. N=4 per group. (E) At 8 weeks post-TAC, CFs were isolated from CTL and GSK-3α^FKO mice. Flow cytometric analysis of pERK^+ve CFs (% total). N=8 per group. (F) WT and GSK-3α KO MEFs were treated with TGF-β1 (10 ng/mL) for 10 min and ERK levels were assessed by Western blotting. Representative blot and quantification, N=3 per group. The original order of lanes was rearranged to make the final representative image. (G) For loss of function studies, CFs were isolated from adult GSK-3α^fl/fl mice and GSK-3α was deleted by adenoviral expression of Cre. After transduction, CFs were treated with TGF-β1 (10 ng/mL) for 10min. Western blotting was carried out; representative Western blot, and quantification of ERK1/2, N=2 for LacZ and Ad-Cre group; N=3 for TGF-β1 treated groups. Data (7D & 7E) were analyzed using the Mann-Whitney test and represented as mean ± SEM. Data (7F & 7G) were analyzed by Kruskal-Wallis followed by the Dunn test and represented as mean ± SEM.

Figure 8:. CF-GSK-3α promotes fibrosis through the RAF-MEK-ERK signaling network

(A) For gain-of-function studies, mutant GSK-3α^S21A was overexpressed in NRVFs, and protein was extracted after 24h of transfection. Western blotting was performed to examine MEK and ERK levels. N=4 per group. (B) At 8 weeks post-TAC, CFs were isolated from CTL and GSK-3α^FKO mice. Flow cytometric analysis of IL-11^+ve CFs (% total). N=8 per group. (C) WT and GSK-3α KO MEFs were treated with TGFβ1 (10 ng/mL, 24h). IL-11 gene expression was analyzed by the qPCR method. The gene expression from each group was normalized to the WT group. N=3 per group. (D) For Western blotting, MEFs were treated with IL-11 for 10 min and ERK levels were assessed. Representative blot and quantification. N=4 per group. (E) Control and mutant GSK-3α^S21A overexpressing NRVFs were treated with ERK inhibitor (U0123, 10μM) for 24h. Culture supernatant was collected, and ELISA was carried out; quantification of collagen-1 and Il-11. N=3 per group. (F) Schematic showing interactions of GSK-3α with IL-11 and ERK signaling pathways in cardiac fibroblast. CF-GSK-3α mediates profibrotic effects through the ERK pathway in the injured heart while classical TGFβ1-SMAD3 signaling remained unaltered. Data (8A & 8B) were analyzed using the Mann-Whitney test and represented as mean ± SEM. Data (8C-8E) were analyzed by Kruskal-Wallis followed by the Dunn test and represented as mean ± SEM.